WO2012039559A2

WO2012039559A2 - Tofi variant proteins and method for producing the same

Info

Publication number: WO2012039559A2
Application number: PCT/KR2011/006797
Authority: WO
Inventors: Sang Kee Rhee; Ji Woung Chung; Sang Heon Yu
Original assignee: Snu R&Db Foundation
Priority date: 2010-09-20
Filing date: 2011-09-15
Publication date: 2012-03-29
Also published as: KR20120030289A; WO2012039559A3; KR101197956B1

Abstract

The present invention discloses a TofI variant. The TofI variant has the same amino acid sequence as that of SEQ ID NO: 1 encoding a wild-type TofI, with the exception that both the histidine (His) at position 91 and the proline (Pro) at position 92 are deleted. In addition, a nucleic acid coding for the TofI variant, a vector carrying the nucleic acid, and a transformant transformed with the vector are disclosed. Methods are also provided for crystallizing the TofI variant and for producing the TofI variant.

Description

TOFI VARIANT PROTEINS AND METHOD FOR PRODUCING THE SAME

The present invention relates to a TofI variant protein. More particularly, the present invention relates to a TofI variant protein devoid of histidine(His) at position 91 and proline(Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1 corresponding to a wild-type TofI, a nucleic acid encoding the TofI variant protein, a vector carrying the nucleic acid, and a transformant transformed with the vector. The present invention is also concerned with methods for crystallizing the TofI variant protein and for producing the TofI variant protein.

Bacteria use quorum sensing to coordinate certain behaviors based on the local density of the bacterial population. Quorum sensing is a type of cell-to-cell communication, featuring the biological phenomenon of bacteria producing low-molecular weight signaling molecules, known as autoinducers or pheromones, intracellularly and excreting them extracellularly to induce active proliferation to a quorum and to alter gene expression. Ever since the discovery of quorum sensing in Vibrio fischeri and Streptococcus pneumonia, bacterial quorum sensing has been recognized as a common model of cell-to-cell communication between bacteria in the same species or between bacteria in different species. The production of autoinducers generally increases in proportion to an increase in bacterial cell density. In response to a threshold concentration of autoinducers reaching a certain level, bacteria are allowed to regulate the expression of genes in charge of various physiological activities including motility, pathogenicity, virulence, etc. (C.T. Parker, V. Sperandio, Cell. Microbiol. 11, 363 (2009), M. Schuster, E. P. Greenberg, Int. J. Med. Microbiol. 296, 73 (2006)). Hence, the regulation of gene expression by quorum sensing is now regarded as playing a pivotal role in the regulation of metabolism according to changes in the density of bacterial cells, especially in the expression of genes relating to pathogenesis.

There are four classes of autoinducers that are known to be biosynthesized by different bacterial species, and their different chemical structures have been identified. AHLs (N-acylhomoserine lactones) are a common class of autoinducers in Gram-negative bacteria, which account for those of many pathogenic bacteria. After being biosynthesized, the autoinducer is recognized by cognate receptors and thereafter regulates gene expression. In this context, the biosynthesis of AHLs is achieved by AHL synthase while AHL receptors are responsible for the recognition of AHLs. In brief, quorum sensing is composed of a protein for synthesizing an autoinducer and a transcription factor for regulating gene expression in response to the signaling molecule. Particularly, AHL synthase catalyzes the production of AHL from SAM (S-adenosyl-L-methionine) and acylated-ACP (acylated acyl-carrier protein) through acylation and lactonization, with the concomitant formation of holo-ACP and MTA (5'-methylthioadenosine) as by-products (FIG. 1) (A. L. Schaefer, D. L. Val, B. L. Hanzelka, J. E. Cronan Jr., E. P. Greenberg, Proc. Natl. Acad. Sci. U. S. A. 93, 9505 (1996), M. I. More et al., Science 272, 1655 (1996)). Primarily produced by Gram-negative bacteria, all of the AHLs are commonly based on a homoserine lactone ring with an acyl chain. However, AHLs are species-specific because their chemical structures differ from one species to another in terms of the length of the acyl side chain bonded via an amide linkage to the homoserine lactone ring and the oxidation of the carbon at position 3 and the saturation of the acyl chain. On the other hand, the AHL receptors, responsible for the recognition of autoinducers, are transcription factors that consist of two domains: a DNA binding domain and an AHL binding domain. Once a species-specific autoinducer binds thereto, the receptors alter gene expression directly or indirectly. Thus, AHL receptors are involved in the regulation of gene expression by quorum sensing.

Most of the currently used antibiotics target the factors indispensable for the subsistence of bacteria, but this has resulted in the acquired resistance in bacteria, leading to the emergence of super-bacteria, the infections of which cannot be cured by antibiotics which are available to date. In contrast, because quorum sensing generally is not an essential factor for bacterial subsistence, drugs designed to inhibit quorum sensing can suppress the growth of pathogens to prevent the onset of the diseases, without causing bacterial resistance, and proteins involved in quorum sensing thus are arising as the next antibiotic targets and are attracting great interest. In most cases the development of quorum sensing inhibitors, AHL receptors have been primarily targeted (G. D. Geske, J. C. O'Neil, H. E. Blackwell, Chem. Soc. Rev. 37, 1432 (2008), D. A. Rasko, V. Sperandio, Nat. Rev. Drug Discov. 9, 117 (2010), Y. Zou, S. K. Nair, Chem. Biol. 16, 961 (2009)). Recent research has been directed toward the development of inhibitors that target enzymes involved in the SAM pathway where MTA formed as a by-product in a biosynthesis of autoinducer is recycled into SAM (J. A. Gutierrez et al., Nat. Chem. Biol. 5, 251 (2009)).

In addition to AHL receptors and enzymes involved in the SAM pathway, AHL synthase, