JP2005245202A - STRUCTURAL CHARACTERISTIC AND FUNCTION OF NEW ZINC BINDING DOMAIN OF HUMAN TFIIEalpha - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To find the structural characteristic and function of zinc binding domain of human TFIIEα. <P>SOLUTION: The method for predicting the stereostructure of protein containing zinc binding domain or a complex between the protein and a substance to be bound with the protein comprises (i) a process of preparing the three-dimensional structure of the zinc binding domain of human TFIIEα or its part defined by atomic coordinates in which the root mean square deviation in relation to a distance from the three-dimensional structure of the atomic coordinates of a table A or defined by the atomic coordinates of the table A is 0.0096±0.0012 angstrom and root mean square deviation in relation to a dihedral angle is 0.75±0.09° and a process (ii) of constructing the three-dimensional structure of protein containing a zinc binding domain or a complex between the protein and a substance to be bound with the protein by homology modeling that uses the three-dimensional structure prepared by the process (i) as a template. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to structural features and functions of a novel zinc-binding domain of human TFIIEα, and more particularly, predicts protein conformation utilizing the structural features and functions of a novel zinc-binding domain of human TFIIEα. The present invention relates to a method, a method for predicting the function of a protein, a method for screening a substance capable of regulating transcriptional activity, and a variant of a zinc binding domain of human TFIIEα.

  In eukaryotes, RNA polymerase (RNA Pol II) and basic transcription factors (GTF) TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH are required to initiate transcription of the gene encoding the protein. RNA Pol II and GTF are assembled on the promoter DNA to form a pre-initiation complex (PIC) (Non-Patent Documents 1 and 2). The in vitro transcription reconstitution system proposed a stepwise assembly model for PIC formation as follows. That is, TATA box binding protein (TBP), one of the components of TFIID, recognizes the TATA box 25-30 bp upstream from the transcription start site. After formation of the TBP (TFIID) -DNA complex, TFIIA and TFIIB enter. RNA Pol II can then be incorporated into the complex by TFIIF. Eventually, TFIIE and TFIIH are incorporated into the complex to complete PIC formation. Another PIC formation model is that a huge holoenzyme pre-assembled by RNA Pol II, GTF, several transcription factors and mediators binds directly to promoter DNA (Non-Patent Documents 3 and 4) . PIC is activated through promoter melting, synthesis of the first phosphodiester bond of the transcript being generated, and promoter clearance (Non-Patent Documents 5, 6, 7). Next, the productive RNA Pol II proceeds to the chain extension stage.

  TFIIE interacts with various GTF, RNA Pol II and promoter DNA (Non-patent Document 8). TFIIE incorporates TFIIH into PIC (Non-Patent Document 9), and exhibits TFIIH enzymatic activity, namely phosphorylation of the C-terminal domain of the largest subunit (CTD) of RNA Pol II, DNA-dependent ATPase activity and DNA helicase activity. Adjust (Non-Patent Documents 5, 6, 10). Furthermore, TFIIE is located near the active site of RNA Pol II (Non-Patent Document 11) and contributes to promoter melting (Non-Patent Documents 5, 6, 10) and promoter clearance (Non-Patent Documents 12, 13, 14). . These data indicated that TFIIE is a multifunctional protein and plays an important role in both the initiation and initiation-to-chain extension transition of transcription.

Human TFIIE is a heterotetramer composed of two large subunits (TFIIEα; 57 kDa, 439 aa) and two small subunits (TFIIEβ; 34 kDa, 291 aa) (Non-patent Documents 15, 16, and 17). , 18). Both of these subunits have several characteristic sequences and putative structural motifs. Despite being a very important protein, there is very little information about the structure. This is mainly due to the low solubility of TFIIE and each subunit at the high concentrations used for structural studies.
Orphanides, G., Lagrange, T. & Reinberg, D. The general transcription factors of RNA polymerase II. Genes Dev. 10, 2657-2683 (1996). Roeder, RG The role of general initiation factors in transcription by RNA polymerase II.Trends Biochem.Sci. 21, 327-335 (1996). Bjorklund, S. & Y.-J. Kim. Mediator of transcriptional regulation.Trends Biochem. Sci. 21, 335-337 (1996). Koleske, AJ & RAYoung.The RNA polymerase II holoenzyme and its implication for gene regulation.Trends Biochem.Sci. 20, 113-116 (1995). Lu, H., Flores, O., Weinmann, R. & Reinberg, D. The nonphosphorylated form of RNA polymerase II preferentially associates with the preinitiation complex.Proc. Natl. Acad. Sci. USA 88, 10004-10008 (1991) . Ohkuma, Y. & Roeder, RG Regulation of TFIIH ATPase and kinase activities by TFIIE during active initiation complex formation.Nature 368, 160-163 (1994). Dvir, A., K. et al. A role for ATP and TFIIH in activation of the RNA polymerase II preinitiation complex prior to transcription initiation.J. Biol. Chem. 271, 7245-7248 (1996). Maxon, ME, Goodrich, JA & Tjian, R. Transcription factor IIE binds preferentially to RNA polymerase IIa and recruits TFIIH: a model for promoter clearance. Genes Dev. 8, 515-524 (1994). Flores, O., Lu, H. & Reinberg, D. Factors involved in specific transcription by mammalian RNA polymerase II. J. Biol. Chem. 267, 2786-2793 (1992). Drapkin, R. et al. Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II.Nature 368, 769-772 (1994). Leuther, KK, Bushnell, DA & Kornberg, RD Two-dimensional crystallography of TFIIB- and IIE-RNA polymerase II complexes: Implications for start site selection and initiation complex formation.Cell 85, 773-779 (1996). Goodrich, JA & Tjian, R. Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II.Cell 77, 145-156 (1994). Dvir, A., Conaway, RC & Conaway, JW A role for TFIIH in controlling the activity of early RNA polymerase II elongation complexes.Proc. Natl. Acad. Sci. USA 94, 9006-9010 (1997). Kumar, KP, Akoulitchev, S. & Reinberg, D. Promoter-proximal stalling results from the inability to recruit transcription factor IIH to the transcription complex and is a regulated event.Proc. Natl. Acad. Sci. USA 95, 9767-9772 (1998). Ohkuma, Y., Sumimoto, H., Horikoshi, M. & Roeder, RG Factors involved in specific transcription by mammalian RNA polymerase II: Purification and characterization of general transcription factor TFIIE. Proc. Natl. Acad. Sci. USA 87, 9163 -9167 (1990). Ohkuma, Y. et al. Structural motifs and potential σ homologies in the large subunit of human general transcription factor TFIIE.Nature 354, 398-402 (1991). Peterson, MG et al. Structure and functional properties of human general transcription factor IIE.Nature 354, 369-373 (1991). Sumimoto, H. et al. Conserved sequence motifs in the small subunit of human general transcription factor TFIIE.Nature 354, 401-404 (1991).

Thus, the inventors focused on the multiple functions of this protein and attempted to identify the structure / function domains within each subunit. Protease limited degradation experiments found a highly conserved zinc-binding region containing the Cx ₂ -Cx _n -Cx ₂ -C (x is any amino acid) sequence within TFIIEα (FIG. 1a). This region is present in the N-terminal half, sufficient for both conserved basal and activated transcription (Ohkuma, Y., Hashimoto, S., Wang, CK, Horikoshi, M. & Roeder, RG Analysis of the Role of TFIIE in basal transcription and TFIIH-mediated carboxy-terminal domain phosphorylation through structure-function studies of TFIIE-α. Mol. Cell. Biol. 15, 4856-4866 (1995)). Analysis of point mutations in the third cysteine residue of the zinc ligand showed that TFIIE transcriptional activity was dependent on zinc binding (Maxon, ME & Tjian, R. Transcriptional activity of transcription factor IIE is dependent on zinc binding. Proc. Natl. Acad. Sci. USA 91, 9529-9533 (1994).). Similarly, deletion mutants of the zinc binding domain show a dominant negative effect on transcription (Ohkuma, Y., Hashimoto, S., Wang, CK, Horikoshi, M. & Roeder, RG Analysis of the Role of TFIIE in basal transcription and TFIIH-mediated carboxy-terminal domain phosphorylation through structure-function studies of TFIIE-α. Mol. Cell. Biol. 15, 4856-4866 (1995)). Homology modeling based on distance geometry predicted that the zinc binding domain would have a zinc ribbon structure (Qian, X. et al. Novel zinc finger motif in the basal transcriptional machinery: Three-dimensional NMR studies of the nucleic acid binding domain of transcriptional e longation factor TFIIS. Biochemistry 32, 9944-9959 (1993).). In this study, we determined the solution structure of the human TFIIE alpha zinc binding domain and provided structural insights about its role in TFIIE function.

  The present invention aims to find the structural features and functions of a novel zinc binding domain of human TFIIEα.

The basic transcription factor TFIIE is a heterotetramer composed of two large subunits (TFIIEα) and two small subunits (TFIIEβ). A highly conserved TFIIEα zinc binding domain with a Cx ₂ Cx _n Cx ₂ C motif is required for transcriptional activity. This domain was predicted to form a zinc ribbon structure. However, the structure determined by the inventors is surprisingly different from the zinc ribbon and other zinc-bonded structures. To elucidate the function of the novel structure, we performed mutation analysis of full-length TFIIEα for transcriptional activity and ability to bind TFIIEβ and other basic transcription factors. Interestingly, the cysteine → alanine point mutations, ie the zinc binding sites C129A, C132A, C154A and C157A, showed asymmetry in both assays. CD, NMR and EDTA titration experiments of zinc-binding domains with mutated Zn ²⁺ -ligands show that these domains have their own partially folded structure that can coordinate with Zn ²⁺ It has been clarified that this is equilibrated to various degrees with the random coil structure. The dependence of structural stability on the position of the ligand explains the above asymmetry. Taken together, our biochemical and structural data, as well as previously reported results, we have linked the role of the zinc binding domain. The present invention has been completed based on these findings.

The gist of the present invention is as follows.
[1] A method for predicting the three-dimensional structure of a protein comprising a zinc-binding domain or a complex of the protein and a substance capable of binding to the protein,
(i) The root mean square deviation for the distance from the three-dimensional structure defined by the atomic coordinates in Table A or the atomic coordinates in Table A is 0.0096 ± 0.0012 angstroms, and the root mean square deviation for the dihedral angle is 0.75 ± 0.09 ° Providing a three-dimensional structure of a human TFIIEα zinc-binding domain or part thereof defined by atomic coordinates of:
(ii) Build a three-dimensional structure of a protein containing a zinc-binding domain or a complex of the protein and a substance capable of binding to the protein by homology modeling using the three-dimensional structure prepared in step (i) as a template Said method comprising the steps.
[2] A method for predicting the function of a protein containing a zinc-binding domain or a part thereof,
(i) The root mean square deviation with respect to the distance from the three-dimensional structure defined by the atomic coordinates in Table A or the atomic coordinates in Table A is 0.0096 ± 0.0012 angstroms, and the root mean square deviation with respect to the dihedral angle is 0.75 ± 0.09 ° Providing a three-dimensional structure of a human TFIIEα zinc-binding domain or part thereof defined by atomic coordinates
(ii) providing a three-dimensional structure of a zinc-binding domain of a protein containing a zinc-binding domain or a part thereof;
(iii) a step of performing structural alignment between the three-dimensional structure prepared in the step (i) and the three-dimensional structure prepared in the step (ii);
(iv) When it is determined that there is structural similarity as a result of the structural alignment performed in the step (iii), the protein containing the zinc-binding domain of (ii) or a part thereof is Said method comprising the step of predicting that it has a function similar to the zinc-binding domain of TFIIEα or a part thereof.
[3] A human TFIIEα zinc-binding domain having the amino acid sequence of SEQ ID NO: 2 or a protein containing the domain is glutamic acid at position 28, aspartic acid at position 52, cysteine at position 17, cysteine at position 20, cysteine at position 42 And at least one amino acid site selected from the group consisting of cysteine at position 45, it is analyzed whether or not it interacts with the test substance, and if it interacts, the test substance can regulate the transcriptional activity. A method of screening for a substance capable of regulating transcriptional activity.
[4] In the presence of zinc ions, the human TFIIEα zinc-binding domain having the amino acid sequence of SEQ ID NO: 2 or a protein containing the domain is glutamic acid at position 28, aspartic acid at position 52, cysteine at position 17, and position 20 The method according to [3], wherein at least one amino acid site selected from the group consisting of cysteine, cysteine at position 42 and cysteine at position 45 is analyzed for interaction with the test substance.
[5] A zinc-binding domain mutant protein of TFIIEα, which is a protein of any one of the following (a), (b), (c) or (d), or a salt thereof.
(a) In the amino acid sequence of SEQ ID NO: 2, substitution of glutamic acid at position 28 with alanine or lysine, substitution of cysteine at position 17 with alanine, substitution of cysteine at position 20 with alanine, cysteine at position 42 with alanine And a protein having an amino acid sequence in which at least one substitution selected from the group consisting of substitution at position 45 and substitution from cysteine to alanine at position 45 is made
(b) In the amino acid sequence of the protein of (a), one or several amino acids other than the 28th amino acid, the 17th amino acid, the 20th amino acid, the 42nd amino acid and the 45th amino acid are deleted or substituted. Alternatively, a protein having an added amino acid sequence and having a transcriptional activity lower than that of the wild-type TFIIEα zinc-binding domain having the amino acid sequence of SEQ ID NO: 2.
(c) a protein having an amino acid sequence in which substitution of aspartic acid at position 52 with alanine or lysine is performed in the amino acid sequence of SEQ ID NO: 2
(d) the amino acid sequence of the protein of (c), which has an amino acid sequence in which one or several amino acids other than the amino acid at position 52 are deleted, substituted or added, and whose transcription activity is SEQ ID NO: 2 A protein higher than the zinc-binding domain of wild-type TFIIEα having [6] The zinc-binding domain mutant protein of TFIIEα or a salt thereof according to [5], which is a protein of any one of the following (1) to (8):
(1) a protein having an amino acid sequence in which glutamic acid at position 28 is substituted with alanine in the amino acid sequence of SEQ ID NO: 2
(2) A protein having an amino acid sequence in which glutamic acid at position 28 is substituted with lysine in the amino acid sequence of SEQ ID NO: 2
(3) A protein having an amino acid sequence in which the cysteine at position 17 is substituted with alanine in the amino acid sequence of SEQ ID NO: 2.
(4) In the amino acid sequence of SEQ ID NO: 2, a protein having an amino acid sequence in which substitution of cysteine to alanine at position 20 is made
(5) A protein having an amino acid sequence in which cysteine is substituted with alanine at position 42 in the amino acid sequence of SEQ ID NO: 2.
(6) A protein having an amino acid sequence in which the cysteine at position 45 is substituted with alanine in the amino acid sequence of SEQ ID NO: 2.
(7) a protein having an amino acid sequence in which aspartic acid is substituted with alanine at position 52 in the amino acid sequence of SEQ ID NO: 2
(8) A protein encoding the protein according to [7] [5], which has an amino acid sequence in which the substitution of aspartic acid to lysine at position 52 in the amino acid sequence of SEQ ID NO: 2 is performed.
[8] A recombinant vector containing the DNA according to [7].
[9] A transformant comprising the recombinant vector according to [8].
[10] A method for producing a TFIIEα zinc-binding domain mutant protein comprising culturing a host transformed with the DNA according to [7] and collecting the TFIIEα zinc-binding domain mutant protein from the culture.
[11] An antibody against the TFIIEα zinc-binding domain mutant protein or a salt thereof according to [5].

  In this specification, “TFIIE” refers to one of six proteins called basic transcription factors, which are cofactors of RNA polymerase II that performs transcription of a gene encoding a protein in eukaryotes. Say.

  “TFIIEα” refers to a large α subunit among the constituent subunits of TFIIE, which is a heterotetramer of α2β2 composed of two types of subunits, α and β.

And and "TFIIEα zinc binding domain of the""TFIIEα zinc binding domain" of the four cysteines coordinating the zinc ion in TFIIEα cysteine -x ₂ - cysteine -x _n - cysteine -x ₂ - cysteine (x is an arbitrary Of amino acid) and a region having a specific three-dimensional structure.

  “Mutant protein” means a protein that is different from the reference protein but retains the essential properties of the reference protein. A typical mutant protein differs in amino acid sequence from the reference protein.

  “Antibody” means a protein that is induced in an organism by an immune reaction as a result of antigen stimulation and has an activity of specifically binding to an immunogen (antigen). Examples include antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, and Fab or Fab fragments.

“Interaction” means that a force works between two or more objects (for example, atoms and molecules). Examples of the interaction include hydrophilic interaction (for example, hydrogen bond, salt bridge), hydrophobic interaction (for example, hydrophobic bond), electrostatic interaction, van der Waals interaction, and the like.

  According to the present invention, the three-dimensional structure and function of a protein having a sequence and / or structure similar to the zinc binding domain of TFIIEα can be predicted.

  In addition, according to the present invention, it has become possible to screen for substances that can regulate transcriptional activity.

Furthermore, the present invention provides a mutant whose transcriptional activity is lower or higher than that of the wild-type human TFIIEα zinc-binding domain.

Hereinafter, the present invention will be described in more detail.
1. Prediction of a three-dimensional structure of a protein containing a zinc-binding domain or a complex thereof by homology modeling The present invention predicts a three-dimensional structure of a protein containing a zinc-binding domain or a complex of the protein and a substance capable of binding to the protein A method,
(i) The root mean square deviation with respect to the distance from the three-dimensional structure defined by the atomic coordinates in Table A or the atomic coordinates in Table A is 0.0096 ± 0.0012 angstroms, and the root mean square deviation with respect to the dihedral angle is 0.75 ± 0.09 ° Providing a three-dimensional structure of a human TFIIEα zinc-binding domain or part thereof defined by atomic coordinates of:
(ii) Build a three-dimensional structure of a protein containing a zinc-binding domain or a complex of the protein and a substance capable of binding to the protein by homology modeling using the three-dimensional structure prepared in step (i) as a template The method is provided comprising the steps of:

  Homology modeling is a technique for predicting the structure of a protein with an unknown structure based on a protein with a similar structure (template) having a similar sequence.

  In homology modeling, first, similar sequences are searched from a structural database, and sequences are aligned. Next, the structure of the corresponding part is selected from the aligned template sequences to construct a predicted structure. Homology modeling includes a fragment-based method and a constraint-based method.

  As a website for carrying out homology modeling, MODELLER (web address: http://guitar.rockefeller.edu/moduller/moduller.html, literature: Sali et al. (1995) Proteins 23: 318-326), SWISS-MODEL (web address: http://www.expasy.ch/swissmod/SWISS-MODEL.html, literature: Peitsch (1996) Biochem. Soc. Trans. 24: 274-279), WHAT IF (web address: http://www.cmbi.kun.nl/whatif/, literature: Rodriguez et al. (1998) Bioinfomatics 14: 523-528), which can be used.

  In addition, calculations of molecular distance, angle, threading, and energy minimization of sequences analyzed and superimposed by these websites are performed by Swiss-PdbViewer (web address: http://www.expasy.ch/spdbv/mainpage .html, literature: Guex and Peitsch, 1977, Electrophoresis 18: 2714-2723), and a protein model can be created.

  Homology modeling is effective when there is a similarity of about 25 to 30% or more with a protein having a known structure.

  Therefore, the three-dimensional structure prediction method of the present invention is effective for predicting the three-dimensional structure of a protein or a complex thereof having a sequence similarity of about 25 to 30% or more compared to the amino acid sequence of SEQ ID NO: 2.

Examples of the protein containing a zinc binding domain include TFIIEα, TFIIB, RNA polymerase II, TFIIS, and the like. Examples of substances that can bind to TFIIEα include TFIIEβ, TFIIF, TFIIH, RNA polymerase II, and the like. The zinc-binding domain of TFIIB and TFIIS can bind to RNA polymerase II.

2. Prediction of the function of a protein containing a zinc binding domain or part thereof The present invention is a method for predicting the function of a protein containing a zinc binding domain or part thereof,
(i) The root mean square deviation with respect to the distance from the three-dimensional structure defined by the atomic coordinates in Table A or the atomic coordinates in Table A is 0.0096 ± 0.0012 angstroms, and the root mean square deviation with respect to the dihedral angle is 0.75 ± 0.09 ° Providing a three-dimensional structure of a human TFIIEα zinc-binding domain or part thereof defined by atomic coordinates
(ii) providing a three-dimensional structure of a zinc-binding domain of a protein containing a zinc-binding domain or a part thereof;
(iii) a step of performing structural alignment between the three-dimensional structure prepared in the step (i) and the three-dimensional structure prepared in the step (ii);
(iv) When it is determined that there is structural similarity as a result of the structural alignment performed in the step (iii), the protein containing the zinc-binding domain of (ii) or a part thereof is The method is provided comprising the step of predicting a function similar to that of a zinc-binding domain of TFIIEα or a part thereof.

  Hereinafter, the zinc-binding domain of human TFIIEα of (i) or a part thereof may be referred to as “reference protein”, and the zinc-binding domain of a protein containing the zinc-binding domain of (ii) or a part thereof may be referred to as “query protein”. .

  The three-dimensional structure coordinates of the query protein may be obtained by techniques such as X-ray crystal structure analysis or nuclear magnetic resonance (NMR), or already registered in the database (Brookhaven Protein Data Bank (PDB)). It may be what has been done. PDB files are readily available from the PDB website (http://www.rcsb.org/pdb/).

  When doing structural alignment, the three-dimensional structure of one protein domain is superimposed on the three-dimensional structure of another protein domain, and atoms are aligned as close as possible to minimize the average deviation between them. Like that. Methods for conducting protein structure comparison are described in Blundel and Johonson, 1993, Protein Sci. 2: 877-883; Holm and Sander, 1994, J. Mol. Biol. 233: 123-138; Holm and Sander, 1996, Science 273: 595-603; Alexandrov and Fischer, 1996, Proteins 25: 354-365; Gibrat et al. 1996, Curr. Opin.Struct. Biol. 6: 377-385; Orengo and Taylor, 1996, Methods Enzymol. 266 : 617-635. In addition, SSAP (Orengo and Taylor, 1996, Methods Enzymol. 266: 617-635), DALI (Vriend and Sander, 1991, Proteins 11: 52-68) are programs for comparing protein structures. . As a result of the structural alignment, the coordinates of the matched region are obtained. The aligned region may be displayed with a molecular display program such as RasMol, Cn3D, Swiss-PbdViewer.

  If the structural alignment results in structural similarity with the reference protein (ie, the human TFIIEα zinc-binding domain of (i) or a portion thereof), the query protein (ie, (ii) zinc-binding) It is predicted that the protein containing the domain or a part thereof has a function similar to that of the reference protein.

  The function of the reference protein (ie, the human TFIIEα zinc-binding domain of (i) or a part thereof) is to control transcription activity, interact with other factors, etc., as an antigen, as an immunogen However, it is not limited to these.

Examples of the protein containing a zinc binding domain include TFIIEα, TFIIB, RNA polymerase II, TFIIS, and the like.

3. Screening method The present invention relates to a human TFIIEα zinc-binding domain having the amino acid sequence of SEQ ID NO: 2 or a protein containing the domain comprising glutamic acid at position 28, aspartic acid at position 52, cysteine at position 17, cysteine at position 20, Whether or not it interacts with the test substance at at least one amino acid site selected from the group consisting of cysteine at the position and cysteine at the 45th position, and if so, the test substance regulates transcriptional activity There is provided a method of screening for a substance capable of regulating transcriptional activity, comprising determining that it can. In the method of the present invention, the zinc binding domain of human TFIIEα having the amino acid sequence of SEQ ID NO: 2 or a protein containing the domain in the presence of zinc ion is glutamic acid at position 28, aspartic acid at position 52, cysteine at position 17 It may be analyzed whether or not it interacts with a test substance at at least one amino acid site selected from the group consisting of cysteine at position 20, cysteine at position 42 and cysteine at position 45.

  An example of a protein containing the human TFIIEα zinc-binding domain having the amino acid sequence of SEQ ID NO: 2 is human TFIIEα. The amino acid sequence of wild type human TFIIE α is shown in SEQ ID NO: 19.

  The human TFIIEα zinc-binding domain having the amino acid sequence of SEQ ID NO: 2 or a protein containing the domain is glutamic acid at position 28, aspartic acid at position 52, cysteine at position 17, cysteine at position 20, cysteine at position 42, and position 45 In order to analyze whether or not it interacts with a test substance at at least one amino acid site selected from the group consisting of cysteine, three-dimensional structures such as X-ray crystal structure analysis, nuclear magnetic resonance (NMR), and neutron diffraction Method of observing the shape of the complex such as analysis, electron microscope and atomic force microscope, and the stable binding mode with the test substance of any structure for the three-dimensional structure of the human TFIIEα zinc binding domain having the amino acid sequence of SEQ ID NO: 2 A method such as a docking study for computer simulation of the human TFIIEα zinc binding disclosed herein by the present inventors It can be used judgment based on the similarity of the main conformation.

A substance capable of regulating transcriptional activity can be used for prevention and / or treatment of diseases involving transcription (for example, cancer, rheumatism, lifestyle-related diseases, genetic diseases, etc.).

4). TFIIEα Zinc-binding Domain Mutant Protein The present invention provides TFIIEα zinc-binding domain mutant protein or a salt thereof, which is one of the following proteins (a), (b), (c) or (d): .
(a) In the amino acid sequence of SEQ ID NO: 2, substitution of glutamic acid at position 28 with alanine or lysine, substitution of cysteine at position 17 with alanine, substitution of cysteine at position 20 with alanine, cysteine at position 42 with alanine And a protein having an amino acid sequence in which at least one substitution selected from the group consisting of substitution at position 45 and substitution from cysteine to alanine at position 45 is made.
(b) In the amino acid sequence of the protein of (a), one or several amino acids other than the 28th amino acid, the 17th amino acid, the 20th amino acid, the 42nd amino acid and the 45th amino acid are deleted or substituted. Alternatively, a protein having an added amino acid sequence and having a transcription activity lower than that of the zinc-binding domain of wild-type TFIIEα having the amino acid sequence of SEQ ID NO: 2.
(c) A protein having an amino acid sequence in which substitution of aspartic acid at position 52 with alanine or lysine is performed in the amino acid sequence of SEQ ID NO: 2.
(d) the amino acid sequence of the protein of (c), which has an amino acid sequence in which one or several amino acids other than the amino acid at position 52 are deleted, substituted or added, and whose transcription activity is SEQ ID NO: 2 A protein higher than the zinc-binding domain of wild-type TFIIEα.

Examples of the TIFIEα zinc-binding domain mutant proteins (a) and (c) include the following proteins (1) to (8).
(1) A protein having an amino acid sequence in which the glutamic acid at position 28 is substituted with alanine in the amino acid sequence of SEQ ID NO: 2.
(2) A protein having an amino acid sequence in which glutamic acid at position 28 is substituted with lysine in the amino acid sequence of SEQ ID NO: 2.
(3) A protein having an amino acid sequence in which cysteine is substituted with alanine at position 17 in the amino acid sequence of SEQ ID NO: 2.
(4) A protein having an amino acid sequence in which substitution of cysteine to alanine at position 20 is made in the amino acid sequence of SEQ ID NO: 2.
(5) A protein having an amino acid sequence in which a cysteine at position 42 is substituted with alanine in the amino acid sequence of SEQ ID NO: 2.
(6) A protein having an amino acid sequence in which cysteine is substituted with alanine at position 45 in the amino acid sequence of SEQ ID NO: 2.
(7) A protein having an amino acid sequence in which substitution of aspartic acid to alanine at position 52 in the amino acid sequence of SEQ ID NO: 2 is performed.
(8) A protein having an amino acid sequence in which substitution of aspartic acid to lysine at position 52 in the amino acid sequence of SEQ ID NO: 2 is performed.

  The amino acid sequence of the protein (1) (hereinafter sometimes referred to as “E140A”) is shown in SEQ ID NO: 4.

  The amino acid sequence of the protein (2) (hereinafter sometimes referred to as “E140K”) is shown in SEQ ID NO: 6.

  The amino acid sequence of the protein (3) (hereinafter sometimes referred to as “C129A”) is shown in SEQ ID NO: 12.

  The amino acid sequence of the protein (4) (hereinafter sometimes referred to as “C132A”) is shown in SEQ ID NO: 14.

  The amino acid sequence of the protein (5) (hereinafter sometimes referred to as “C154A”) is shown in SEQ ID NO: 16.

  The amino acid sequence of the protein (6) (hereinafter sometimes referred to as “C157A”) is shown in SEQ ID NO: 18.

  The amino acid sequence of the protein (7) (hereinafter sometimes referred to as “D164A”) is shown in SEQ ID NO: 8.

  The amino acid sequence of the protein (8) (hereinafter sometimes referred to as “D164K”) is shown in SEQ ID NO: 10.

  (b) and (d) TFIIE α zinc binding domain mutant proteins include non-human organisms (eg, non-human mammals (eg, mice), amphibians (eg, Xenopus), insects (eg, Drosophila, etc.) ), Mutants of TFIIEα zinc-binding domain derived from animals such as linear animals (eg nematodes), fungi (eg fission yeast, budding yeast, etc.), SEQ ID NOs: 4, 6, 8, 10, 12 An amino acid sequence in which methionine is added to the N-terminus (ie, position 1) of amino acid sequences 14, 14, and 18, an amino acid sequence in which two or more extra amino acid residues are added, or an extra amino acid sequence such as a His tag Examples include proteins having added amino acid sequences.

  The TFIIEα zinc-binding domain mutant protein of the present invention may be a human-derived wild-type TFIIEα zinc-binding domain mutant protein, even if it is a non-human organism (eg, a non-human mammal (eg, mouse), amphibian ( Wild type TFIIEα zinc-binding domain derived from animals such as Xenopus, insects (eg, Drosophila), linear animals (eg, nematodes), fungi (eg, fission yeast, budding yeast, etc.) It may be a mutant.

  The amino acid sequence of wild type human TFIIE α is shown in SEQ ID NO: 19. In the amino acid sequence of SEQ ID NO: 19, positions 113 to 174 are zinc binding domains. The amino acid sequence of SEQ ID NO: 2 is a fragment from arginine at position 113 to arginine at position 174 in the amino acid sequence of SEQ ID NO: 19. The amino acid positions at positions 17, 20, 28, 42, 45 and 52 in the amino acid sequence of SEQ ID NO: 2 correspond to the amino acid positions at positions 129, 132, 140, 154, 157 and 164 in the amino acid sequence of SEQ ID NO: 19, respectively. To do.

  The TFIIE α zinc-binding domain mutant protein and salts thereof of the present invention can be produced by known methods. For example, as described in 5 below, a DNA encoding a TFIIE α zinc binding domain mutant protein is obtained, and the obtained DNA is incorporated into an appropriate expression vector and then introduced into an appropriate host to obtain a recombinant protein. As a result, it is possible to produce a TFIIE α zinc-binding domain mutant protein (for example, Jun Saigo, Yoko Sano, CURRENT PROTOCOLS Compact Edition, Molecular Biology Experiment Protocol, I, II, III, Maruzen Co., Ltd.) : See, Original, Ausubel, FM et al., Short Protocols in Molecular Biology, Third Edition, John Wiley & Sons, Inc., New York).

  Alternatively, the TFIIE α zinc-binding domain mutant protein and salts thereof of the present invention can also be produced according to known peptide synthesis methods.

  The TFIIE α zinc binding domain mutant protein of the present invention can also be obtained in the form of a salt by a known method.

The salt of the TFIIE α zinc-binding domain mutant protein of the present invention may be a pharmacologically acceptable salt, and a pharmacologically acceptable acid addition salt is particularly preferable. Pharmacologically acceptable acid addition salts include salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid). Examples thereof include salts with acid, succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid.

5). DNA encoding TFIIEα zinc binding domain mutant protein
The DNA encoding the TFIIE α zinc-binding domain mutant protein of the present invention may be any DNA as long as it contains a base sequence encoding the TFIIE α zinc-binding domain mutant protein of the present invention. Examples of the DNA encoding the TFIIE α zinc binding domain mutant protein of the present invention include DNA having any one of the nucleotide sequences of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15 or 17.

  The DNA encoding the TFIIE α zinc-binding domain mutant protein of the present invention can be produced, for example, as follows.

  Wild-type human TFIIEα is a reverse-phase high-speed liquid from TFIIE (Ohkuma, Y. et al., Proc Natl Acad Sci USA. 87, 9163-9167, 1990) purified from transcriptional activity from HeLa cells, which are cultured human cells. The two subunits α and β are separated and purified by chromatography, and the partial sequence of the cDNA is amplified by PCR based on the amino acid sequence of the peptide fragment obtained by restricting the α subunit. The full-length cDNA is isolated by screening a NAM cell cDNA library (Ohkuma, Y. et al., Nature, 354, 398-401, 1991). This human TFIIEα cDNA is incorporated into pBluescriptII-SK (-) and has an NdeI cleavage site that spans the 289th and 290th codons of the protein coding region. In order to crush the site and create a new NdeI recognition site at the N-terminus and a BamHI recognition site 3 'downstream of the C-terminal stop codon, mutations were introduced using the Mutan-K mutagenesis kit (TaKaRa). To do. This was excised at the NdeI recognition site and the BamHI recognition site and incorporated into a pET-11d vector (Novagen) capable of expressing a 6His-tagged protein with 6 histidine residues added to the N-terminal side, resulting in a wild-type human TFIIEα expression plasmid .

  Zinc-binding domain mutants (E140A, E140K, D164A, D164K, C129A, C132A, C154A, C157A) were also synthesized with oligo DNAs to mutate each amino acid, and the above Mutan-K mutagenesis kit Using (TaKaRa), a mutation was introduced into wild-type human TFIIEα incorporated into pBluescriptII-SK (-), and this was also excised at the NdeI recognition site and the BamHI recognition site as described above. It is incorporated into a pET-11d vector (Novagen) capable of expressing a 6His-tagged protein with one histidine residue added.

In the amino acid sequence of SEQ ID NO: 2, an example of a base sequence of DNA encoding a protein (E140A) having an amino acid sequence in which glutamic acid at position 28 is substituted with alanine is shown in SEQ ID NO: 3. In the amino acid sequence of SEQ ID NO: 2, an example of the base sequence of DNA encoding a protein (E140K) having an amino acid sequence in which glutamic acid at position 28 is substituted with lysine is shown in SEQ ID NO: 5. In the amino acid sequence of SEQ ID NO: 2, an example of the base sequence of DNA encoding a protein (C129A) having an amino acid sequence in which cysteine is substituted with alanine at position 17 is shown in SEQ ID NO: 11. In the amino acid sequence of SEQ ID NO: 2, an example of the base sequence of DNA encoding a protein (C132A) having an amino acid sequence in which substitution of cysteine at position 20 with alanine is made is shown in SEQ ID NO: 13. In the amino acid sequence of SEQ ID NO: 2, an example of a base sequence of DNA encoding a protein (C154A) having an amino acid sequence in which cysteine is substituted with alanine at position 42 is shown in SEQ ID NO: 15. In the amino acid sequence of SEQ ID NO: 2, an example of a base sequence of DNA encoding a protein (C157A) having an amino acid sequence in which cysteine is substituted with alanine at position 45 is shown in SEQ ID NO: 17. An example of the base sequence of DNA encoding a protein (D164A) having an amino acid sequence in which the substitution of aspartic acid at position 52 with alanine in the amino acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 7. An example of the base sequence of DNA encoding a protein (D164K) having an amino acid sequence in which the substitution of aspartic acid to lysine at position 52 in the amino acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 9.

6). Recombinant Vector A recombinant vector containing DNA encoding the TFIIE α zinc binding domain mutant protein of the present invention can be obtained by a known method (for example, Molecular Cloning 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). By inserting the DNA encoding the TFIIE α zinc-binding domain mutant protein of the present invention into an appropriate expression vector.

  As expression vectors, plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), yeast-derived plasmids (eg, pSH19, pSH15), λ phage, etc. Bacteriophages, animal viruses such as retroviruses and vaccinia viruses, insect pathogenic viruses such as baculoviruses, and the like can be used.

  A promoter, enhancer, splicing signal, poly A addition signal, selection marker, SV40 replication origin, and the like may be added to the expression vector.

The expression vector may be a fusion protein expression vector. Various fusion protein expression vectors are commercially available: pGEX series (Amersham Pharmacia Biotech), pET CBD Fusion System 34b-38b (Novagen), pET Dsb Fusion Systems 39b and 40b (Novagen), pET GST Fusion System 41 and 42 (Novagen).

7). Transformant A transformant can be obtained by introducing a recombinant vector containing DNA encoding the TFIIE α zinc-binding domain mutant protein of the present invention into a host.

  Hosts include bacterial cells (eg, Escherichia, Bacillus, Bacillus, etc.), fungal cells (eg, yeast, Aspergillus, etc.), insect cells (eg, S2 cells, Sf cells, etc.), animal cells (eg, CHO cells, COS cells, HeLa cells, C127 cells, 3T3 cells, BHK cells, HEK293 cells, etc.), plant cells and the like.

  In order to introduce a recombinant vector into a host, the method described in Molecular Cloning 2nd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989 (for example, calcium phosphate method, DEAE-dextran method, transfection method, Injection method, lipofection method, electroporation method, transduction method, scrape loading method, shotgun method, etc.) or infection.

  The transformant can be cultured in a medium, and the TFIIE α zinc-binding domain mutant protein can be collected from the culture. When the TFIIEα zinc-binding domain mutant protein is secreted into the medium, the medium may be recovered, and the TFIIE α zinc-binding domain mutant protein may be separated from the medium and purified. When the TFIIEα zinc-binding domain mutant protein is produced in a transformed cell, the cell may be lysed, and the TFIIE α zinc-binding domain mutant protein may be separated from the lysate and purified.

  If the TFIIEα zinc-binding domain mutant protein is expressed in the form of a fusion protein with another protein (functioning as a tag), the fusion protein is isolated and purified, and then treated with FactorXa or an enzyme (enterokinase) By this, another protein can be cut | disconnected and the target TFIIE alpha zinc binding domain mutant protein can be obtained.

Separation and purification of the TFIIEα zinc-binding domain mutant protein can be performed by known methods. Known separation and purification methods include differences in molecular weight such as methods utilizing solubility such as salting out and solvent precipitation, dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. Methods that use, methods that use charge differences such as ion exchange chromatography, methods that use specific affinity such as affinity chromatography, methods that use hydrophobic differences such as reversed-phase high-performance liquid chromatography, etc. A method using the difference in isoelectric point, such as electric point electrophoresis, is used.

8). Antibody The antibody against the TFIIE α zinc-binding domain mutant protein or a salt thereof of the present invention can be used for detection and / or quantification of the TFIIE α zinc-binding domain mutant protein or a salt thereof of the present invention.

  The antibody against the TFIIE α zinc-binding domain mutant protein or a salt thereof of the present invention can be obtained by administering a fragment containing the TFIIE α zinc-binding domain mutant protein of the present invention or a salt thereof or an epitope thereof to an animal using a conventional protocol. can get.

  The antibody of the present invention may be any of a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a single chain antibody, and a humanized antibody.

  In order to produce a polyclonal antibody, it can be produced according to a known method or a method analogous thereto. For example, a complex of an immunizing antigen (protein antigen) and a carrier protein is prepared, administered (immunized) to an animal, and an antibody-containing substance against the protein of the present invention is collected from the immunized animal, and the antibody is separated and purified. Can be manufactured. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. Administration is usually performed once every about 2 to 6 weeks, about 3 to 10 times in total. Polyclonal antibodies can be collected from blood, ascites, etc., preferably from blood of immunized animals. Polyclonal antibodies can be separated and purified by immunoglobulin separation and purification methods (for example, salting-out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, adsorption / desorption method using ion exchanger, ultracentrifugation method, gel filtration method, A specific purification method in which only an antibody is collected using an antigen-binding solid phase or an active adsorbent such as protein A or protein G, and the antibody is obtained by dissociating the binding.

  Monoclonal antibodies can be prepared by the hybridoma method of G. Koehler and C. Milstein described in Nature (1975) 256: 495, Science (1980) 208: 692-. That is, after immunizing an animal, antibody-producing cells are isolated from the spleen of the immunized animal and fused with myeloma cells to prepare monoclonal antibody-producing cells. In addition, cell lines that produce antibodies that react specifically with the interleukin-18 mutant protein or salt thereof but do not substantially cross-react with other antigenic proteins may be isolated. This cell line can be cultured, and the desired monoclonal antibody can be obtained from the culture. Purification of the monoclonal antibody can be performed according to the above-described method for separating and purifying immunoglobulin.

  Techniques for making single chain antibodies are described in US Pat. No. 4,946,778.

Techniques for making humanized antibodies are described in Biotechnology 10, 1121-, 1992; Biotechnology 10, 169-, 1992.

9. Use of TFIIEα zinc-binding domain mutant protein or salt thereof The TFIIEα zinc-binding domain mutant protein and salts thereof of the present invention can be used for events involving TFIIEα (eg, transcriptional activity, binding to TFIIEβ and other basic transcription factors). Can be used to control For example, the prevention and / or treatment of diseases involving transcription (for example, cancer, rheumatism, lifestyle-related diseases, genetic diseases, etc.) can be carried out using the TFIIE α zinc-binding domain mutant protein and salts thereof of the present invention. I may be able to do it. In addition, the transcription mechanism may be elucidated using the TFIIE α zinc-binding domain mutant protein of the present invention and salts thereof.

The TFIIE α zinc-binding domain mutant protein and salts thereof of the present invention can be used as pharmaceuticals for the purpose of preventing and / or treating diseases, or as experimental reagents.

Hereinafter, the present invention will be specifically described by way of examples. These examples are for explaining the present invention and do not limit the scope of the present invention.

Description of structure
N-terminal 12 residues and C-terminal 11 residues did not converge because of limited distance. In fact, ¹ H- ¹⁵ N heteronuclear NOE and ¹⁵ NT ₁ , T ₂ values revealed backbone flexibility in these regions (data not shown). However, with only the remaining 40 amino acids, it is possible to design a compact structure consisting of 6 β chains and 1 short α helix (FIGS. 1b and 1c); S1 chain (126-129aa) , T1 turn (130-133aa), S2 chain (134-136aa), α helix (138-142aa), S3 chain (144-146aa), loop (147-150aa), S4 chain (151-154aa), t2 turn (155-158aa), S5 chain (159-161aa), S6 chain (162-164aa). S1, S2, S6 chains and S3, S4, S5 chains each form an antiparallel β sheet. The two β sheets separated by an α helix are equal, including the position of the cysteine residue in the topology diagram (FIG. 1d). In this conformation, the six β chains draw a semicircle with an α helix as the diameter. Zn ²⁺ coordinates with 129C and 132C in the t1 turn and 154C and 157C in the t2 turn. In HD exchange experiments, the exchange rate of 131V, 132C, 156F and 157C amide protons was relatively slow. Judging from the structure, hydrogen bonds are probably formed in 129C: Sγ-131V: H _N , -132 C: H _N and 154C: Sγ-156F: H _N , -157C: H _N (Fig. 1e). These bifurcated to split the hydrogen bond is consistent with the hydrogen bonding pattern between the H _N of Sγ and i + 2, i + 3 residues of many observed in zinc finger domains, i residues (Qian, X. et al. Novel zinc finger motif in the basal transcriptional machinery: Three-dimensional NMR studies of the nucleic acid binding domain of transcriptional elongation factor TFIIS.Biochemistry 32, 9944-9959 (1993) .; Zhu, W. et al. The N-terminal domain of TFIIB from Pyrococcus furiosus forms a zinc ribbon. Nat.Struct. Biol. 3, 122-124 (1996) .; Wang, B., Jones, DN, Kaine, BP & Weiss, MA High-resolution structure of an archaeal zinc ribbon defines a general architectural motif in eukaryotic RNA polymerases.Structure 6, 555-569 (1998) .; Chen, HT, Legault, P., Glushka, J., Omichinski, JG & Scott, RA Structure of a (Cys3His) zinc ribbon, a ubiquitous motif in archaeal and eucaryal transcription. Protein Sci. 9, 1743-1752 (2000).). Almost all of the amide protons were exchanged for deuterium within 40 minutes. For this reason, little information on hydrogen bonding was obtained in HD exchange experiments. Fortunately, we were able to directly observe a larger number of hydrogen bonds in the β-sheet by measuring the trans hydrogen bond ^3h J _{NC ′} bond (Cordier, F. & Grzesiek, S Direct observation of hydrogen bonds in proteins by interresidue ³ hJ _{NC '} Scalar Couplings. J. Am. Chem. Soc. 121, 1601-1602 (1999) .; Cornilescu, G., Hu, JS. & Bax, A. Identification of the hydrogen bonding network in a protein by scalar couplings. J. Am. Chem. Soc. 121, 2949-2950 (1999).).
The current structure can explain how the structure can be maintained despite such a short region. The coordination bond between Zn ²⁺ and the four cysteines and the two auxiliary branched hydrogen bonds tightly fix the t1 and t2 turns. This brings the two antiparallel β sheets closer to one side of the α helix. A hydrophobic core is formed by the hydrophobic amino acids in each secondary structure (FIG. 1f). Within this hydrophobic core, five phenylalanine residues are stacked on top of each other and arranged in an L shape. Aromatic-aromatic interaction networks contribute significantly to structural stabilization. Besides them, hydrophobic amino acids such as 141A, 144L and 161V are also important for the formation of the hydrophobic core. The TFIIEα zinc binding domain is highly conserved, including residues involved in the hydrophobic core and the four cysteine residues in the zinc binding site (FIG. 1a). The electrostatic potential on the molecular surface indicates that this zinc binding domain is conserved 138D, 160E, 162E, 163E, 164D and 165E (which are located in a wide range of recessed surfaces opposite the zinc binding site) It has a negative potential cluster consisting of (Fig. 1g). These observations strongly suggest that the structure of the zinc binding domain in the homologue will be similar to that of human TFIIEα.

Novel structure of zinc binding motif
TFIIEα zinc binding domain is the C-terminal domain of transcription elongation factor TFIIS (TFIISc) (Qian, X. et al. Novel zinc finger motif in the basal transcriptional machinery: Three-dimensional NMR studies of the nucleic acid binding domain of transcriptional elongation factor TFIIS. Biochemistry 32, 9944-9959 (1993) .; Olmsted, VK et al. Yeast transcript elongation factor (TFIIS), structure and function.J. Biol. Chem. 273, 22589-22594 (1998).), TFIIB ( The N-terminal domain of TFIIBn) (Zhu, W. et al. The N-terminal domain of TFIIB from Pyrococcus furiosus forms a zinc ribbon. Nat. Struct. Biol. 3, 122-124 (1996) .; Chen, HT, Legault , P., Glushka, J., Omichinski, JG & Scott, RA Structure of a (Cys3His) zinc ribbon, a ubiquitous motif in archaeal and eucaryal transcription. Protein Sci. 9, 1743-1752 (2000).) And RNA Pol C-terminal domain (RPB9c) of the RPB9 subunit of II (Wang, B., Jones, DN, Kaine, BP & Weiss, MA High-resolution structure of an archaeal zinc ribbon defines a general architectural motif in eukaryotic RNA polymerases.Structure 6, 555-569 (1998) .; Cramer, P., Bushnell, DA & Kornberg, RD Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.Science 292, 1863-1875 (2001) .; Gnatt, AL, Cramer, P., Fu, J., Bushnell, DA, & Kornberg, RD Structural basis of transcription: An RNA polymerase II elongation complex at 3.3 Å resolution. Science 292, 1876-1882 ( 2001).) Was predicted to form a zinc ribbon structure. Surprisingly, the TFIIEα zinc binding domain is quite different from the zinc ribbon structure (FIG. 2a). The topologies of TFIISc, TFIIBn and RPB9c are βββ, βββ and ββββ (β: β chain), respectively, which form antiparallel β sheets. On the other hand, the topology of TFIIEα zinc binding domain is more complex. It is ββαββββ (α: α helix), forming two antiparallel β sheets and one α helix. On the other hand, these zinc binding sites are very similar to each other (Figure 2b). Comparing the backbones of the two turns (CxxCxx and CxxCxx, 12aa regions), the root mean square deviations (rmsd) for TFIIEα zinc binding domain TFIISc, TFIIBn and RPB9c are 0.87Å, 1.05Å and 0.72Å, respectively. The present inventors tried to search for a similar structure, but no similar structure was detected by a search using a DALI server (Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. Biol. 233, 123-138 (1993).). Therefore, TFIIE alpha zinc binding domains should be classified into a new kind of zinc binding structure.

Mutation analysis To elucidate the function of the zinc binding domain, two highly conserved amino acids located on the negative potential surface, 140E and 164D, were mutated to alanine or lysine, respectively (E140A, E140K, D164A, D164K), and each of the four Zn ²⁺ ligands was mutated from cysteine to alanine (C129A, C132A, C154A, C157A). We assayed the effect of mutated full-length TFIIEα on basal transcriptional activity using a supercoiled template (FIG. 3a). The E140A mutant had a slight increase in transcriptional activity, and the transcriptional activity of the E140K mutant decreased to 50% of the wild type transcriptional activity. Surprisingly, for D164A and D164K, transcriptional activity increased from about 200 to 250%. On the other hand, the activity decreased dramatically in all Zn ²⁺ ligand mutants, but to varying degrees. The inventors predicted that all Zn ²⁺ ligand mutants would lose transcriptional activity equally. This is because they seem to lose the coordinated Zn ²⁺ and destroy the zinc binding site, resulting in a random coil structure. In practice, C154A and C157A almost completely lost activity, while C129A and C132A retained 12-25% activity.
Next, we examined the effect of these mutations on binding to TFIIEβ and other GTFs by GST pull-down assay (FIG. 3b). Despite transcriptional effects, almost all mutants, including C154A and C157A, had the same binding ability as wild-type. In contrast, C129A and C132A bound to TFIIFβ more strongly than the wild type. Thus, these assays suggested asymmetry of the contribution of each Zn ²⁺ ligand (ie, cysteine) to the structure.

Asymmetry of Zn ²⁺ ligands. As for the cause of this asymmetry, we first destroyed the structure of C154A and C157A completely into a random coil structure, but the structure of C129A and C132A changes partially. I estimated. To test this idea, far-UV CD spectra were recorded for the zinc-binding domains of wild-type and Zn ²⁺ ligand point mutants (FIG. 4a). The secondary structure calculated from the CD spectrum suggested that the relative amount of the random coil structure was in the order of C129A>C132A>C157A> C154A, which was contrary to our estimation. In order to study their structures in more detail, we recorded ¹ H NMR spectra (FIG. 4b). Comparison of the spectrum with the wild type showed that all structures were significantly changed. Especially in C129A, the signal dispersion was dramatically reduced. This is consistent with the results of point mutant analysis at the zinc binding site of yeast TFIIEα (Kuldell, NH, & Buratowski, S. Genetic analysis of the large subunit of yeast transcription factor IIE reveals two regions with distinct functions. Mol. Cell. Biol. 17, 5288-5298 (1997).). In this analysis, the strain with the C124F (129C in human) substitution was lethal, while the C127F (132C in human) mutant was temperature sensitive. However, the C129A spectrum was not identical to the wild-type spectrum denatured with EDTA and the signal was broader. This suggested a partially folded structure similar to C132A, C154A and C157A. Also, each different spectrum indicated that the mutants had different structures.
All Zn ²⁺ ligand point mutants lack one of the four cysteines. Can these mutants coordinate with Zn ²⁺ through the remaining 3 cysteines? In order to find out this, the present inventors conducted an EDTA titration experiment while monitoring NMR ¹ H- ¹⁵ N HSQC. The addition of EDTA first changed the spectrum of all mutants and wild type. Next, an equal amount of EDTA completely erased the signal dispersion, indicating that the folded structure spread into a random coil structure. The spectrum before and after adding a 2-fold excess of EDTA is shown in Figure 4c. In the spectrum of C129A before addition of EDTA, most but not all of the signals were consistent with the signal of the random coil structure from which Zn ²⁺ was removed. The zinc binding domain has two asparagines and one glutamine, so three pairs of their side chain signals are observed in the ¹ H- ¹⁵ N HSQC spectrum. However, the C129A spectrum had 4 pairs of side chain signals, 2 of which were consistent with that of the random coil structure (Figure 4d). These results indicate that the random coil structure is dominant in C129A, but is equilibrated with the partially folded structure coordinated with Zn ²⁺ . In the case of C132A, 57 signals were observed in the wild type, whereas 104 main chain signals were observed. Of note, the C132A spectrum was similar to the C129A spectrum. In fact, the C132A spectrum contained a number of identical signals (FIG. 4d). In addition to these signals, the C132A spectrum also had numerous dispersed signals and concentrated signals corresponding to random coil structures (FIGS. 4c, 4d). Six pairs of asparagine and glutamine side-chain signals were observed, two of which were consistent with that of the random coil structure and that of the partially folded structure in C129A, respectively (Figure 4d) . A comparison of the spectra of C132A and C129A shows that the structure is identical or very similar to the partially folded structure of C129A, and other partially folded structures coordinated with Zn ²⁺ and randomly It was clarified that C132A was equilibrated between coil structures. The spectra of C154A and C157A were also similar to each other, including a large number of dispersed signals and less concentrated signal compared to the C129A, C132A spectra (Figure 4c). For asparagine and glutamine side chain signals, 5 pairs were observed with C154A. Two of them matched the random coil structure, and the other pair corresponded to the partially folded structure (Fig. 4d). Thus, C154A is coordinated with Zn ²⁺ and is balanced between a partially folded structure and a random coil structure (which is not as prevalent as the random coil structure of C129A and C132A). For the C157A spectrum, 5 pairs of side chain signals were observed. Two of them matched the random coil structure, and the other two matched that of C154A. For the main chain, 77 signals were observed. These signals corresponded to signals of a random coil structure, a partially folded structure of C154A, and other partially folded structures (FIGS. 4c, 4d). These results indicate that C157A is identical or very similar to C154A, and coordinated with other Zn ²⁺ , partially folded structures, and equilibrated with random coil structures. After the EDTA titration experiment, the inventors conversely performed a ZnCl ₂ titration experiment using a sample from which EDTA and ZnCl ₂ had been removed by dialysis. Addition of ZnCl ₂ greatly dispersed the signal, and addition of an equal amount of ZnCl ₂ returned the spectrum of each sample to the same spectrum as before EDTA addition (data not shown).
In summary, coordinate binding to Zn ²⁺ is possible even if one of the four cysteines at the zinc binding site is lost. However, the structure is partially destroyed and becomes unstable. This is especially true when the first or second cysteine is replaced. All the structures were equilibrated with the random coil structure not coordinated with Zn ²⁺ . The predominance of the random coil structure is the order of C129A>C132A> C154A≈C157A. The structures of C129A and C132A are similar to each other. The structures of C154A and C157A are similar. In addition, C132A and C157A were also equilibrated with other partially folded structures. All structures show reversibility of structure formation depending on the Zn ²⁺ bond.

In order to clarify the function of the zinc-binding domain, the structure of the zinc-binding domain was determined by using human TFIIEα (hTFIIEα) with mutations in the cysteine (C129, C132, C154, C157) that coordinates Zn2 +. The effect of transcriptional activity on decay was observed. In the transcription assay, a supercoiled type and a linear type of adenovirus major late promoter were used as DNA templates. The need for the basic transcription factors TFIIE and TFIII during promoter melting depends on the type and topology of the promoter. In the case of the adenovirus major late promoter template, TFIIH is not necessarily required for the supercoiled type, and TFIIE can perform promoter melting, but TFIIH is essential for the linear type template. As a result of the transcription assay, the activity of C129A and C132A was only reduced to about 15 to 30% in the transcription using the supercoiled template, whereas C154A and C157A were almost completely inactivated. On the other hand, in the linear type, transcription was completely inactivated by C129A, C132A, C154A, and C157A. Thus, in the case of using the supercoil type and the straight chain type, asymmetry between two cysteines on the N-terminal side and two on the C-terminal side of the zinc binding domain structure was observed with respect to transcriptional activity. From the analysis reported so far, when transcription with a supercoiled template is not seen, it has no transcription initiation activity, and transcription is seen with a supercoiled template. When it is not seen, considering that there is a defect in the transition (transition) activity from transcription initiation to elongation, C154A and C157A fail to initiate transcription, while C129A and C132A also affect transcription initiation. However, there is a more important defect in the transition. These results also correspond to the lack of Zn domain being dominant negative in transcription.
Next, in order to investigate the cause of asymmetry and function of the zinc-binding domain structure, binding of basic transcription factors including hTFIIEβ subunit involved in in vitro transcription reconstitution reaction and RNA polymerase II (Pol II) factors Specificity was analyzed. The binding of each basic transcription factor to each subunit was examined by a GST pull-down assay using a fusion protein of the N-terminal GST of these subunits. As a result, all the mutants were similarly strongly bound to the p62 subunit of hTFIIEβ and TFIIH, which are known to bind hTFIIEα strongly. However, the binding between TFIIFβ and TFIIH XPB subunit, which binds strongly next to these, is that C129A and C132A bind more strongly to TFIIFβ, while C154A and C157A have very weak binding to XPB. It was revealed.
Therefore, the present inventors were interested in the asymmetry of Zn2 + ligand cysteine residues with respect to transcriptional activity, and investigated their structures. All mutants were broken in structure, but coordinated with Zn2 + and had a partial structure. From the NMR signal, it was shown that C129A and C132A are similar, and C154A and C157A have similar structures. As a result of structural analysis, it was suggested that C129A and C132A, which remained active in the supercoiled transcription system, were almost completely denatured. On the other hand, it was shown that the structure of C154A and C157A is in equilibrium with the characteristic Zn2 + coordinated structure, which is different from the random coil structure and the native structure. Therefore, with a supercoiled template, the complete conformation of the zinc binding domain is not absolutely required for transition from promoter melting. Rather, it became clear that partial structures different from the characteristic native structures such as C154A and C157A hindered conversely.

  Let us consider the molecular mechanism. Zinc binding domains do not bind to DNA from negatively charged clusters on the surface of the molecule. The single-stranded and double-stranded DNA binding domain of TFIIE is found in TFIIEβ. It is likely that TFIIEβ will contact DNA directly during promoter melting. As described above, the difference between the supercoiled type and the linear type transcription system is the necessity of involvement with factors such as TFIIH through promoter melting and transition. It is the C-terminal region of TFIIEα that interacts directly with TFIIH. In fact, it binds strongly to the p62 subunit of TFIIH, and its binding ability did not change between the wild type and all mutants. Therefore, the zinc-binding domain does not have a role of directly contacting DNA to promote melting, stabilize its state, or recruit TFIIH. This is consistent with almost no change in the interaction with GTF in all mutants. However, C129A and C132A originally increased the ability to bind to weak TFIIFβ in the wild type by about 3 times. This is probably because the structure of the zinc-binding domain was broken and the interaction became stronger. This explains that although the structure is broken, about 15-30% of transcription initiation activity is observed in C129A and C132A. On the other hand, C154A and C157A, mutations on the C-terminal side of the zinc-binding domain, are unable to bind to functioning XPB by cleaving DNA with its helicase activity during promoter melting at the start of transcription. It can be well explained that there is no transcription initiation activity. Although data are not shown, binding assay using a column with PolII as a ligand revealed that Pol II binds to TFIIEβ but not TFIIEα. That is, the zinc-binding domain may contribute to the positioning of the domain that performs them during promoter melting and transition. In promoter melting and transition following PIC formation, the arrangement of domains within the TFIIE molecule is affected by the disruption of the structure of the zinc-binding domain. The C-terminal domain that interacts directly with TFIIH may also be unable to position correctly in PIC. However, a transcription system using supercoiled DNA as a template does not necessarily require TFIII promoter melting or transition, so efficiency is low unless TFIIE promoter melting or transition is completely impossible. I think we can clear this stage. However, in linear transcription, coordinated work with TFIIH and other factors is important at the stage of promoter melting and transition, so the structure of the zinc-binding domain must be complete. C129A and C132A will be completely deactivated.

On the other hand, the possibility that the zinc-binding domain is an interaction domain with other proteins was suggested by another mutant. Two acidic residues E140 and D164, which are highly conserved from human to yeast, were substituted with alanine and lysine residues. As a result, in the transcription with the supercoiled template, E140A was almost the same as the wild type, and E140K was reduced to about 50%. In the linear form, the activity decreased even with E140A, and further decreased with E140K. Interesting is D164. In the supercoiled type, the activities of D164A and D164K were both about two times higher than in the wild type. In the linear type, the activity of D164K was increased by about 2 times, but in D164A, the activity increase was markedly 3.5 times that of the wild type. E140 and D164 mutations should not affect the conformation. D164 is located on the zinc binding surface following the negative potential cluster on the opposite side of the zinc ion binding site. When K is selected, the activity increases, and when A is selected, the activity increases further. Therefore, there is a possibility that the area around this negative charge is a surface that interacts with other factors. The present inventors analyzed the binding to GTF and Pol II and could hardly confirm the change in binding ability between the wild type and the mutant, but a large structural change was expected as PIC was activated at the transcription initiation stage. Is done. Therefore, it is conceivable that the zinc binding domain specifically binds to a complex structure at a certain stage. In vivo, many factors such as chromatin remodeling factors, mediators, transcriptional regulatory factors, and transcription elongation factors that function ubiquitously interact with the transcription apparatus and regulate transcription. As a function is also conceivable. Based on the above, mutant experiments show that the zinc-binding domain of TFIIEα is an important functional domain that is involved in the regulation of very subtle transcription, in addition to its distinctive structure. It was done.

Method Sample preparation Plasmids were introduced into E. coli BL21 (DE3) pLysS (Novagen) and transformed. The resulting E. coli cells were cultured in LB liquid medium or M 9 minimal medium containing [ ¹⁵ N] ammonium chloride at 37 ° C. in the presence or absence of [ ¹³ C] glucose. When OD600 became 0.3-0.4, 1 mM IPTG was added. Bacteria were collected after growth for 4-7 hours. The cell pellet was resuspended in buffer A (20 mM Tris-HCl, pH 7.0, 10% glycerin, 30 μM ZnCl ₂ , 1 mM PMSF, 1 mM benzamidine and 0.1 M NaCl). The cells were lysed by sonication on ice, centrifuged, and the supernatant was introduced onto a Ni-nitrilotriacetic acid (NTA) agarose (Qiagen) column equilibrated with buffer A containing 20 mM imidazole. Protein samples were eluted with an imidazole linear gradient from 20 mM to 0.35 M. The peak fractions were pooled and the buffer was replaced with 50 mM Tris-HCl, pH 8.0, 30 μM ZnCl ₂ , 2.5 mM CaCl ₂ and 0.15 M NaCl. Samples were cleaved for 16 hours at room temperature using thrombin protease. Next, this was introduced into a Ni-NTA agarose (Qiagen) column equilibrated with buffer B (20 mM potassium phosphate buffer, pH 7.0, 30 μM ZnCl ₂ and 0.2 M NaCl), and washed with buffer B. The sample from which the 6 N-terminal histidine tags had been removed was passed through the column. Concentrate the sample using a Centriprep Membrane (Amicon) with a cutoff of MW3000 until the volume is less than 2.5 ml, and add this to 20 mM potassium phosphate buffer, pH 7.0, 30 μM ZnCl ₂ , 5.0 mM DTT and 50 mM NaCl. It was introduced into an equilibrated Superdex 30 column (Amersham Pharmacia Biotech). The final sample fraction was collected.

NMR spectroscopy
Protein concentration for NMR experiments is 20 mM potassium phosphate buffer, pH 6.0, 30 μM ZnCl ₂ , 5.0 mM perdeuterated 1, dissolved in 90% H ₂ O / 10% D ₂ O or 99.9% D ₂ O. 4-dithiothreitol, about 3-4 mM in 50 mM NaCl. All NMR experiments were performed at 32 ° C. using a Bruker DRX-500 or DRX-600 spectrometer equipped with a triple resonance gradient probe. The following NMR experiments were used to determine the assignment of main chain and side chain resonances: 3D CBCA (CO) NH, 3D CBCANH, 3D HN (CA) CO, 3D HNCO, 2D DQF-COSY, 2D TOCSY, 3D HBHA TOCSY-HSQC, 3D HCCH-COSY, 3D HCCH-TOCSY edited with (CO) NH, 3D ¹⁵ N (Cavanagh, J., Fairbroher, WJ, Palmer III, AG & Skelton, NJ Protein NMR spectroscopy (Academic Press, San Diego; 1996).), 2D (Hβ) Cβ (CγCδ) Hδ, 2D (Hβ) Cβ (CγCδCε) Hε (Yamazaki, T., Forman-Kay, JD & Kay, LE Two-dimensional NMR experiments for correlating ¹³ Cβ and ¹ Hδ / ε chemical shifts of aromatic residues in ¹³ C-labeled proteins via scalar coupling.J. Am. Chem. Soc. 115, 11054-11055 (1993)), 2D CG (CB) H, 2D CG (CD ) H and 2D CG (CDCE) H (Prompers, JJ, Groenewegen, A., Hilbers, CW & Pepermans, HAM Two-dimensional NMR experiments for the assignment of aromatic side chains in ¹³ C-labaled proteins. J. Magn. Reson 130, 68-75 (1998).) Experiment. Stereospecific assignments are 3D HNHB, 3D HN (CO) HB, 3D HNCG, 3D HN (CO) CG (Krichna, NR & Berliner, LJ Biological Magnetic Resonance 16 (Kluwer Academic / Plenum Publishers, New York; 1998)), NOESY-HSQC edited with 2D DQF-COSY and 3D ¹⁵ N (mixing time 50 ms) (Yamazaki, T., Forman-Kay, JD & Kay, LE Two-dimensional NMR experiments for correlating ¹³ Cβ and ¹ Hδ / ε chemical ^{shifts of aromatic residues in 13 C-} labeled proteins via scalar coupling. J. Am. Chem. Soc. 115, 11054-11055 (1993).) obtained from the combination experiments. Distance information is NOESY-HSQC edited with 2D NOESY, 3D ¹⁵ N (mixing time 50 ms and 150 ms) and NOESY-HSQC edited with 3D ¹³ C (Yamazaki, T., Forman-Kay, JD & Kay, LE Two -dimensional NMR experiments for correlating ¹³ Cβ and ¹ Hδ / ε chemical shifts of aromatic residues in ¹³ C-labeled proteins via scalar coupling. J. Am. Chem. Soc. 115, 11054-11055 (1993)) . The main chain twist angle φ is 3D HNHA and 3D HNCA-J (Yamazaki, T., Forman-Kay, JD & Kay, LE Two-dimensional NMR experiments for correlating ¹³ Cβ and ¹ Hδ / ε chemical shifts of aromatic residues in ¹³ C -labeled proteins via scalar coupling. J. Am. Chem. Soc. 115, 11054-11055 (1993)). The side chain twist angles χ ₁ and χ ₂ are 3D HNHB, 3D HN (CO) HB, 3D HNCG, 3D HN (CO) CG (Lui, A., Hu, W., Majumdar, A., Rosen, MK & Patel, DJ Detection of very weak side chain-main chain hydrogen bonding interactions in medium-size ¹³ C / ¹⁵ N-labeled proteins by sensitivity-enhanced NMR spectroscopy.J. Biomol.NMR 17, 79-82 (2000). ), NOESY-HSQC edited with 3D ¹⁵ N and NOESY-HSQC edited with 3D ¹³ C (Yamazaki, T., Forman-Kay, JD & Kay, LE Two-dimensional NMR experiments for correlating ¹³ Cβ and ¹ Hδ / ε Chemical shifts of aromatic residues in ¹³ C-labeled proteins via scalar coupling. J. Am. Chem. Soc. 115, 11054-11055 (1993)). Hydrogen bond constraints include main chain amide H / D exchange experiments and 2D CPD-H (N) CO experiments to detect trans hydrogen bond ^3h J _{NC '} bonds (Lui, A., Hu, W., Majumdar, A ., Rosen, MK & Patel, DJ Detection of very weak side chain-main chain hydrogen bonding interactions in medium-size ¹³ C / ¹⁵ N-labeled proteins by sensitivity-enhanced NMR spectroscopy. J. Biomol. NMR 17, 79-82 (2000).). NMRPipe (Delaglio, F. et al. NMRPipe: A multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277-293 (1995)) is used to process the spectrum and program PIPP, CAPP, STAPP ( Garrett, DS, Powers, R., Gronenborn, AM & Clore, GM A common sense approach to peak picking in two-, three-, and four-dimensional spectra using automatic computer analysis of contour diagrams. J. Magn. Reson. 95 , 214-220 (1991)) and NMRView (Johnson, BA & Blevins, RA NMRView: a computer program for the visualization and analysis of NMR data.J. Biomol. NMR 4, 603-614 (1994).) And analyzed.

Structural calculation
Interproton distance constraints derived from NOE intensity were classified into three distance ranges 1.8-3.0 mm, 1.8-4.0 mm, and 1.8-5.0 mm, corresponding to strong NOE, moderate NOE, and weak NOE, respectively. The distance including the methyl group was further increased by 0.5% in consideration of the higher apparent intensity of methyl resonance. A 2.0 Å pseudoatom correction was added to the degenerate phenylalanine ring proton. ^The φ angle estimated from the ³ J _HNα coupling constant is -90 ^o <φ <-40 ^o for ³ J _HNα <7.0 Hz and -160 ^o <φ <-80 for ³ J _HNα > 10.0 Hz. ^o . The χ ₁ angle was constrained ± 30 ° for the three side chain rotamers. Zinc was constrained as follows to be tetrahedrally coordinated by four cysteine residues. That is, the Zn-Sγ and Sγ-Sγ bond lengths were set to 2.3 and 3.8, and the Zn-Sγ-Cβ and Sγ-Zn-Sγ angles were set to 108 ° and 109 °. In the final stage of refinement, hydrogen bond constraints were introduced in the regular secondary structure region. Structural calculations are simulated using distance geometry and the program X-PLOR (Brunger, AT X-PLOR Version 3.1: A system for X-ray crystallography and NMR (Yale University Press, New Haven, Connecticut; 1993)). Carried out by annealing. A total of 100 structures were calculated. Twenty of them had no NOE violation greater than 0.5 cm and dihedral angle biolation greater than 5 °. A summary of the structural statistics is shown in Table I. PROCHECK-NMR (Laskowski, RA, Rullmann, JAC, MacArthur, MW, Kaptein, R. & Thornton, JM AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR.J. Biomol.NMR 8, 477 -486 (1996).), GRASP (Nicholls, A., Sharp, KA & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons.Prot.Struct. Funct. Genet. 11, 281- 296 (1991).), MOLMOL (Koradi, R., Billeter, M. & Wuthrich, K. MOLMOL: A program for display and analysis of macromolecular structures.J. Mol. Graphics 14, 51-55 (1996).) And the structure was analyzed and displayed using SYBYL (Tripos Inc., St. Louis).

Generation of a site-specific mutant Wild-type human TFIIEα is TFIIE (Ohkuma, Y. et al., Proc Natl Acad Sci USA. 87, 9163-9167) purified from HeLa cells, which are cultured human cells, using transcriptional activity as an indicator. , 1990) using reverse-phase high-performance liquid chromatography to separate and purify the two subunits α and β, of which the α subunit is subjected to limited digestion, based on the amino acid sequence of the peptide fragment obtained by PCR. After amplification of the partial sequence, the full-length cDNA was isolated by screening a human NAM cell cDNA library (Ohkuma, Y. et al., Nature, 354, 398-401, 1991). This human TFIIEα cDNA is incorporated into pBluescriptII-SK (-) and has an NdeI cleavage site that spans the 289th and 290th codons of the protein coding region. In order to crush the site and create a new NdeI recognition site at the N-terminus and a BamHI recognition site 3 'downstream of the C-terminal stop codon, mutations were introduced using the Mutan-K mutagenesis kit (TaKaRa). did. This was excised at the NdeI recognition site and the BamHI recognition site, and incorporated into a pET-11d vector (Novagen) capable of expressing a 6His-tagged fusion protein with 6 histidine residues added to the N-terminal side, resulting in a wild-type human TFIIEα expression plasmid .
Zinc-binding domain mutants (E140A, E140K, D164A, D164K, C129A, C132A, C154A, C157A) were also synthesized with oligo DNAs to mutate each amino acid, and the above Mutan-K mutagenesis kit Using (TaKaRa), a mutation was introduced into wild-type human TFIIEα incorporated into pBluescriptII-SK (-), and this was also excised at the NdeI recognition site and the BamHI recognition site as described above. It was incorporated into a pET-11d vector (Novagen) capable of expressing a 6His-tag fusion protein with one histidine residue added.

In vitro transcription assay (supercoil type)
The promoter of the DNA template is pML (C2AT) Δ-50 in which a template containing no G base (G free cassette) is inserted downstream from the -50 to +10 region of the adenovirus major late promoter (AdML). Supercoiled DNA and linear DNA were used as templates. The G-free cassette downstream of the promoter produces a transcription product of about 390 nucleotides that does not contain a G base. When a G base appears, 3′-O-methyl GTP is incorporated, and subsequent transcription stops. Using this, basic transcriptional activity was measured in the presence or absence of a transcript of a certain length.
The transcription reaction was performed at 25 ° C. for 45 minutes in 25 μl of a reaction solution containing basic transcription factor and RNA polymerase II (Pol II). At this time, TFIIEα wild type or point mutant was added at 6,12,18 ng. After extraction with phenol / chloroform, the transcription reaction was stopped by ethanol precipitation, and developed on a 4% acrylamide-urea modified gel (4% acrylamide, 50% urea, 1 × TBE). After drying the gel, uptake of [α- ³² P] CTP was detected by autoradiography, and the activity was measured with BAS2000.
Mold: 100 ng pML (C2AT) Δ-50 (super coil type)
Factor: Pol II, TFIIB 20 ng, TBP 10 ng, TFIIEβ 8 ng, TFIIF 16 ng
Reaction solution: 12 mM Tris-HCl (pH 7.9 at 4 ° C), 12% Glycerol, 60 mM KCl, 0.12 mM EDTA, 0.12 mM PMSF, 0.6 mM β-mercaptothanol, 12 mg / ml BSA, 125 μM ATP, 125 μM UTP, 25 μM CTP, 40 mM Hepes (pH 8.4), 8 mM MgCl2, 5.6 mM DTT, 50 μM 3'-O-methyl GTP, 0.8 U RNasin, 0.15 URNaseT1, 0.5 μCi [α- ³² P] CTP
(Linear type)
A transcription reaction was performed in the same manner as when using a supercoiled template, and [α- ³² P] CTP incorporation was detected by autoradiography, and the activity was measured with BAS2000. Here, TFIIEα wild type or point mutants were added at 12,18 ng and 2 points.
Template: 100 ng pML (C2AT) Δ-50 (linear type)
Factors: Pol II, TFIIB 20 ng, TBP 10 ng, TFIIEβ 8 ng, TFIIF 16 ng, TFIH 78 ng
Reaction solution: the same as when using a supercoiled mold.

GST pull-down assay The interaction between human TFIIEα wild-type and point mutants of each basic transcription factor was examined. Glutathione-S-transferase (GST)-basic transcription factor [TFIIB, TBP, RAP74, RAP30, TFIIAα, β, γ, TFIIH (XPB, XPD, p62, p52, p44, p34, cdk7 / MO15, cyclinH, MAT1) Glutathion-sepharose (Amersham Pharmacia Biotech) 7.5 μl and Sepharose CL-4B (Amersham Pharmacia Biotech) 7.5 μl in Buffer C (0.1) + BSA 500 μl The reaction was carried out at 4 ° C. for 4 hours. Collect the resin, wash the resin twice with Buffer C (0.2), and once with Buffer C (0.1), separate the protein bound to Glutathion-sepharose by SDS-PAGE, and PVDF membrane (Immobion-P, Millipore) Were blotted and blocked. The primary antibody is anti-TFIIEα antibody (MBL) diluted 5000-fold with Rinse Buffer and allowed to react overnight at 4 ° C. The secondary antibody is anti-mouse Ig G goat Fab-POD diluted 5000-fold with Rinse Buffer and diluted to 30 at room temperature. Reacted for 1 minute. Detection was performed by chemiluminescence using ECL (Amersham Pharmacia Biotech) and exposed to Fuji New RX Film.
Buffer C (0.1) + BSA: 20 mM Tris-HCl (pH 7.9 at 4 ° C), 20% Glycerol, 0.1 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, 10 mM β-mercaptoethanol, 0.1% NP40, 0.2 mg / ml BSA
Buffer C (0.2): 20 mM Tris HCl (pH 7.9 at 4 ° C), 20% Glycerol, 0.2 M KCl, 0.2 mM EDTA, 0.2 mM PMSF, 10 mM β-mercaptoethanol
Blocking solution: 5% nonfat skim milk, 25 mM Tris-HCl (pH 7.5 at rt), 150 mM NaCl
Rinse Buffer: 10 mM Tris-HCl (pH 7.5 at rt), 1 mM EDTA, 150 mM NaCl, 0.1% NP40, 0.4% nonfat skim milk

CD spectroscopy
15 μM wild type and mutant zinc binding domains were dissolved in 10 mM potassium phosphate buffer, pH 6.0, 30 μM ZnCl ₂ , 1.0 mM 1,4-dithiothreitol. CD spectra were obtained at 25 ° using a Jasco J-720W spectropolarimeter with a 1 mm path length cell. Each spectrum is an average of the results of 16 scans in the wavelength range of 185-260 nm.

EDTA titration
1 mM wild-type in 20 mM potassium phosphate buffer, pH 6.0, 30 μM ZnCl ₂ , 5.0 mM perdeuterated 1,4-dithiothreitol, 50 mM NaCl, 90% H ₂ O / 10% D ₂ O Alternatively, EDTA was added to the mutant zinc binding domain in increments of 0.5 mM to 2.0 mM. A ¹ H- ¹⁵ N HSQC spectrum was recorded after each EDTA addition.

Coordinate
The coordinates of the TFIIEα zinc binding domain structure are shown in Table A.

[Table A]
Coordinates of wild-type human TFIIE alpha zinc-binding domain structure

MODEL 1
ATOM 1 CA ARG 1 -13.547 .248 -24.620 1.00 .00
ATOM 2 HA ARG 1 -12.896 -.589 -24.815 1.00 .00
ATOM 3 CB ARG 1 -12.761 1.378 -23.954 1.00 .00
ATOM 4 HB1 ARG 1 -13.423 1.954 -23.323 1.00 .00
ATOM 5 HB2 ARG 1 -12.338 2.019 -24.714 1.00 .00
ATOM 6 CG ARG 1 -11.637 .785 -23.101 1.00 .00
ATOM 7 HG1 ARG 1 -11.923 -.200 -22.763 1.00 .00
ATOM 8 HG2 ARG 1 -11.460 1.422 -22.247 1.00 .00
ATOM 9 CD ARG 1 -10.359 .683 -23.937 1.00 .00
ATOM 10 HD1 ARG 1 -10.140 1.629 -24.408 1.00 .00
ATOM 11 HD2 ARG 1 -10.458 -.097 -24.680 1.00 .00
ATOM 12 NE ARG 1 -9.288 .341 -22.959 1.00 .00
ATOM 13 HE ARG 1 -9.526 .091 -22.042 1.00 .00
ATOM 14 CZ ARG 1 -8.035 .372 -23.324 1.00 .00
ATOM 15 NH1 ARG 1 -7.249 1.313 -22.876 1.00 .00
ATOM 16 HH11 ARG 1 -7.608 2.010 -22.254 1.00 .00
ATOM 17 HH12 ARG 1 -6.289 1.338 -23.154 1.00 .00
ATOM 18 NH2 ARG 1 -7.568 -.539 -24.134 1.00 .00
ATOM 19 HH21 ARG 1 -8.170 -1.260 -24.476 1.00 .00
ATOM 20 HH22 ARG 1 -6.608 -.516 -24.412 1.00 .00
ATOM 21 C ARG 1 -14.723 -.181 -23.739 1.00 .00
ATOM 22 O ARG 1 -15.720 .505 -23.641 1.00 .00
ATOM 23 N ARG 1 -14.049 .828 -25.899 1.00 .00
ATOM 24 HT1 ARG 1 -14.781 .206 -26.299 1.00 .00
ATOM 25 HT2 ARG 1 -13.261 .915 -26.573 1.00 .00
ATOM 26 HT3 ARG 1 -14.457 1.766 -25.718 1.00 .00
ATOM 27 N ILE 2 -14.614 -1.313 -23.098 1.00 .00
ATOM 28 HN ILE 2 -13.801 -1.852 -23.191 1.00 .00
ATOM 29 CA ILE 2 -15.727 -1.785 -22.226 1.00 .00
ATOM 30 HA ILE 2 -16.643 -1.855 -22.791 1.00 .00
ATOM 31 CB ILE 2 -15.297 -3.175 -21.756 1.00 .00
ATOM 32 HB ILE 2 -15.162 -3.819 -22.614 1.00 .00
ATOM 33 CG1 ILE 2 -16.375 -3.761 -20.840 1.00 .00
ATOM 34 HG11 ILE 2 -17.324 -3.293 -21.056 1.00 .00
ATOM 35 HG12 ILE 2 -16.109 -3.578 -19.809 1.00 .00
ATOM 36 CG2 ILE 2 -13.979 -3.071 -20.987 1.00 .00
ATOM 37 HG21 ILE 2 -14.186 -2.925 -19.937 1.00 .00
ATOM 38 HG22 ILE 2 -13.409 -2.234 -21.361 1.00 .00
ATOM 39 HG23 ILE 2 -13.413 -3.981 -21.119 1.00 .00
ATOM 40 CD1 ILE 2 -16.486 -5.268 -21.080 1.00 .00
ATOM 41 HD11 ILE 2 -16.141 -5.797 -20.205 1.00 .00
ATOM 42 HD12 ILE 2 -15.879 -5.543 -21.930 1.00 .00
ATOM 43 HD13 ILE 2 -17.516 -5.527 -21.274 1.00 .00
ATOM 44 C ILE 2 -15.904 -.841 -21.033 1.00 .00
ATOM 45 O ILE 2 -15.218 -.948 -20.036 1.00 .00
ATOM 46 N GLU 3 -16.820 .084 -21.129 1.00 .00
ATOM 47 HN GLU 3 -17.362 .153 -21.943 1.00 .00
ATOM 48 CA GLU 3 -17.043 1.037 -20.003 1.00 .00
ATOM 49 HA GLU 3 -16.238 1.752 -19.949 1.00 .00
ATOM 50 CB GLU 3 -18.352 1.750 -20.344 1.00 .00
ATOM 51 HB1 GLU 3 -19.157 1.324 -19.764 1.00 .00
ATOM 52 HB2 GLU 3 -18.564 1.629 -21.397 1.00 .00
ATOM 53 CG GLU 3 -18.225 3.238 -20.017 1.00 .00
ATOM 54 HG1 GLU 3 -17.253 3.593 -20.325 1.00 .00
ATOM 55 HG2 GLU 3 -18.342 3.383 -18.953 1.00 .00
ATOM 56 CD GLU 3 -19.309 4.019 -20.763 1.00 .00
ATOM 57 OE1 GLU 3 -19.277 5.237 -20.710 1.00 .00
ATOM 58 OE2 GLU 3 -20.154 3.384 -21.373 1.00 .00
ATOM 59 C GLU 3 -17.175 .276 -18.681 1.00 .00
ATOM 60 O GLU 3 -18.069 -.528 -18.504 1.00 .00
ATOM 61 N THR 4 -16.292 .522 -17.752 1.00 .00
ATOM 62 HN THR 4 -15.578 1.174 -17.915 1.00 .00
ATOM 63 CA THR 4 -16.368 -.189 -16.443 1.00 .00
ATOM 64 HA THR 4 -17.314 -.700 -16.348 1.00 .00
ATOM 65 CB THR 4 -15.225 -1.208 -16.469 1.00 .00
ATOM 66 HB THR 4 -14.591 -1.059 -15.608 1.00 .00
ATOM 67 OG1 THR 4 -14.460 -1.038 -17.656 1.00 .00
ATOM 68 HG1 THR 4 -13.745 -.427 -17.463 1.00 .00
ATOM 69 CG2 THR 4 -15.801 -2.624 -16.429 1.00 .00
ATOM 70 HG21 THR 4 -15.231 -3.225 -15.737 1.00 .00
ATOM 71 HG22 THR 4 -15.748 -3.062 -17.415 1.00 .00
ATOM 72 HG23 THR 4 -16.831 -2.584 -16.107 1.00 .00
ATOM 73 C THR 4 -16.177 .798 -15.288 1.00 .00
ATOM 74 O THR 4 -16.248 1.998 -15.465 1.00 .00
ATOM 75 N ASP 5 -15.933 .300 -14.107 1.00 .00
ATOM 76 HN ASP 5 -15.880 -.671 -13.987 1.00 .00
ATOM 77 CA ASP 5 -15.736 1.205 -12.938 1.00 .00
ATOM 78 HA ASP 5 -15.260 2.122 -13.247 1.00 .00
ATOM 79 CB ASP 5 -17.146 1.495 -12.423 1.00 .00
ATOM 80 HB1 ASP 5 -17.100 1.763 -11.379 1.00 .00
ATOM 81 HB2 ASP 5 -17.761 .614 -12.543 1.00 .00
ATOM 82 CG ASP 5 -17.753 2.654 -13.219 1.00 .00
ATOM 83 OD1 ASP 5 -18.624 2.395 -14.033 1.00 .00
ATOM 84 OD2 ASP 5 -17.337 3.779 -13.000 1.00 .00
ATOM 85 C ASP 5 -14.903 .502 -11.862 1.00 .00
ATOM 86 O ASP 5 -15.418 .067 -10.851 1.00 .00
ATOM 87 N GLU 6 -13.620 .387 -12.072 1.00 .00
ATOM 88 HN GLU 6 -13.224 .743 -12.895 1.00 .00
ATOM 89 CA GLU 6 -12.757 -.290 -11.061 1.00 .00
ATOM 90 HA GLU 6 -13.345 -.952 -10.446 1.00 .00
ATOM 91 CB GLU 6 -11.747 -1.096 -11.878 1.00 .00
ATOM 92 HB1 GLU 6 -10.797 -.584 -11.883 1.00 .00
ATOM 93 HB2 GLU 6 -12.106 -1.201 -12.892 1.00 .00
ATOM 94 CG GLU 6 -11.574 -2.480 -11.250 1.00 .00
ATOM 95 HG1 GLU 6 -12.350 -2.642 -10.518 1.00 .00
ATOM 96 HG2 GLU 6 -10.608 -2.539 -10.771 1.00 .00
ATOM 97 CD GLU 6 -11.671 -3.552 -12.337 1.00 .00
ATOM 98 OE1 GLU 6 -11.854 -4.707 -11.988 1.00 .00
ATOM 99 OE2 GLU 6 -11.560 -3.201 -13.500 1.00 .00
ATOM 100 C GLU 6 -12.039 .749 -10.197 1.00 .00
ATOM 101 O GLU 6 -12.513 1.852 -10.013 1.00 .00
ATOM 102 N ARG 7 -10.897 .405 -9.665 1.00 .00
ATOM 103 HN ARG 7 -10.531 -.490 -9.826 1.00 .00
ATOM 104 CA ARG 7 -10.150 1.374 -8.813 1.00 .00
ATOM 105 HA ARG 7 -10.418 2.387 -9.071 1.00 .00
ATOM 106 CB ARG 7 -10.591 1.067 -7.380 1.00 .00
ATOM 107 HB1 ARG 7 -11.654 1.232 -7.288 1.00 .00
ATOM 108 HB2 ARG 7 -10.066 1.716 -6.695 1.00 .00
ATOM 109 CG ARG 7 -10.275 -.393 -7.046 1.00 .00
ATOM 110 HG1 ARG 7 -9.621 -.432 -6.188 1.00 .00
ATOM 111 HG2 ARG 7 -9.790 -.859 -7.892 1.00 .00
ATOM 112 CD ARG 7 -11.574 -1.136 -6.726 1.00 .00
ATOM 113 HD1 ARG 7 -12.070 -1.435 -7.636 1.00 .00
ATOM 114 HD2 ARG 7 -12.224 -.513 -6.127 1.00 .00
ATOM 115 NE ARG 7 -11.151 -2.339 -5.957 1.00 .00
ATOM 116 HE ARG 7 -11.420 -2.441 -5.021 1.00 .00
ATOM 117 CZ ARG 7 -10.422 -3.257 -6.531 1.00 .00
ATOM 118 NH1 ARG 7 -10.994 -4.227 -7.190 1.00 .00
ATOM 119 HH11 ARG 7 -11.991 -4.267 -7.256 1.00 .00
ATOM 120 HH12 ARG 7 -10.436 -4.931 -7.630 1.00 .00
ATOM 121 NH2 ARG 7 -9.121 -3.206 -6.444 1.00 .00
ATOM 122 HH21 ARG 7 -8.682 -2.464 -5.937 1.00 .00
ATOM 123 HH22 ARG 7 -8.563 -3.909 -6.885 1.00 .00
ATOM 124 C ARG 7 -8.642 1.164 -8.969 1.00 .00
ATOM 125 O ARG 7 -8.049 .335 -8.308 1.00 .00
ATOM 126 N ASP 8 -8.017 1.911 -9.838 1.00 .00
ATOM 127 HN ASP 8 -8.514 2.575 -10.360 1.00 .00
ATOM 128 CA ASP 8 -6.547 1.757 -10.036 1.00 .00
ATOM 129 HA ASP 8 -6.181 2.493 -10.734 1.00 .00
ATOM 130 CB ASP 8 -5.935 1.997 -8.655 1.00 .00
ATOM 131 HB1 ASP 8 -5.182 1.248 -8.459 1.00 .00
ATOM 132 HB2 ASP 8 -6.708 1.937 -7.903 1.00 .00
ATOM 133 CG ASP 8 -5.291 3.384 -8.615 1.00 .00
ATOM 134 OD1 ASP 8 -5.532 4.154 -9.531 1.00 .00
ATOM 135 OD2 ASP 8 -4.568 3.653 -7.670 1.00 .00
ATOM 136 C ASP 8 -6.222 .344 -10.527 1.00 .00
ATOM 137 O ASP 8 -6.033 .112 -11.704 1.00 .00
ATOM 138 N SER 9 -6.154 -.601 -9.630 1.00 .00
ATOM 139 HN SER 9 -6.309 -.392 -8.686 1.00 .00
ATOM 140 CA SER 9 -5.840 -2.000 -10.041 1.00 .00
ATOM 141 HA SER 9 -6.506 -2.322 -10.825 1.00 .00
ATOM 142 CB SER 9 -4.405 -1.947 -10.562 1.00 .00
ATOM 143 HB1 SER 9 -4.213 -.970 -10.986 1.00 .00
ATOM 144 HB2 SER 9 -4.267 -2.698 -11.322 1.00 .00
ATOM 145 OG SER 9 -3.506 -2.197 -9.489 1.00 .00
ATOM 146 HG SER 9 -3.388 -1.377 -9.004 1.00 .00
ATOM 147 C SER 9 -5.940 -2.936 -8.833 1.00 .00
ATOM 148 O SER 9 -6.598 -2.636 -7.857 1.00 .00
ATOM 149 N THR 10 -5.291 -4.066 -8.891 1.00 .00
ATOM 150 HN THR 10 -4.765 -4.290 -9.687 1.00 .00
ATOM 151 CA THR 10 -5.349 -5.018 -7.744 1.00 .00
ATOM 152 HA THR 10 -5.963 -4.618 -6.953 1.00 .00
ATOM 153 CB THR 10 -5.987 -6.290 -8.310 1.00 .00
ATOM 154 HB THR 10 -5.384 -7.144 -8.041 1.00 .00
ATOM 155 OG1 THR 10 -6.066 -6.194 -9.726 1.00 .00
ATOM 156 HG1 THR 10 -5.172 -6.139 -10.070 1.00 .00
ATOM 157 CG2 THR 10 -7.391 -6.463 -7.728 1.00 .00
ATOM 158 HG21 THR 10 -8.094 -5.884 -8.307 1.00 .00
ATOM 159 HG22 THR 10 -7.400 -6.121 -6.703 1.00 .00
ATOM 160 HG23 THR 10 -7.668 -7.506 -7.762 1.00 .00
ATOM 161 C THR 10 -3.937 -5.313 -7.232 1.00 .00
ATOM 162 O THR 10 -3.755 -5.996 -6.244 1.00 .00
ATOM 163 N ASN 11 -2.937 -4.806 -7.900 1.00 .00
ATOM 164 HN ASN 11 -3.106 -4.262 -8.697 1.00 .00
ATOM 165 CA ASN 11 -1.537 -5.062 -7.455 1.00 .00
ATOM 166 HA ASN 11 -1.523 -5.756 -6.630 1.00 .00
ATOM 167 CB ASN 11 -.850 -5.685 -8.671 1.00 .00
ATOM 168 HB1 ASN 11.079 -5.169 -8.864 1.00 .00
ATOM 169 HB2 ASN 11 -1.496 -5.597 -9.533 1.00 .00
ATOM 170 CG ASN 11 -.563 -7.161 -8.395 1.00 .00
ATOM 171 OD1 ASN 11 -1.149 -8.031 -9.008 1.00 .00
ATOM 172 ND2 ASN 11 .320 -7.483 -7.490 1.00 .00
ATOM 173 HD21 ASN 11 .793 -6.781 -6.996 1.00 .00
ATOM 174 HD22 ASN 11 .511 -8.426 -7.306 1.00 .00
ATOM 175 C ASN 11 -.851 -3.750 -7.065 1.00 .00
ATOM 176 O ASN 11 .234 -3.447 -7.519 1.00 .00
ATOM 177 N ARG 12 -1.475 -2.969 -6.226 1.00 .00
ATOM 178 HN ARG 12 -2.349 -3.231 -5.870 1.00 .00
ATOM 179 CA ARG 12 -.855 -1.678 -5.810 1.00 .00
ATOM 180 HA ARG 12 .172 -1.631 -6.134 1.00 .00
ATOM 181 CB ARG 12 -1.678 -.602 -6.521 1.00 .00
ATOM 182 HB1 ARG 12 -2.162 .024 -5.787 1.00 .00
ATOM 183 HB2 ARG 12 -2.426 -1.074 -7.141 1.00 .00
ATOM 184 CG ARG 12 -.758 .256 -7.392 1.00 .00
ATOM 185 HG1 ARG 12 .194 .379 -6.900 1.00 .00
ATOM 186 HG2 ARG 12 -1.212 1.224 -7.547 1.00 .00
ATOM 187 CD ARG 12 -.545 -.431 -8.744 1.00 .00
ATOM 188 HD1 ARG 12 -1.462 -.436 -9.312 1.00 .00
ATOM 189 HD2 ARG 12 -.183 -1.440 -8.600 1.00 .00
ATOM 190 NE ARG 12 .480 .398 -9.438 1.00 .00
ATOM 191 HE ARG 12.201 1.117 -10.043 1.00 .00
ATOM 192 CZ ARG 12 1.749 .166 -9.242 1.00 .00
ATOM 193 NH1 ARG 12 2.186 -1.062 -9.172 1.00 .00
ATOM 194 HH11 ARG 12 1.548 -1.826 -9.268 1.00 .00
ATOM 195 HH12 ARG 12 3.159 -1.239 -9.023 1.00 .00
ATOM 196 NH2 ARG 12 2.582 1.163 -9.116 1.00 .00
ATOM 197 HH21 ARG 12 2.248 2.104 -9.170 1.00 .00
ATOM 198 HH22 ARG 12 3.555 .986 -8.965 1.00 .00
ATOM 199 C ARG 12 -.949 -1.510 -4.290 1.00 .00
ATOM 200 O ARG 12 -2.008 -1.267 -3.747 1.00 .00
ATOM 201 N ALA 13 .152 -1.638 -3.598 1.00 .00
ATOM 202 HN ALA 13 .997 -1.835 -4.054 1.00 .00
ATOM 203 CA ALA 13 .121 -1.486 -2.114 1.00 .00
ATOM 204 HA ALA 13 -.658 -2.087 -1.690 1.00 .00
ATOM 205 CB ALA 13 1.483 -1.982 -1.628 1.00 .00
ATOM 206 HB1 ALA 13 1.625 -1.690 -.598 1.00 .00
ATOM 207 HB2 ALA 13 2.262 -1.547 -2.235 1.00 .00
ATOM 208 HB3 ALA 13 1.524 -3.058 -1.706 1.00 .00
ATOM 209 C ALA 13 -.080 -.023 -1.733 1.00 .00
ATOM 210 O ALA 13 -.256 .831 -2.580 1.00 .00
ATOM 211 N SER 14 -.044 .273 -.463 1.00 .00
ATOM 212 HN SER 14 .106 -.435 .198 1.00 .00
ATOM 213 CA SER 14 -.225 1.681 -.012 1.00 .00
ATOM 214 HA SER 14 .553 2.309 -.416 1.00 .00
ATOM 215 CB SER 14 -1.583 2.107 -.568 1.00 .00
ATOM 216 HB1 SER 14 -1.525 2.176 -1.646 1.00 .00
ATOM 217 HB2 SER 14 -1.852 3.068 -.166 1.00 .00
ATOM 218 OG SER 14 -2.565 1.151 -.193 1.00 .00
ATOM 219 HG SER 14 -2.415 .916 .726 1.00 .00
ATOM 220 C SER 14 -.224 1.745 1.517 1.00 .00
ATOM 221 O SER 14 -1.104 2.322 2.123 1.00 .00
ATOM 222 N PHE 15 .758 1.151 2.145 1.00 .00
ATOM 223 HN PHE 15 1.455 .691 1.634 1.00 .00
ATOM 224 CA PHE 15 .813 1.176 3.639 1.00 .00
ATOM 225 HA PHE 15 .153 .430 4.054 1.00 .00
ATOM 226 CB PHE 15 2.264 .860 4.016 1.00 .00
ATOM 227 HB1 PHE 15 2.317 .632 5.071 1.00 .00
ATOM 228 HB2 PHE 15 2.882 1.720 3.806 1.00 .00
ATOM 229 CG PHE 15 2.766 -.322 3.225 1.00 .00
ATOM 230 CD1 PHE 15 4.108 -.372 2.831 1.00 .00
ATOM 231 HD1 PHE 15 4.778 .432 3.094 1.00 .00
ATOM 232 CD2 PHE 15 1.896 -1.364 2.885 1.00 .00
ATOM 233 HD2 PHE 15 .860 -1.324 3.189 1.00 .00
ATOM 234 CE1 PHE 15 4.581 -1.464 2.097 1.00 .00
ATOM 235 HE1 PHE 15 5.616 -1.501 1.795 1.00 .00
ATOM 236 CE2 PHE 15 2.370 -2.457 2.150 1.00 .00
ATOM 237 HE2 PHE 15 1.699 -3.262 1.887 1.00 .00
ATOM 238 CZ PHE 15 3.713 -2.507 1.756 1.00 .00
ATOM 239 HZ PHE 15 4.080 -3.349 1.189 1.00 .00
ATOM 240 C PHE 15 .439 2.565 4.150 1.00 .00
ATOM 241 O PHE 15 .671 3.558 3.492 1.00 .00
ATOM 242 N LYS 16 -.129 2.647 5.319 1.00 .00
ATOM 243 HN LYS 16 -.303 1.833 5.838 1.00 .00
ATOM 244 CA LYS 16 -.506 3.980 5.866 1.00 .00
ATOM 245 HA LYS 16 -.240 4.759 5.169 1.00 .00
ATOM 246 CB LYS 16 -2.025 3.930 6.035 1.00 .00
ATOM 247 HB1 LYS 16 -2.309 2.985 6.475 1.00 .00
ATOM 248 HB2 LYS 16 -2.496 4.031 5.068 1.00 .00
ATOM 249 CG LYS 16 -2.478 5.075 6.946 1.00 .00
ATOM 250 HG1 LYS 16 -1.625 5.674 7.226 1.00 .00
ATOM 251 HG2 LYS 16 -2.939 4.668 7.835 1.00 .00
ATOM 252 CD LYS 16 -3.488 5.952 6.201 1.00 .00
ATOM 253 HD1 LYS 16 -3.811 5.447 5.304 1.00 .00
ATOM 254 HD2 LYS 16 -3.024 6.892 5.940 1.00 .00
ATOM 255 CE LYS 16 -4.699 6.212 7.100 1.00 .00
ATOM 256 HE1 LYS 16 -4.586 5.699 8.042 1.00 .00
ATOM 257 HE2 LYS 16 -5.608 5.899 6.604 1.00 .00
ATOM 258 NZ LYS 16 -4.704 7.685 7.319 1.00 .00
ATOM 259 HZ1 LYS 16 -5.682 8.013 7.453 1.00 .00
ATOM 260 HZ2 LYS 16 -4.289 8.160 6.491 1.00 .00
ATOM 261 HZ3 LYS 16 -4.146 7.912 8.166 1.00 .00
ATOM 262 C LYS 16 .185 4.211 7.211 1.00 .00
ATOM 263 O LYS 16 .269 3.326 8.040 1.00 .00
ATOM 264 N CYS 17.685 5.396 7.432 1.00 .00
ATOM 265 HN CYS 17 .609 6.094 6.748 1.00 .00
ATOM 266 CA CYS 17 1.376 5.687 8.719 1.00 .00
ATOM 267 HA CYS 17 1.941 4.829 9.045 1.00 .00
ATOM 268 HB1 CYS 17 1.757 7.758 8.329 1.00 .00
ATOM 269 HB2 CYS 17 2.822 6.652 7.463 1.00 .00
ATOM 270 C CYS 17 .360 6.104 9.788 1.00 .00
ATOM 271 O CYS 17 -.437 6.995 9.572 1.00 .00
ATOM 272 ZN CYS 17 4.829 8.852 9.299 1.00 .00
ATOM 273 CB CYS 17 2.321 6.842 8.401 1.00 .00
ATOM 274 SG CYS 17 3.547 6.992 9.720 1.00 .00
ATOM 275 N PRO 18 .423 5.438 10.910 1.00 .00
ATOM 276 CA PRO 18 -.504 5.737 12.027 1.00 .00
ATOM 277 HA PRO 18 -1.514 5.845 11.666 1.00 .00
ATOM 278 CB PRO 18 -.398 4.503 12.916 1.00 .00
ATOM 279 HB1 PRO 18 -1.168 3.791 12.663 1.00 .00
ATOM 280 HB2 PRO 18 -.473 4.786 13.957 1.00 .00
ATOM 281 CG PRO 18 .952 3.921 12.628 1.00 .00
ATOM 282 HG1 PRO 18 .904 2.844 12.673 1.00 .00
ATOM 283 HG2 PRO 18 1.668 4.288 13.349 1.00 .00
ATOM 284 CD PRO 18 1.352 4.352 11.239 1.00 .00
ATOM 285 HD2 PRO 18 2.373 4.710 11.235 1.00 .00
ATOM 286 HD1 PRO 18 1.232 3.538 10.541 1.00 .00
ATOM 287 C PRO 18 -.061 6.990 12.794 1.00 .00
ATOM 288 O PRO 18 -.710 7.410 13.731 1.00 .00
ATOM 289 N VAL 19 1.039 7.589 12.417 1.00 .00
ATOM 290 HN VAL 19 1.559 7.240 11.663 1.00 .00
ATOM 291 CA VAL 19 1.501 8.806 13.152 1.00 .00
ATOM 292 HA VAL 19.921 8.935 14.052 1.00 .00
ATOM 293 CB VAL 19 2.961 8.529 13.520 1.00 .00
ATOM 294 HB VAL 19 3.451 8.026 12.699 1.00 .00
ATOM 295 CG1 VAL 19 3.685 9.845 13.814 1.00 .00
ATOM 296 HG11 VAL 19 4.007 10.293 12.886 1.00 .00
ATOM 297 HG12 VAL 19 4.544 9.652 14.438 1.00 .00
ATOM 298 HG13 VAL 19 3.013 10.519 14.324 1.00 .00
ATOM 299 CG2 VAL 19 3.006 7.644 14.768 1.00 .00
ATOM 300 HG21 VAL 19 2.522 8.154 15.587 1.00 .00
ATOM 301 HG22 VAL 19 4.035 7.441 15.029 1.00 .00
ATOM 302 HG23 VAL 19 2.495 6.714 14.570 1.00 .00
ATOM 303 C VAL 19 1.383 10.055 12.271 1.00 .00
ATOM 304 O VAL 19 1.123 11.137 12.758 1.00 .00
ATOM 305 N CYS 20 1.557 9.927 10.982 1.00 .00
ATOM 306 HN CYS 20 1.753 9.047 10.587 1.00 .00
ATOM 307 CA CYS 20 1.433 11.131 10.111 1.00 .00
ATOM 308 HA CYS 20 .966 11.933 10.663 1.00 .00
ATOM 309 HB1 CYS 20 3.504 11.408 10.611 1.00 .00
ATOM 310 HB2 CYS 20 2.885 12.559 9.430 1.00 .00
ATOM 311 C CYS 20 .586 10.789 8.881 1.00 .00
ATOM 312 O CYS 20 .547 11.515 7.907 1.00 .00
ATOM 313 CB CYS 20 2.869 11.526 9.744 1.00 .00
ATOM 314 SG CYS 20 3.487 10.487 8.402 1.00 .00
ATOM 315 N SER 21 -.113 9.691 8.951 1.00 .00
ATOM 316 HN SER 21 -.069 9.145 9.763 1.00 .00
ATOM 317 CA SER 21 -1.002 9.264 7.834 1.00 .00
ATOM 318 HA SER 21 -1.443 8.312 8.073 1.00 .00
ATOM 319 CB SER 21 -2.093 10.330 7.772 1.00 .00
ATOM 320 HB1 SER 21 -2.203 10.788 8.746 1.00 .00
ATOM 321 HB2 SER 21 -3.027 9.876 7.485 1.00 .00
ATOM 322 OG SER 21 -1.735 11.313 6.810 1.00 .00
ATOM 323 HG SER 21 -2.542 11.640 6.406 1.00 .00
ATOM 324 C SER 21 -.261 9.176 6.495 1.00 .00
ATOM 325 O SER 21 -.876 9.061 5.453 1.00 .00
ATOM 326 N SER 22 1.041 9.213 6.496 1.00 .00
ATOM 327 HN SER 22 1.534 9.298 7.338 1.00 .00
ATOM 328 CA SER 22 1.773 9.112 5.200 1.00 .00
ATOM 329 HA SER 22 1.449 9.886 4.522 1.00 .00
ATOM 330 CB SER 22 3.246 9.304 5.550 1.00 .00
ATOM 331 HB1 SER 22 3.858 8.834 4.792 1.00 .00
ATOM 332 HB2 SER 22 3.454 8.853 6.505 1.00 .00
ATOM 333 OG SER 22 3.533 10.693 5.613 1.00 .00
ATOM 334 HG SER 22 3.592 11.024 4.713 1.00 .00
ATOM 335 C SER 22 1.543 7.728 4.584 1.00 .00
ATOM 336 O SER 22 1.813 6.716 5.199 1.00 .00
ATOM 337 N THR 23 1.041 7.672 3.380 1.00 .00
ATOM 338 HN THR 23 .823 8.497 2.898 1.00 .00
ATOM 339 CA THR 23 .793 6.345 2.743 1.00 .00
ATOM 340 HA THR 23 .493 5.626 3.487 1.00 .00
ATOM 341 CB THR 23 -.351 6.576 1.751 1.00 .00
ATOM 342 HB THR 23 .020 6.480 .743 1.00 .00
ATOM 343 OG1 THR 23 -.887 7.879 1.936 1.00 .00
ATOM 344 HG1 THR 23 -1.593 8.003 1.297 1.00 .00
ATOM 345 CG2 THR 23 -1.448 5.535 1.984 1.00 .00
ATOM 346 HG21 THR 23 -.997 4.591 2.252 1.00 .00
ATOM 347 HG22 THR 23 -2.028 5.413 1.081 1.00 .00
ATOM 348 HG23 THR 23 -2.094 5.865 2.784 1.00 .00
ATOM 349 C THR 23 2.052 5.863 2.013 1.00 .00
ATOM 350 O THR 23 2.986 6.612 1.810 1.00 .00
ATOM 351 N PHE 24 2.085 4.618 1.619 1.00 .00
ATOM 352 HN PHE 24 1.322 4.028 1.794 1.00 .00
ATOM 353 CA PHE 24 3.287 4.092 .906 1.00 .00
ATOM 354 HA PHE 24 3.721 4.856 .282 1.00 .00
ATOM 355 CB PHE 24 4.265 3.703 2.016 1.00 .00
ATOM 356 HB1 PHE 24 5.141 3.246 1.579 1.00 .00
ATOM 357 HB2 PHE 24 3.789 3.001 2.685 1.00 .00
ATOM 358 CG PHE 24 4.675 4.935 2.787 1.00 .00
ATOM 359 CD1 PHE 24 5.617 5.819 2.248 1.00 .00
ATOM 360 HD1 PHE 24 6.048 5.623 1.277 1.00 .00
ATOM 361 CD2 PHE 24 4.117 5.190 4.046 1.00 .00
ATOM 362 HD2 PHE 24 3.390 4.508 4.462 1.00 .00
ATOM 363 CE1 PHE 24 6.000 6.958 2.967 1.00 .00
ATOM 364 HE1 PHE 24 6.727 7.640 2.551 1.00 .00
ATOM 365 CE2 PHE 24 4.499 6.329 4.764 1.00 .00
ATOM 366 HE2 PHE 24 4.068 6.526 5.735 1.00 .00
ATOM 367 CZ PHE 24 5.442 7.213 4.225 1.00 .00
ATOM 368 HZ PHE 24 5.738 8.091 4.780 1.00 .00
ATOM 369 C PHE 24 2.916 2.862 .071 1.00 .00
ATOM 370 O PHE 24 2.575 1.823 .599 1.00 .00
ATOM 371 N THR 25 2.983 2.968 -1.227 1.00 .00
ATOM 372 HN THR 25 3.262 3.813 -1.638 1.00 .00
ATOM 373 CA THR 25 2.634 1.799 -2.087 1.00 .00
ATOM 374 HA THR 25 1.759 1.296 -1.704 1.00 .00
ATOM 375 CB THR 25 2.333 2.393 -3.464 1.00 .00
ATOM 376 HB THR 25 2.045 1.604 -4.142 1.00 .00
ATOM 377 OG1 THR 25 3.491 3.049 -3.960 1.00 .00
ATOM 378 HG1 THR 25 4.253 2.503 -3.752 1.00 .00
ATOM 379 CG2 THR 25 1.187 3.398 -3.346 1.00 .00
ATOM 380 HG21 THR 25 .657 3.452 -4.285 1.00 .00
ATOM 381 HG22 THR 25 1.586 4.371 -3.100 1.00 .00
ATOM 382 HG23 THR 25 .508 3.081 -2.568 1.00 .00
ATOM 383 C THR 25 3.810 .824 -2.165 1.00 .00
ATOM 384 O THR 25 4.777 .938 -1.437 1.00 .00
ATOM 385 N ASP 26 3.727 -.140 -3.037 1.00 .00
ATOM 386 HN ASP 26 2.931 -.215 -3.606 1.00 .00
ATOM 387 CA ASP 26 4.831 -1.136 -3.162 1.00 .00
ATOM 388 HA ASP 26 4.959 -1.671 -2.235 1.00 .00
ATOM 389 CB ASP 26 4.375 -2.100 -4.258 1.00 .00
ATOM 390 HB1 ASP 26 3.373 -2.440 -4.044 1.00 .00
ATOM 391 HB2 ASP 26 5.044 -2.948 -4.291 1.00 .00
ATOM 392 CG ASP 26 4.391 -1.384 -5.610 1.00 .00
ATOM 393 OD1 ASP 26 3.364 -.841 -5.983 1.00 .00
ATOM 394 OD2 ASP 26 5.430 -1.392 -6.250 1.00 .00
ATOM 395 C ASP 26 6.139 -.447 -3.563 1.00 .00
ATOM 396 O ASP 26 7.205 -.811 -3.107 1.00 .00
ATOM 397 N LEU 27 6.072 .540 -4.415 1.00 .00
ATOM 398 HN LEU 27 5.206 .819 -4.777 1.00 .00
ATOM 399 CA LEU 27 7.321 1.238 -4.840 1.00 .00
ATOM 400 HA LEU 27 8.014 .533 -5.271 1.00 .00
ATOM 401 CB LEU 27 6.872 2.243 -5.908 1.00 .00
ATOM 402 HB1 LEU 27 6.122 1.786 -6.536 1.00 .00
ATOM 403 HB2 LEU 27 7.721 2.528 -6.512 1.00 .00
ATOM 404 CG LEU 27 6.282 3.487 -5.240 1.00 .00
ATOM 405 HG LEU 27 5.904 3.225 -4.263 1.00 .00
ATOM 406 CD1 LEU 27 7.370 4.553 -5.098 1.00 .00
ATOM 407 HD11 LEU 27 7.607 4.958 -6.070 1.00 .00
ATOM 408 HD12 LEU 27 8.256 4.109 -4.668 1.00 .00
ATOM 409 HD13 LEU 27 7.016 5.345 -4.455 1.00 .00
ATOM 410 CD2 LEU 27 5.142 4.037 -6.101 1.00 .00
ATOM 411 HD21 LEU 27 4.694 4.885 -5.605 1.00 .00
ATOM 412 HD22 LEU 27 4.396 3.269 -6.244 1.00 .00
ATOM 413 HD23 LEU 27 5.531 4.345 -7.060 1.00 .00
ATOM 414 C LEU 27 7.964 1.950 -3.644 1.00 .00
ATOM 415 O LEU 27 9.135 2.275 -3.659 1.00 .00
ATOM 416 N GLU 28 7.208 2.190 -2.607 1.00 .00
ATOM 417 HN GLU 28 6.267 1.917 -2.614 1.00 .00
ATOM 418 CA GLU 28 7.774 2.874 -1.409 1.00 .00
ATOM 419 HA GLU 28 8.609 3.497 -1.687 1.00 .00
ATOM 420 CB GLU 28 6.633 3.737 -.867 1.00 .00
ATOM 421 HB1 GLU 28 7.004 4.371 -.076 1.00 .00
ATOM 422 HB2 GLU 28 5.853 3.097 -.480 1.00 .00
ATOM 423 CG GLU 28 6.070 4.606 -1.992 1.00 .00
ATOM 424 HG1 GLU 28 5.005 4.448 -2.070 1.00 .00
ATOM 425 HG2 GLU 28 6.544 4.337 -2.924 1.00 .00
ATOM 426 CD GLU 28 6.344 6.079 -1.686 1.00 .00
ATOM 427 OE1 GLU 28 5.745 6.591 -.756 1.00 .00
ATOM 428 OE2 GLU 28 7.148 6.669 -2.389 1.00 .00
ATOM 429 C GLU 28 8.200 1.836 -.368 1.00 .00
ATOM 430 O GLU 28 8.860 2.148 .605 1.00 .00
ATOM 431 N ALA 29 7.825 .603 -.566 1.00 .00
ATOM 432 HN ALA 29 7.293 .376 -1.357 1.00 .00
ATOM 433 CA ALA 29 8.201 -.462 .406 1.00 .00
ATOM 434 HA ALA 29 7.957 -.160 1.414 1.00 .00
ATOM 435 CB ALA 29 7.356 -1.671 .003 1.00 .00
ATOM 436 HB1 ALA 29 7.078 -2.227 .885 1.00 .00
ATOM 437 HB2 ALA 29 7.928 -2.305 -.657 1.00 .00
ATOM 438 HB3 ALA 29 6.465 -1.333 -.506 1.00 .00
ATOM 439 C ALA 29 9.692 -.789 .286 1.00 .00
ATOM 440 O ALA 29 10.374 -.996 1.268 1.00 .00
ATOM 441 N ASN 30 10.200 -.843 -.914 1.00 .00
ATOM 442 HN ASN 30 9.629 -.678 -1.693 1.00 .00
ATOM 443 CA ASN 30 11.644 -1.165 -1.104 1.00 .00
ATOM 444 HA ASN 30 11.837 -2.193 -.843 1.00 .00
ATOM 445 CB ASN 30 11.895 -.954 -2.597 1.00 .00
ATOM 446 HB1 ASN 30 12.957 -.951 -2.788 1.00 .00
ATOM 447 HB2 ASN 30 11.471 -.008 -2.903 1.00 .00
ATOM 448 CG ASN 30 11.239 -2.087 -3.388 1.00 .00
ATOM 449 OD1 ASN 30 11.215 -3.218 -2.944 1.00 .00
ATOM 450 ND2 ASN 30 10.702 -1.831 -4.549 1.00 .00
ATOM 451 HD21 ASN 30 10.721 -.920 -4.908 1.00 .00
ATOM 452 HD22 ASN 30 10.279 -2.551 -5.063 1.00 .00
ATOM 453 C ASN 30 12.528 -.228 -.273 1.00 .00
ATOM 454 O ASN 30 13.371 -.667 .484 1.00 .00
ATOM 455 N GLN 31 12.354 1.058 -.414 1.00 .00
ATOM 456 HN GLN 31 11.675 1.395 -1.036 1.00 .00
ATOM 457 CA GLN 31 13.198 2.015 .361 1.00 .00
ATOM 458 HA GLN 31 14.240 1.759 .265 1.00 .00
ATOM 459 CB GLN 31 12.938 3.378 -.280 1.00 .00
ATOM 460 HB1 GLN 31 13.156 3.328 -1.336 1.00 .00
ATOM 461 HB2 GLN 31 13.571 4.121 .185 1.00 .00
ATOM 462 CG GLN 31 11.470 3.764 -.083 1.00 .00
ATOM 463 HG1 GLN 31 11.363 4.318 .837 1.00 .00
ATOM 464 HG2 GLN 31 10.866 2.869 -.037 1.00 .00
ATOM 465 CD GLN 31 11.009 4.631 -1.255 1.00 .00
ATOM 466 OE1 GLN 31 10.905 5.836 -1.131 1.00 .00
ATOM 467 NE2 GLN 31 10.727 4.066 -2.396 1.00 .00
ATOM 468 HE21 GLN 31 10.812 3.095 -2.496 1.00 .00
ATOM 469 HE22 GLN 31 10.430 4.613 -3.154 1.00 .00
ATOM 470 C GLN 31 12.787 2.035 1.838 1.00 .00
ATOM 471 O GLN 31 13.584 2.327 2.707 1.00 .00
ATOM 472 N LEU 32 11.551 1.740 2.128 1.00 .00
ATOM 473 HN LEU 32 10.919 1.516 1.414 1.00 .00
ATOM 474 CA LEU 32 11.095 1.757 3.549 1.00 .00
ATOM 475 HA LEU 32 11.642 2.498 4.110 1.00 .00
ATOM 476 CB LEU 32 9.620 2.148 3.476 1.00 .00
ATOM 477 HB1 LEU 32 9.196 2.151 4.468 1.00 .00
ATOM 478 HB2 LEU 32 9.090 1.436 2.859 1.00 .00
ATOM 479 CG LEU 32 9.496 3.545 2.865 1.00 .00
ATOM 480 HG LEU 32 9.967 3.555 1.893 1.00 .00
ATOM 481 CD1 LEU 32 8.019 3.911 2.719 1.00 .00
ATOM 482 HD11 LEU 32 7.743 4.611 3.494 1.00 .00
ATOM 483 HD12 LEU 32 7.417 3.019 2.808 1.00 .00
ATOM 484 HD13 LEU 32 7.854 4.361 1.751 1.00 .00
ATOM 485 CD2 LEU 32 10.184 4.564 3.778 1.00 .00
ATOM 486 HD21 LEU 32 9.449 5.256 4.162 1.00 .00
ATOM 487 HD22 LEU 32 10.929 5.107 3.215 1.00 .00
ATOM 488 HD23 LEU 32 10.658 4.049 4.600 1.00 .00
ATOM 489 C LEU 32 11.252 .375 4.192 1.00 .00
ATOM 490 O LEU 32 11.183 .231 5.396 1.00 .00
ATOM 491 N PHE 33 11.458 -.641 3.402 1.00 .00
ATOM 492 HN PHE 33 11.508 -.506 2.432 1.00 .00
ATOM 493 CA PHE 33 11.612 -2.012 3.973 1.00 .00
ATOM 494 HA PHE 33 10.706 -2.314 4.472 1.00 .00
ATOM 495 CB PHE 33 11.867 -2.916 2.766 1.00 .00
ATOM 496 HB1 PHE 33 12.791 -2.625 2.289 1.00 .00
ATOM 497 HB2 PHE 33 11.054 -2.816 2.065 1.00 .00
ATOM 498 CG PHE 33 11.968 -4.354 3.218 1.00 .00
ATOM 499 CD1 PHE 33 13.178 -4.846 3.721 1.00 .00
ATOM 500 HD1 PHE 33 14.036 -4.195 3.794 1.00 .00
ATOM 501 CD2 PHE 33 10.854 -5.198 3.126 1.00 .00
ATOM 502 HD2 PHE 33 9.919 -4.819 2.741 1.00 .00
ATOM 503 CE1 PHE 33 13.276 -6.181 4.131 1.00 .00
ATOM 504 HE1 PHE 33 14.210 -6.560 4.519 1.00 .00
ATOM 505 CE2 PHE 33 10.951 -6.533 3.537 1.00 .00
ATOM 506 HE2 PHE 33 10.092 -7.184 3.466 1.00 .00
ATOM 507 CZ PHE 33 12.162 -7.025 4.039 1.00 .00
ATOM 508 HZ PHE 33 12.238 -8.055 4.355 1.00 .00
ATOM 509 C PHE 33 12.798 -2.063 4.942 1.00 .00
ATOM 510 O PHE 33 13.842 -1.493 4.693 1.00 .00
ATOM 511 N ASP 34 12.642 -2.747 6.043 1.00 .00
ATOM 512 HN ASP 34 11.791 -3.201 6.219 1.00 .00
ATOM 513 CA ASP 34 13.756 -2.846 7.031 1.00 .00
ATOM 514 HA ASP 34 14.537 -2.142 6.793 1.00 .00
ATOM 515 CB ASP 34 13.123 -2.491 8.378 1.00 .00
ATOM 516 HB1 ASP 34 13.238 -3.320 9.060 1.00 .00
ATOM 517 HB2 ASP 34 12.071 -2.285 8.237 1.00 .00
ATOM 518 CG ASP 34 13.813 -1.256 8.959 1.00 .00
ATOM 519 OD1 ASP 34 14.509 -1.402 9.950 1.00 .00
ATOM 520 OD2 ASP 34 13.632 -.184 8.405 1.00 .00
ATOM 521 C ASP 34 14.307 -4.275 7.057 1.00 .00
ATOM 522 O ASP 34 13.556 -5.229 7.025 1.00 .00
ATOM 523 N PRO 35 15.608 -4.372 7.108 1.00 .00
ATOM 524 CA PRO 35 16.272 -5.698 7.131 1.00 .00
ATOM 525 HA PRO 35 15.886 -6.332 6.349 1.00 .00
ATOM 526 CB PRO 35 17.737 -5.369 6.859 1.00 .00
ATOM 527 HB1 PRO 35 17.952 -5.451 5.806 1.00 .00
ATOM 528 HB2 PRO 35 18.380 -6.027 7.429 1.00 .00
ATOM 529 CG PRO 35 17.908 -3.952 7.308 1.00 .00
ATOM 530 HG1 PRO 35 18.645 -3.458 6.696 1.00 .00
ATOM 531 HG2 PRO 35 18.213 -3.931 8.346 1.00 .00
ATOM 532 CD PRO 35 16.573 -3.269 7.147 1.00 .00
ATOM 533 HD2 PRO 35 16.378 -2.619 7.990 1.00 .00
ATOM 534 HD1 PRO 35 16.540 -2.714 6.222 1.00 .00
ATOM 535 C PRO 35 16.114 -6.369 8.501 1.00 .00
ATOM 536 O PRO 35 15.994 -7.573 8.599 1.00 .00
ATOM 537 N MET 36 16.119 -5.602 9.559 1.00 .00
ATOM 538 HN MET 36 16.223 -4.632 9.461 1.00 .00
ATOM 539 CA MET 36 15.978 -6.206 10.919 1.00 .00
ATOM 540 HA MET 36 16.924 -6.603 11.250 1.00 .00
ATOM 541 CB MET 36 15.551 -5.056 11.837 1.00 .00
ATOM 542 HB1 MET 36 16.419 -4.482 12.116 1.00 .00
ATOM 543 HB2 MET 36 15.089 -5.461 12.726 1.00 .00
ATOM 544 CG MET 36 14.553 -4.144 11.118 1.00 .00
ATOM 545 HG1 MET 36 14.285 -4.576 10.167 1.00 .00
ATOM 546 HG2 MET 36 15.003 -3.175 10.958 1.00 .00
ATOM 547 SD MET 36 13.068 -3.956 12.136 1.00 .00
ATOM 548 CE MET 36 13.364 -2.247 12.652 1.00 .00
ATOM 549 HE1 MET 36 14.357 -1.947 12.348 1.00 .00
ATOM 550 HE2 MET 36 13.280 -2.173 13.724 1.00 .00
ATOM 551 HE3 MET 36 12.630 -1.601 12.190 1.00 .00
ATOM 552 C MET 36 14.916 -7.309 10.912 1.00 .00
ATOM 553 O MET 36 15.070 -8.335 11.544 1.00 .00
ATOM 554 N THR 37 13.843 -7.106 10.202 1.00 .00
ATOM 555 HN THR 37 13.740 -6.272 9.700 1.00 .00
ATOM 556 CA THR 37 12.772 -8.141 10.154 1.00 .00
ATOM 557 HA THR 37 13.185 -9.121 10.334 1.00 .00
ATOM 558 CB THR 37 11.808 -7.762 11.278 1.00 .00
ATOM 559 HB THR 37 12.314 -7.839 12.228 1.00 .00
ATOM 560 OG1 THR 37 10.694 -8.643 11.260 1.00 .00
ATOM 561 HG1 THR 37 11.026 -9.542 11.320 1.00 .00
ATOM 562 CG2 THR 37 11.330 -6.324 11.077 1.00 .00
ATOM 563 HG21 THR 37 11.053 -5.901 12.032 1.00 .00
ATOM 564 HG22 THR 37 10.475 -6.319 10.419 1.00 .00
ATOM 565 HG23 THR 37 12.125 -5.738 10.640 1.00 .00
ATOM 566 C THR 37 12.064 -8.095 8.799 1.00 .00
ATOM 567 O THR 37 11.769 -9.113 8.205 1.00 .00
ATOM 568 N GLY 38 11.797 -6.918 8.302 1.00 .00
ATOM 569 HN GLY 38 12.051 -6.110 8.795 1.00 .00
ATOM 570 CA GLY 38 11.117 -6.804 6.981 1.00 .00
ATOM 571 HA1 GLY 38 10.773 -7.778 6.668 1.00 .00
ATOM 572 HA2 GLY 38 11.816 -6.418 6.254 1.00 .00
ATOM 573 C GLY 38 9.919 -5.857 7.087 1.00 .00
ATOM 574 O GLY 38 8.952 -5.986 6.361 1.00 .00
ATOM 575 N THR 39 9.970 -4.903 7.976 1.00 .00
ATOM 576 HN THR 39 10.757 -4.807 8.551 1.00 .00
ATOM 577 CA THR 39 8.827 -3.954 8.107 1.00 .00
ATOM 578 HA THR 39 7.921 -4.406 7.737 1.00 .00
ATOM 579 CB THR 39 8.696 -3.673 9.606 1.00 .00
ATOM 580 HB THR 39 8.790 -2.612 9.785 1.00 .00
ATOM 581 OG1 THR 39 9.714 -4.366 10.314 1.00 .00
ATOM 582 HG1 THR 39 9.345 -4.664 11.149 1.00 .00
ATOM 583 CG2 THR 39 7.324 -4.143 10.088 1.00 .00
ATOM 584 HG21 THR 39 7.168 -5.167 9.782 1.00 .00
ATOM 585 HG22 THR 39 6.558 -3.517 9.655 1.00 .00
ATOM 586 HG23 THR 39 7.279 -4.077 11.164 1.00 .00
ATOM 587 C THR 39 9.126 -2.662 7.343 1.00 .00
ATOM 588 O THR 39 10.251 -2.392 6.976 1.00 .00
ATOM 589 N PHE 40 8.123 -1.864 7.099 1.00 .00
ATOM 590 HN PHE 40 7.223 -2.103 7.402 1.00 .00
ATOM 591 CA PHE 40 8.346 -.592 6.357 1.00 .00
ATOM 592 HA PHE 40 9.320 -.592 5.895 1.00 .00
ATOM 593 CB PHE 40 7.259 -.570 5.282 1.00 .00
ATOM 594 HB1 PHE 40 7.369 .315 4.675 1.00 .00
ATOM 595 HB2 PHE 40 6.287 -.568 5.753 1.00 .00
ATOM 596 CG PHE 40 7.398 -1.797 4.414 1.00 .00
ATOM 597 CD1 PHE 40 6.575 -2.908 4.634 1.00 .00
ATOM 598 HD1 PHE 40 5.837 -2.886 5.422 1.00 .00
ATOM 599 CD2 PHE 40 8.354 -1.827 3.392 1.00 .00
ATOM 600 HD2 PHE 40 8.990 -.970 3.222 1.00 .00
ATOM 601 CE1 PHE 40 6.708 -4.048 3.832 1.00 .00
ATOM 602 HE1 PHE 40 6.073 -4.905 4.001 1.00 .00
ATOM 603 CE2 PHE 40 8.487 -2.966 2.590 1.00 .00
ATOM 604 HE2 PHE 40 9.223 -2.988 1.801 1.00 .00
ATOM 605 CZ PHE 40 7.664 -4.076 2.809 1.00 .00
ATOM 606 HZ PHE 40 7.767 -4.955 2.191 1.00 .00
ATOM 607 C PHE 40 8.202 .602 7.302 1.00 .00
ATOM 608 O PHE 40 7.155 .832 7.874 1.00 .00
ATOM 609 N ARG 41 9.250 1.360 7.476 1.00 .00
ATOM 610 HN ARG 41 10.087 1.155 7.010 1.00 .00
ATOM 611 CA ARG 41 9.174 2.534 8.390 1.00 .00
ATOM 612 HA ARG 41 8.397 2.390 9.124 1.00 .00
ATOM 613 CB ARG 41 10.537 2.587 9.081 1.00 .00
ATOM 614 HB1 ARG 41 10.794 3.615 9.291 1.00 .00
ATOM 615 HB2 ARG 41 11.286 2.153 8.435 1.00 .00
ATOM 616 CG ARG 41 10.475 1.802 10.393 1.00 .00
ATOM 617 HG1 ARG 41 9.829 .946 10.270 1.00 .00
ATOM 618 HG2 ARG 41 10.084 2.439 11.173 1.00 .00
ATOM 619 CD ARG 41 11.879 1.329 10.777 1.00 .00
ATOM 620 HD1 ARG 41 12.604 2.104 10.586 1.00 .00
ATOM 621 HD2 ARG 41 12.133 .429 10.233 1.00 .00
ATOM 622 NE ARG 41 11.803 1.050 12.238 1.00 .00
ATOM 623 HE ARG 41 11.061 .520 12.598 1.00 .00
ATOM 624 CZ ARG 41 12.723 1.512 13.041 1.00 .00
ATOM 625 NH1 ARG 41 13.850 .870 13.179 1.00 .00
ATOM 626 HH11 ARG 41 14.010 .024 12.671 1.00 .00
ATOM 627 HH12 ARG 41 14.555 1.223 13.795 1.00 .00
ATOM 628 NH2 ARG 41 12.514 2.615 13.707 1.00 .00
ATOM 629 HH21 ARG 41 11.649 3.107 13.602 1.00 .00
ATOM 630 HH22 ARG 41 13.219 2.969 14.321 1.00 .00
ATOM 631 C ARG 41 8.928 3.818 7.595 1.00 .00
ATOM 632 O ARG 41 9.385 3.969 6.479 1.00 .00
ATOM 633 N CYS 42 8.211 4.743 8.168 1.00 .00
ATOM 634 HN CYS 42 7.857 4.596 9.070 1.00 .00
ATOM 635 CA CYS 42 7.931 6.027 7.463 1.00 .00
ATOM 636 HA CYS 42 7.178 5.895 6.704 1.00 .00
ATOM 637 HB1 CYS 42 8.256 7.321 9.130 1.00 .00
ATOM 638 HB2 CYS 42 6.757 6.398 9.207 1.00 .00
ATOM 639 C CYS 42 9.215 6.600 6.868 1.00 .00
ATOM 640 O CYS 42 10.284 6.045 7.023 1.00 .00
ATOM 641 CB CYS 42 7.419 6.949 8.558 1.00 .00
ATOM 642 SG CYS 42 6.524 8.341 7.834 1.00 .00
ATOM 643 N THR 43 9.124 7.715 6.198 1.00 .00
ATOM 644 HN THR 43 8.259 8.155 6.086 1.00 .00
ATOM 645 CA THR 43 10.346 8.316 5.612 1.00 .00
ATOM 646 HA THR 43 11.168 7.625 5.688 1.00 .00
ATOM 647 CB THR 43 10.018 8.570 4.141 1.00 .00
ATOM 648 HB THR 43 9.981 9.632 3.957 1.00 .00
ATOM 649 OG1 THR 43 8.761 7.988 3.825 1.00 .00
ATOM 650 HG1 THR 43 8.679 7.960 2.869 1.00 .00
ATOM 651 CG2 THR 43 11.107 7.945 3.269 1.00 .00
ATOM 652 HG21 THR 43 11.704 8.726 2.823 1.00 .00
ATOM 653 HG22 THR 43 10.649 7.352 2.491 1.00 .00
ATOM 654 HG23 THR 43 11.738 7.313 3.878 1.00 .00
ATOM 655 C THR 43 10.697 9.623 6.325 1.00 .00
ATOM 656 O THR 43 11.826 10.069 6.275 1.00 .00
ATOM 657 N PHE 44 9.764 10.247 7.003 1.00 .00
ATOM 658 HN PHE 44 8.850 9.885 7.058 1.00 .00
ATOM 659 CA PHE 44 10.135 11.514 7.704 1.00 .00
ATOM 660 HA PHE 44 11.207 11.635 7.663 1.00 .00
ATOM 661 CB PHE 44 9.478 12.660 6.928 1.00 .00
ATOM 662 HB1 PHE 44 9.846 12.654 5.912 1.00 .00
ATOM 663 HB2 PHE 44 9.744 13.597 7.392 1.00 .00
ATOM 664 CG PHE 44 7.977 12.531 6.912 1.00 .00
ATOM 665 CD1 PHE 44 7.344 11.861 5.859 1.00 .00
ATOM 666 HD1 PHE 44 7.933 11.410 5.073 1.00 .00
ATOM 667 CD2 PHE 44 7.215 13.114 7.930 1.00 .00
ATOM 668 HD2 PHE 44 7.705 13.629 8.743 1.00 .00
ATOM 669 CE1 PHE 44 5.949 11.776 5.824 1.00 .00
ATOM 670 HE1 PHE 44 5.460 11.259 5.011 1.00 .00
ATOM 671 CE2 PHE 44 5.820 13.025 7.899 1.00 .00
ATOM 672 HE2 PHE 44 5.232 13.472 8.686 1.00 .00
ATOM 673 CZ PHE 44 5.187 12.357 6.844 1.00 .00
ATOM 674 HZ PHE 44 4.111 12.297 6.815 1.00 .00
ATOM 675 C PHE 44 9.698 11.485 9.171 1.00 .00
ATOM 676 O PHE 44 10.319 12.103 10.013 1.00 .00
ATOM 677 N CYS 45 8.674 10.750 9.508 1.00 .00
ATOM 678 HN CYS 45 8.193 10.223 8.834 1.00 .00
ATOM 679 CA CYS 45 8.284 10.685 10.947 1.00 .00
ATOM 680 HA CYS 45 8.840 11.424 11.506 1.00 .00
ATOM 681 HB1 CYS 45 6.482 11.473 10.064 1.00 .00
ATOM 682 HB2 CYS 45 6.596 11.715 11.800 1.00 .00
ATOM 683 C CYS 45 8.628 9.276 11.466 1.00 .00
ATOM 684 O CYS 45 8.132 8.823 12.479 1.00 .00
ATOM 685 CB CYS 45 6.774 11.015 10.999 1.00 .00
ATOM 686 SG CYS 45 5.760 9.538 11.282 1.00 .00
ATOM 687 N HIS 46 9.501 8.608 10.749 1.00 .00
ATOM 688 HN HIS 46 9.875 9.031 9.950 1.00 .00
ATOM 689 CA HIS 46 9.969 7.231 11.106 1.00 .00
ATOM 690 HA HIS 46 10.106 6.656 10.204 1.00 .00
ATOM 691 CB HIS 46 11.332 7.453 11.755 1.00 .00
ATOM 692 HB1 HIS 46 11.672 6.536 12.212 1.00 .00
ATOM 693 HB2 HIS 46 11.255 8.228 12.504 1.00 .00
ATOM 694 CG HIS 46 12.300 7.873 10.684 1.00 .00
ATOM 695 ND1 HIS 46 13.274 7.021 10.186 1.00 .00
ATOM 696 HD1 HIS 46 13.449 6.104 10.484 1.00 .00
ATOM 697 CD2 HIS 46 12.429 9.043 9.976 1.00 .00
ATOM 698 HD2 HIS 46 11.817 9.921 10.109 1.00 .00
ATOM 699 CE1 HIS 46 13.936 7.683 9.219 1.00 .00
ATOM 700 HE1 HIS 46 14.744 7.262 8.638 1.00 .00
ATOM 701 NE2 HIS 46 13.463 8.920 9.052 1.00 .00
ATOM 702 C HIS 46 9.017 6.480 12.043 1.00 .00
ATOM 703 O HIS 46 9.428 5.918 13.039 1.00 .00
ATOM 704 N THR 47 7.761 6.423 11.707 1.00 .00
ATOM 705 HN THR 47 7.454 6.855 10.887 1.00 .00
ATOM 706 CA THR 47 6.798 5.662 12.549 1.00 .00
ATOM 707 HA THR 47 7.217 5.464 13.523 1.00 .00
ATOM 708 CB THR 47 5.566 6.561 12.672 1.00 .00
ATOM 709 HB THR 47 5.290 6.929 11.697 1.00 .00
ATOM 710 OG1 THR 47 5.867 7.656 13.527 1.00 .00
ATOM 711 HG1 THR 47 6.524 7.364 14.164 1.00 .00
ATOM 712 CG2 THR 47 4.401 5.755 13.257 1.00 .00
ATOM 713 HG21 THR 47 3.466 6.213 12.970 1.00 .00
ATOM 714 HG22 THR 47 4.478 5.742 14.334 1.00 .00
ATOM 715 HG23 THR 47 4.434 4.744 12.883 1.00 .00
ATOM 716 C THR 47 6.442 4.354 11.834 1.00 .00
ATOM 717 O THR 47 6.176 4.342 10.648 1.00 .00
ATOM 718 N GLU 48 6.439 3.252 12.535 1.00 .00
ATOM 719 HN GLU 48 6.659 3.274 13.489 1.00 .00
ATOM 720 CA GLU 48 6.102 1.957 11.873 1.00 .00
ATOM 721 HA GLU 48 6.872 1.685 11.169 1.00 .00
ATOM 722 CB GLU 48 6.043 .929 13.003 1.00 .00
ATOM 723 HB1 GLU 48 5.024 .596 13.135 1.00 .00
ATOM 724 HB2 GLU 48 6.398 1.378 13.918 1.00 .00
ATOM 725 CG GLU 48 6.925 -.270 12.647 1.00 .00
ATOM 726 HG1 GLU 48 6.912 -.421 11.578 1.00 .00
ATOM 727 HG2 GLU 48 6.548 -1.154 13.141 1.00 .00
ATOM 728 CD GLU 48 8.360 -.001 13.104 1.00 .00
ATOM 729 OE1 GLU 48 9.059 -.959 13.391 1.00 .00
ATOM 730 OE2 GLU 48 8.736 1.158 13.159 1.00 .00
ATOM 731 C GLU 48 4.748 2.064 11.165 1.00 .00
ATOM 732 O GLU 48 3.718 2.224 11.791 1.00 .00
ATOM 733 N VAL 49 4.745 1.980 9.862 1.00 .00
ATOM 734 HN VAL 49 5.589 1.854 9.380 1.00 .00
ATOM 735 CA VAL 49 3.464 2.080 9.106 1.00 .00
ATOM 736 HA VAL 49 2.891 2.928 9.445 1.00 .00
ATOM 737 CB VAL 49 3.883 2.281 7.651 1.00 .00
ATOM 738 HB VAL 49 3.003 2.408 7.037 1.00 .00
ATOM 739 CG1 VAL 49 4.766 3.526 7.541 1.00 .00
ATOM 740 HG11 VAL 49 5.488 3.387 6.751 1.00 .00
ATOM 741 HG12 VAL 49 5.282 3.685 8.477 1.00 .00
ATOM 742 HG13 VAL 49 4.150 4.385 7.320 1.00 .00
ATOM 743 CG2 VAL 49 4.669 1.057 7.176 1.00 .00
ATOM 744 HG21 VAL 49 5.492 1.376 6.554 1.00 .00
ATOM 745 HG22 VAL 49 4.018 .408 6.609 1.00 .00
ATOM 746 HG23 VAL 49 5.053 .522 8.033 1.00 .00
ATOM 747 C VAL 49 2.652 .789 9.258 1.00 .00
ATOM 748 O VAL 49 2.988 -.078 10.040 1.00 .00
ATOM 749 N GLU 50 1.586 .658 8.516 1.00 .00
ATOM 750 HN GLU 50 1.333 1.370 7.892 1.00 .00
ATOM 751 CA GLU 50 .751 -.575 8.616 1.00 .00
ATOM 752 HA GLU 50 1.367 -1.431 8.839 1.00 .00
ATOM 753 CB GLU 50 -.212 -.306 9.774 1.00 .00
ATOM 754 HB1 GLU 50 -1.064 -.964 9.694 1.00 .00
ATOM 755 HB2 GLU 50 -.545 .721 9.733 1.00 .00
ATOM 756 CG GLU 50 .503 -.560 11.103 1.00 .00
ATOM 757 HG1 GLU 50 1.088 .308 11.369 1.00 .00
ATOM 758 HG2 GLU 50 1.154 -1.417 11.003 1.00 .00
ATOM 759 CD GLU 50 -.531 -.828 12.198 1.00 .00
ATOM 760 OE1 GLU 50 -.465 -.169 13.223 1.00 .00
ATOM 761 OE2 GLU 50 -1.373 -1.688 11.994 1.00 .00
ATOM 762 C GLU 50 -.024 -.795 7.313 1.00 .00
ATOM 763 O GLU 50 -.492 .139 6.694 1.00 .00
ATOM 764 N GLU 51 -.161 -2.023 6.891 1.00 .00
ATOM 765 HN GLU 51 .226 -2.764 7.404 1.00 .00
ATOM 766 CA GLU 51 -.905 -2.299 5.626 1.00 .00
ATOM 767 HA GLU 51 -.284 -2.089 4.770 1.00 .00
ATOM 768 CB GLU 51 -1.237 -3.792 5.678 1.00 .00
ATOM 769 HB1 GLU 51 -2.274 -3.921 5.946 1.00 .00
ATOM 770 HB2 GLU 51 -.611 -4.275 6.414 1.00 .00
ATOM 771 CG GLU 51 -.987 -4.420 4.305 1.00 .00
ATOM 772 HG1 GLU 51 -.346 -3.775 3.724 1.00 .00
ATOM 773 HG2 GLU 51 -1.929 -4.550 3.792 1.00 .00
ATOM 774 CD GLU 51 -.310 -5.781 4.481 1.00 .00
ATOM 775 OE1 GLU 51 .022 -6.390 3.478 1.00 .00
ATOM 776 OE2 GLU 51 -.137 -6.191 5.617 1.00 .00
ATOM 777 C GLU 51 -2.188 -1.465 5.575 1.00 .00
ATOM 778 O GLU 51 -3.000 -1.503 6.478 1.00 .00
ATOM 779 N ASP 52 -2.378 -.714 4.523 1.00 .00
ATOM 780 HN ASP 52 -1.711 -.699 3.804 1.00 .00
ATOM 781 CA ASP 52 -3.611 .119 4.417 1.00 .00
ATOM 782 HA ASP 52 -3.667 .814 5.241 1.00 .00
ATOM 783 CB ASP 52 -3.459 .883 3.100 1.00 .00
ATOM 784 HB1 ASP 52 -3.079 .218 2.340 1.00 .00
ATOM 785 HB2 ASP 52 -2.769 1.702 3.240 1.00 .00
ATOM 786 CG ASP 52 -4.818 1.432 2.661 1.00 .00
ATOM 787 OD1 ASP 52 -5.519 1.968 3.504 1.00 .00
ATOM 788 OD2 ASP 52 -5.133 1.311 1.489 1.00 .00
ATOM 789 C ASP 52 -4.853 -.781 4.391 1.00 .00
ATOM 790 O ASP 52 -4.751 -1.990 4.328 1.00 .00
ATOM 791 N GLU 53 -6.021 -.203 4.449 1.00 .00
ATOM 792 HN GLU 53 -6.083 .773 4.508 1.00 .00
ATOM 793 CA GLU 53 -7.264 -1.028 4.440 1.00 .00
ATOM 794 HA GLU 53 -7.128 -1.918 5.033 1.00 .00
ATOM 795 CB GLU 53 -8.330 -.137 5.078 1.00 .00
ATOM 796 HB1 GLU 53 -9.265 -.674 5.130 1.00 .00
ATOM 797 HB2 GLU 53 -8.458 .755 4.482 1.00 .00
ATOM 798 CG GLU 53 -7.888 .251 6.490 1.00 .00
ATOM 799 HG1 GLU 53 -8.309 1.210 6.749 1.00 .00
ATOM 800 HG2 GLU 53 -6.809 .308 6.525 1.00 .00
ATOM 801 CD GLU 53 -8.376 -.803 7.485 1.00 .00
ATOM 802 OE1 GLU 53 -9.393 -.568 8.116 1.00 .00
ATOM 803 OE2 GLU 53 -7.724 -1.828 7.600 1.00 .00
ATOM 804 C GLU 53 -7.661 -1.397 3.006 1.00 .00
ATOM 805 O GLU 53 -8.690 -2.002 2.776 1.00 .00
ATOM 806 N SER 54 -6.858 -1.043 2.041 1.00 .00
ATOM 807 HN SER 54 -6.032 -.556 2.242 1.00 .00
ATOM 808 CA SER 54 -7.202 -1.382 .629 1.00 .00
ATOM 809 HA SER 54 -8.268 -1.510 .522 1.00 .00
ATOM 810 CB SER 54 -6.736 -.182 -.194 1.00 .00
ATOM 811 HB1 SER 54 -6.825 .717 .401 1.00 .00
ATOM 812 HB2 SER 54 -7.349 -.087 -1.075 1.00 .00
ATOM 813 OG SER 54 -5.384 -.377 -.585 1.00 .00
ATOM 814 HG SER 54 -5.381 -.764 -1.464 1.00 .00
ATOM 815 C SER 54 -6.466 -2.652 .194 1.00 .00
ATOM 816 O SER 54 -6.113 -2.812 -.959 1.00 .00
ATOM 817 N ALA 55 -6.228 -3.558 1.104 1.00 .00
ATOM 818 HN ALA 55 -6.518 -3.412 2.029 1.00 .00
ATOM 819 CA ALA 55 -5.513 -4.813 .734 1.00 .00
ATOM 820 HA ALA 55 -5.236 -4.793 -.308 1.00 .00
ATOM 821 CB ALA 55 -4.256 -4.827 1.604 1.00 .00
ATOM 822 HB1 ALA 55 -4.363 -5.572 2.379 1.00 .00
ATOM 823 HB2 ALA 55 -4.119 -3.856 2.055 1.00 .00
ATOM 824 HB3 ALA 55 -3.397 -5.065 .994 1.00 .00
ATOM 825 C ALA 55 -6.384 -6.038 1.030 1.00 .00
ATOM 826 O ALA 55 -6.441 -6.969 .251 1.00 .00
ATOM 827 N MET 56 -7.060 -6.052 2.148 1.00 .00
ATOM 828 HN MET 56 -7.003 -5.296 2.768 1.00 .00
ATOM 829 CA MET 56 -7.917 -7.228 2.476 1.00 .00
ATOM 830 HA MET 56 -7.736 -8.025 1.773 1.00 .00
ATOM 831 CB MET 56 -7.451 -7.665 3.865 1.00 .00
ATOM 832 HB1 MET 56 -8.311 -7.870 4.485 1.00 .00
ATOM 833 HB2 MET 56 -6.865 -6.875 4.312 1.00 .00
ATOM 834 CG MET 56 -6.598 -8.930 3.748 1.00 .00
ATOM 835 HG1 MET 56 -6.019 -9.058 4.650 1.00 .00
ATOM 836 HG2 MET 56 -5.933 -8.838 2.902 1.00 .00
ATOM 837 SD MET 56 -7.676 -10.365 3.515 1.00 .00
ATOM 838 CE MET 56 -8.286 -10.470 5.216 1.00 .00
ATOM 839 HE1 MET 56 -8.119 -11.468 5.597 1.00 .00
ATOM 840 HE2 MET 56 -9.341 -10.252 5.235 1.00 .00
ATOM 841 HE3 MET 56 -7.761 -9.751 5.830 1.00 .00
ATOM 842 C MET 56 -9.425 -6.884 2.509 1.00 .00
ATOM 843 O MET 56 -10.190 -7.626 3.093 1.00 .00
ATOM 844 N PRO 57 -9.830 -5.792 1.890 1.00 .00
ATOM 845 CA PRO 57 -11.272 -5.436 1.893 1.00 .00
ATOM 846 HA PRO 57 -11.688 -5.529 2.883 1.00 .00
ATOM 847 CB PRO 57 -11.287 -3.979 1.448 1.00 .00
ATOM 848 HB1 PRO 57 -11.277 -3.324 2.305 1.00 .00
ATOM 849 HB2 PRO 57 -12.157 -3.785 .835 1.00 .00
ATOM 850 CG PRO 57 -10.035 -3.798 .652 1.00 .00
ATOM 851 HG1 PRO 57 -9.650 -2.800 .793 1.00 .00
ATOM 852 HG2 PRO 57 -10.238 -3.970 -.395 1.00 .00
ATOM 853 CD PRO 57 -9.029 -4.803 1.153 1.00 .00
ATOM 854 HD2 PRO 57 -8.517 -5.269 .322 1.00 .00
ATOM 855 HD1 PRO 57 -8.325 -4.327 1.816 1.00 .00
ATOM 856 C PRO 57 -12.037 -6.312 .893 1.00 .00
ATOM 857 O PRO 57 -12.591 -5.826 -.072 1.00 .00
ATOM 858 N LYS 58 -12.070 -7.597 1.115 1.00 .00
ATOM 859 HN LYS 58 -11.615 -7.971 1.898 1.00 .00
ATOM 860 CA LYS 58 -12.796 -8.500 .174 1.00 .00
ATOM 861 HA LYS 58 -12.566 -9.531 .394 1.00 .00
ATOM 862 CB LYS 58 -14.281 -8.234 .435 1.00 .00
ATOM 863 HB1 LYS 58 -14.519 -7.218 .157 1.00 .00
ATOM 864 HB2 LYS 58 -14.494 -8.380 1.484 1.00 .00
ATOM 865 CG LYS 58 -15.127 -9.200 -.397 1.00 .00
ATOM 866 HG1 LYS 58 -14.487 -9.754 -1.067 1.00 .00
ATOM 867 HG2 LYS 58 -15.852 -8.641 -.972 1.00 .00
ATOM 868 CD LYS 58 -15.854 -10.175 .532 1.00 .00
ATOM 869 HD1 LYS 58 -16.898 -9.904 .593 1.00 .00
ATOM 870 HD2 LYS 58 -15.412 -10.132 1.516 1.00 .00
ATOM 871 CE LYS 58 -15.731 -11.595 -.023 1.00 .00
ATOM 872 HE1 LYS 58 -14.703 -11.922 .002 1.00 .00
ATOM 873 HE2 LYS 58 -16.118 -11.638 -1.032 1.00 .00
ATOM 874 NZ LYS 58 -16.557 -12.436 .889 1.00 .00
ATOM 875 HZ1 LYS 58 -17.559 -12.179 .786 1.00 .00
ATOM 876 HZ2 LYS 58 -16.256 -12.276 1.872 1.00 .00
ATOM 877 HZ3 LYS 58 -16.431 -13.438 .645 1.00 .00
ATOM 878 C LYS 58 -12.416 -8.170 -1.279 1.00 .00
ATOM 879 O LYS 58 -11.291 -8.376 -1.689 1.00 .00
ATOM 880 N LYS 59 -13.334 -7.663 -2.063 1.00 .00
ATOM 881 HN LYS 59 -14.238 -7.501 -1.726 1.00 .00
ATOM 882 CA LYS 59 -13.002 -7.333 -3.480 1.00 .00
ATOM 883 HA LYS 59 -13.885 -7.013 -4.010 1.00 .00
ATOM 884 CB LYS 59 -11.999 -6.183 -3.393 1.00 .00
ATOM 885 HB1 LYS 59 -11.361 -6.195 -4.264 1.00 .00
ATOM 886 HB2 LYS 59 -11.397 -6.297 -2.503 1.00 .00
ATOM 887 CG LYS 59 -12.752 -4.852 -3.336 1.00 .00
ATOM 888 HG1 LYS 59 -13.679 -4.985 -2.799 1.00 .00
ATOM 889 HG2 LYS 59 -12.961 -4.513 -4.340 1.00 .00
ATOM 890 CD LYS 59 -11.894 -3.814 -2.614 1.00 .00
ATOM 891 HD1 LYS 59 -11.314 -3.261 -3.337 1.00 .00
ATOM 892 HD2 LYS 59 -11.229 -4.315 -1.926 1.00 .00
ATOM 893 CE LYS 59 -12.797 -2.849 -1.843 1.00 .00
ATOM 894 HE1 LYS 59 -12.622 -2.935 -.782 1.00 .00
ATOM 895 HE2 LYS 59 -13.836 -3.045 -2.072 1.00 .00
ATOM 896 NZ LYS 59 -12.403 -1.493 -2.317 1.00 .00
ATOM 897 HZ1 LYS 59 -12.464 -1.457 -3.354 1.00 .00
ATOM 898 HZ2 LYS 59 -11.427 -1.293 -2.019 1.00 .00
ATOM 899 HZ3 LYS 59 -13.044 -.783 -1.909 1.00 .00
ATOM 900 C LYS 59 -12.370 -8.544 -4.172 1.00 .00
ATOM 901 O LYS 59 -12.082 -9.547 -3.549 1.00 .00
ATOM 902 N ASP 60 -12.152 -8.459 -5.457 1.00 .00
ATOM 903 HN ASP 60 -12.391 -7.641 -5.940 1.00 .00
ATOM 904 CA ASP 60 -11.538 -9.606 -6.188 1.00 .00
ATOM 905 HA ASP 60 -10.770 -10.068 -5.588 1.00 .00
ATOM 906 CB ASP 60 -12.686 -10.591 -6.416 1.00 .00
ATOM 907 HB1 ASP 60 -12.598 -11.024 -7.401 1.00 .00
ATOM 908 HB2 ASP 60 -13.629 -10.069 -6.334 1.00 .00
ATOM 909 CG ASP 60 -12.623 -11.701 -5.365 1.00 .00
ATOM 910 OD1 ASP 60 -13.672 -12.087 -4.877 1.00 .00
ATOM 911 OD2 ASP 60 -11.526 -12.146 -5.068 1.00 .00
ATOM 912 C ASP 60 -10.962 -9.134 -7.526 1.00 .00
ATOM 913 O ASP 60 -10.732 -7.959 -7.733 1.00 .00
ATOM 914 N ALA 61 -10.725 -10.040 -8.435 1.00 .00
ATOM 915 HN ALA 61 -10.917 -10.983 -8.249 1.00 .00
ATOM 916 CA ALA 61 -10.162 -9.641 -9.758 1.00 .00
ATOM 917 HA ALA 61 -9.865 -8.605 -9.744 1.00 .00
ATOM 918 CB ALA 61 -8.936 -10.534 -9.949 1.00 .00
ATOM 919 HB1 ALA 61 -8.233 -10.356 -9.148 1.00 .00
ATOM 920 HB2 ALA 61 -8.468 -10.306 -10.895 1.00 .00
ATOM 921 HB3 ALA 61 -9.240 -11.570 -9.937 1.00 .00
ATOM 922 C ALA 61 -11.187 -9.891 -10.868 1.00 .00
ATOM 923 O ALA 61 -12.380 -9.879 -10.639 1.00 .00
ATOM 924 N ARG 62 -10.729 -10.117 -12.070 1.00 .00
ATOM 925 HN ARG 62 -9.763 -10.121 -12.232 1.00 .00
ATOM 926 CA ARG 62 -11.673 -10.368 -13.198 1.00 .00
ATOM 927 HA ARG 62 -12.066 -9.437 -13.575 1.00 .00
ATOM 928 CB ARG 62 -10.827 -11.052 -14.272 1.00 .00
ATOM 929 HB1 ARG 62 -11.065 -12.105 -14.302 1.00 .00
ATOM 930 HB2 ARG 62 -9.780 -10.925 -14.040 1.00 .00
ATOM 931 CG ARG 62 -11.127 -10.426 -15.636 1.00 .00
ATOM 932 HG1 ARG 62 -12.003 -9.799 -15.560 1.00 .00
ATOM 933 HG2 ARG 62 -11.305 -11.209 -16.360 1.00 .00
ATOM 934 CD ARG 62 -9.933 -9.579 -16.082 1.00 .00
ATOM 935 HD1 ARG 62 -9.472 -9.100 -15.232 1.00 .00
ATOM 936 HD2 ARG 62 -10.248 -8.841 -16.807 1.00 .00
ATOM 937 NE ARG 62 -8.984 -10.546 -16.699 1.00 .00
ATOM 938 HE ARG 62 -8.839 -10.540 -17.668 1.00 .00
ATOM 939 CZ ARG 62 -8.344 -11.402 -15.950 1.00 .00
ATOM 940 NH1 ARG 62 -7.101 -11.180 -15.622 1.00 .00
ATOM 941 HH11 ARG 62 -6.638 -10.354 -15.944 1.00 .00
ATOM 942 HH12 ARG 62 -6.609 -11.837 -15.049 1.00 .00
ATOM 943 NH2 ARG 62 -8.948 -12.480 -15.529 1.00 .00
ATOM 944 HH21 ARG 62 -9.900 -12.651 -15.781 1.00 .00
ATOM 945 HH22 ARG 62 -8.457 -13.136 -14.955 1.00 .00
ATOM 946 C ARG 62 -12.811 -11.286 -12.744 1.00 .00
ATOM 947 OT1 ARG 62 -13.909 -11.130 -13.251 1.00 .00
ATOM 948 OT2 ARG 62 -12.563 -12.130 -11.899 1.00 .00
ENDMDL

Table A describes the three-dimensional coordinates of each atom except for the first line (ie, MODEL 1 indication) as well as the last line (ie, ENDMDL indication). ATOM in the first column indicates that this row is a row of atomic coordinates, the second column shows the order of the atoms, the third column shows the distinction of atoms in amino acid residues, etc., and the fourth column shows the amino acid residues. The fifth column shows the number of the amino acid residue containing the atom, and the sixth, seventh and eighth columns show the coordinates of the atom (in units of Å). The last line indicates the last line of this table. This table is described according to the format of the protein data bank, which is a notation commonly used by those skilled in the art. (The ninth and tenth columns are numbers for matching with the database format. In the present invention, the numbers are not particularly meaningful, and the ninth column uses all 1.00 and the tenth column uses all 0.00.)

  According to the present invention, the structure and function of the basic transcription factor TFIIEα zinc-binding domain have been analyzed, and it has become possible to screen for substances that can regulate transcriptional activity.

In addition, the present invention provides a TFIIEα zinc-binding domain mutant that has higher or lower transcriptional activity than the wild type. The mutants of the present invention can be used to control events involving TFIIEα (eg, transcriptional activity, binding to TFIIEβ and other basic transcription factors). For example, the prevention and / or treatment of diseases involving transcription (for example, cancer, rheumatism, lifestyle-related diseases, genetic diseases, etc.) can be carried out using the TFIIE α zinc-binding domain mutant protein and salts thereof of the present invention. I may be able to do it. In addition, the transcription mechanism may be elucidated using the TFIIE α zinc-binding domain mutant protein of the present invention and salts thereof.

Sequence alignment of TFIIEα zinc binding domain. Conserved amino acids are shown in bold. Cysteine that coordinates to Zn ²⁺ to form a chelate is shown enclosed in a red (solid line) frame. Residues contributing to the formation of the hydrophobic core are shown surrounded by a yellow (dotted line) frame. The secondary structure of the human zinc binding domain is shown above the sequence. Arrows and rectangles represent β chain and α helix, respectively. Superposition of the final 20 NMR structural families of TFIIEα zinc binding domains. The structures were superimposed by minimizing the r.m.s.d. of the main chain atoms of residues 127-145 and 152-163. Ribbon representation of the average structure of the TFIIEα zinc binding domain. Six β-strands, one α-helix and two turns are indicated by S1-S6, H1 and t1-t2, respectively. Zn ²⁺ is shown as a pink sphere. Topology diagram of TFIIEα zinc binding domain. The first and second β sheets are shown in yellow and green, and the α helix is shown in light blue. The four Cs on the turn represent cysteines that coordinate to Zn ²⁺ to form chelates. ZFI binding site of TFIIEα zinc binding domain. Pink spheres indicate Zn ²⁺ . Residues involved in Zn ²⁺ coordination bonds (shown in red dotted line) and bifurcated hydrogen bonds (shown in blue dotted line) are shown with side chains. Hydrophobic core of TFIIEα zinc binding domain. Residues involved in the hydrophobic core and zinc binding domain are shown with side chains. Cysteine that coordinates to Zn ²⁺ to form a chelate is shown in green. Five phenylalanines are shown in red. Others are shown in blue. Electrostatic potential of the molecular surface of TFIIEα zinc binding domain. Positive potential is shown in blue and negative potential is shown in red. Conserved negative potential clusters are marked. Structural comparison of TFIIEα zinc binding domain and zinc ribbon domain. TFIIE: zinc binding domain of human TFIIEα, TFIISc: C-terminal domain of human transcription elongation factor TFIIS, TFIIBn: N-terminal region of archaeal basic transcription factor TFIIB, RPB9c: C-terminal region of yeast RNA polymerase II-derived RPB9. Zn ²⁺ is shown as a pink sphere. Structural comparison of zinc binding domains. Show two turns by overlapping with a minimal r.m.s.d. Red: TFIIE, blue-green: TFIISc, green: TFIIBn, orange: RBP9c. SDS-polyacrylamide electrophoresis of purified wild-type (IIEαwt) and point mutant TFIIEα In vitro transcription assay using supercoiled DNA template. Results for wild type (IIEαwt) and cysteine point mutation TFIIEα (C129A, C132A, C154A, C157A). In vitro transcription assay using supercoiled DNA template. Results for wild type (IIEαwt) and highly conserved acidic residue point mutations TFIIEα (E140A, E140K, D164A, C164K). In vitro transcription assay using linear DNA template. Results for wild type (IIEαwt) and cysteine point mutation TFIIEα (C129A, C132A, C154A, C157A). In vitro transcription assay using linear DNA template. Results for wild type (IIEαwt) and highly conserved acidic residue point mutations TFIIEα (E140A, E140K, D164A, C164K). GST pull-down assay. Binding results of wild type (IIEαwt) and point mutation TFIIEα with basic transcription factors (except TFIIH). GST pull-down assay. Results of binding of wild type (IIEαwt) and point mutation TFIIEα to the basic transcription factor TFIIH subunit. Far ultraviolet CD spectra of wild-type and point-mutated zinc binding domains. The far ultraviolet CD spectra of wild type (WT) and point mutant zinc binding domains C129A, C132A, C154A, C157A are shown. 1D ¹ H NMR spectra of wild-type and point-mutated zinc binding domains. The spectrum after adding a 2-fold excess of EDTA to the wild-type zinc binding domain is shown at the bottom (WT + EDTA). EDTA titration experiment. Graph plotting ¹⁵ N- ¹ H HSQC spectra and signal distribution before addition of EDTA (-EDTA) and after addition of 2-fold excess EDTA to ¹⁵ N-labeled wild-type and point mutant zinc binding domains (+ EDTA) Are shown on the right and left sides, respectively. In the graph, the numbers on the horizontal axis indicate the spectral range defined as follows.

縦軸は、規定された領域内で観察された主鎖シグナルの数を示す。右側のグラフにおいて、左側のグラフと同一の色は左側のスペクトルで観察されたシグナルと一致するシグナルを意味する。グラフの上に記載されている”N”、”Ave.”および”S.D.”は、それぞれ主鎖シグナルの数、平均ケミカルシフト、および標準偏差を示す。
亜鉛結合部位の点突然変異体間の構造的類似。 アスパラギンおよびグルタミンの側鎖シグナルを上段のパネルに示す。対をなすシグナルを線で結んである。黒色の線：EDTA添加後のスペクトル内のシグナルと一致するシグナル。赤色の線：C129AおよびC132Aの両方のスペクトルに観察された共通シグナル。青色の線：C132Aのスペクトルにのみ観察されたシグナル。橙色の線：C154AおよびC157Aに共通のシグナル。緑色の線：C157Aのスペクトルにのみ観察されたシグナル。分散した主鎖シグナルを中段パネルに示す。下段左側のグラフはC132Aについてプロットしたもので、ピンク色および赤色はC129Aのスペクトルのシグナルと一致したシグナルを意味する。下段右側のグラフはC157Aについてプロットしたもので、淡い橙色および橙色はC154Aのスペクトルのシグナルと一致したシグナルを意味する。軸と着色はパネルcと同じものを表す。

The vertical axis shows the number of main chain signals observed within the defined region. In the right graph, the same color as in the left graph means a signal that matches the signal observed in the left spectrum. “N”, “Ave.” and “SD” described above the graph indicate the number of main chain signals, the average chemical shift, and the standard deviation, respectively.
Structural similarity between point mutants at the zinc binding site. The asparagine and glutamine side chain signals are shown in the upper panel. Paired signals are connected by a line. Black line: signal consistent with the signal in the spectrum after EDTA addition. Red line: common signal observed in both C129A and C132A spectra. Blue line: signal observed only in the spectrum of C132A. Orange line: signal common to C154A and C157A. Green line: signal observed only in the spectrum of C157A. Dispersed backbone signals are shown in the middle panel. The lower left graph is plotted for C132A, and pink and red mean signals that are consistent with the signals in the spectrum of C129A. The graph on the lower right is plotted for C157A, and light orange and orange colors represent signals consistent with the C154A spectrum signal. The axis and color represent the same as panel c.

<SEQ ID NO: 1>
SEQ ID NO: 1 shows the DNA sequence of the wild type human TFIIE α zinc binding domain.
<SEQ ID NO: 2>
SEQ ID NO: 2 shows the amino acid sequence of the wild type human TFIIE α zinc binding domain.
<SEQ ID NO: 3>
The DNA sequence of mutant E140A is shown.
<SEQ ID NO: 4>
The amino acid sequence of mutant E140A is shown.
<SEQ ID NO: 5>
The DNA sequence of mutant E140K is shown.
<SEQ ID NO: 6>
The amino acid sequence of mutant E140K is shown.
<SEQ ID NO: 7>
The DNA sequence of mutant D164A is shown.
<SEQ ID NO: 8>
The amino acid sequence of mutant D164A is shown.
<SEQ ID NO: 9>
The DNA sequence of mutant D164K is shown.
<SEQ ID NO: 10>
The amino acid sequence of mutant D164K is shown.
<SEQ ID NO: 11>
The DNA sequence of mutant C129A is shown.
<SEQ ID NO: 12>
The amino acid sequence of mutant C129A is shown.
<SEQ ID NO: 13>
The DNA sequence of mutant C132A is shown.
<SEQ ID NO: 14>
The amino acid sequence of mutant C132A is shown.
<SEQ ID NO: 15>
The DNA sequence of mutant C154A is shown.
<SEQ ID NO: 16>
The amino acid sequence of mutant C154A is shown.
<SEQ ID NO: 17>
The DNA sequence of mutant C157A is shown.
<SEQ ID NO: 18>
The amino acid sequence of mutant C157A is shown.
<SEQ ID NO: 19>
The amino acid sequence of wild type human TFIIEα is shown.

Claims (11)

A method for predicting the three-dimensional structure of a protein containing a zinc-binding domain or a complex of the protein and a substance capable of binding to the protein,
(i) The root mean square deviation for the distance from the three-dimensional structure defined by the atomic coordinates in Table A or the atomic coordinates in Table A is 0.0096 ± 0.0012 angstroms, and the root mean square deviation for the dihedral angle is 0.75 ± 0.09 ° Providing a three-dimensional structure of a human TFIIEα zinc-binding domain or part thereof defined by atomic coordinates of:
(ii) Build a three-dimensional structure of a protein containing a zinc-binding domain or a complex of the protein and a substance capable of binding to the protein by homology modeling using the three-dimensional structure prepared in step (i) as a template Said method comprising the steps. A method for predicting the function of a protein comprising a zinc binding domain or a part thereof,
(i) The root mean square deviation with respect to the distance from the three-dimensional structure defined by the atomic coordinates in Table A or the atomic coordinates in Table A is 0.0096 ± 0.0012 angstroms, and the root mean square deviation with respect to the dihedral angle is 0.75 ± 0.09 ° Providing a three-dimensional structure of a human TFIIEα zinc-binding domain or part thereof defined by atomic coordinates
(ii) providing a three-dimensional structure of a zinc-binding domain of a protein containing a zinc-binding domain or a part thereof;
(iii)
a step of performing structural alignment of the three-dimensional structure prepared in the step (i) and the three-dimensional structure prepared in the step (ii);
(iv) When it is determined that there is structural similarity as a result of the structural alignment performed in the step (iii), the protein containing the zinc-binding domain of (ii) or a part thereof is Said method comprising the step of predicting that it has a function similar to the zinc-binding domain of TFIIEα or a part thereof. The human TFIIEα zinc-binding domain having the amino acid sequence of SEQ ID NO: 2 or a protein containing the domain is glutamic acid at position 28, aspartic acid at position 52, cysteine at position 17, cysteine at position 20, cysteine at position 42, and position 45 Whether or not it interacts with the test substance at at least one amino acid site selected from the group consisting of cysteine, and if it interacts, determines that the test substance can regulate transcriptional activity Screening a substance capable of regulating transcriptional activity. In the presence of zinc ion, the human TFIIEα zinc-binding domain having the amino acid sequence of SEQ ID NO: 2 or a protein containing the domain is 28th glutamic acid, 52nd aspartic acid, 17th cysteine, 20th cysteine, 4. The method according to claim 3, wherein at least one amino acid site selected from the group consisting of cysteine at position 42 and cysteine at position 45 is analyzed for interaction with the test substance. A zinc-binding domain mutant protein of TFIIEα, which is a protein of any of the following (a), (b), (c) or (d), or a salt thereof.
(a) In the amino acid sequence of SEQ ID NO: 2, substitution of glutamic acid at position 28 with alanine or lysine, substitution of cysteine at position 17 with alanine, substitution of cysteine at position 20 with alanine, cysteine at position 42 with alanine And a protein having an amino acid sequence in which at least one substitution selected from the group consisting of substitution at position 45 and substitution from cysteine to alanine at position 45 is made
(b) In the amino acid sequence of the protein of (a), one or several amino acids other than the 28th amino acid, the 17th amino acid, the 20th amino acid, the 42nd amino acid and the 45th amino acid are deleted or substituted. Alternatively, a protein having an added amino acid sequence and having a transcriptional activity lower than that of the wild-type TFIIEα zinc-binding domain having the amino acid sequence of SEQ ID NO: 2.
(c) a protein having an amino acid sequence in which substitution of aspartic acid at position 52 with alanine or lysine is performed in the amino acid sequence of SEQ ID NO: 2
(d) the amino acid sequence of the protein of (c), which has an amino acid sequence in which one or several amino acids other than the amino acid at position 52 are deleted, substituted or added, and whose transcription activity is SEQ ID NO: 2 Protein higher than the zinc-binding domain of wild-type TFIIEα 6. The TFIIEα zinc-binding domain mutant protein or a salt thereof according to claim 5, which is a protein of any one of the following (1) to (8).
(1) a protein having an amino acid sequence in which glutamic acid at position 28 is substituted with alanine in the amino acid sequence of SEQ ID NO: 2
(2) A protein having an amino acid sequence in which glutamic acid at position 28 is substituted with lysine in the amino acid sequence of SEQ ID NO: 2
(3) A protein having an amino acid sequence in which the cysteine at position 17 is substituted with alanine in the amino acid sequence of SEQ ID NO: 2.
(4) In the amino acid sequence of SEQ ID NO: 2, a protein having an amino acid sequence in which substitution of cysteine to alanine at position 20 is made
(5) A protein having an amino acid sequence in which cysteine is substituted with alanine at position 42 in the amino acid sequence of SEQ ID NO: 2.
(6) A protein having an amino acid sequence in which the cysteine at position 45 is substituted with alanine in the amino acid sequence of SEQ ID NO: 2.
(7) a protein having an amino acid sequence in which aspartic acid is substituted with alanine at position 52 in the amino acid sequence of SEQ ID NO: 2
(8) A protein having an amino acid sequence in which the substitution of aspartic acid to lysine at position 52 in the amino acid sequence of SEQ ID NO: 2 DNA encoding the protein according to claim 5. A recombinant vector containing the DNA according to claim 7. A transformant comprising the recombinant vector according to claim 8. A method for producing a TFIIEα zinc-binding domain mutant protein, comprising culturing a host transformed with the DNA according to claim 7, and collecting the TFIIEα zinc-binding domain mutant protein from the culture. 6. An antibody against the TFIIEα zinc-binding domain mutant protein or a salt thereof according to claim 5.

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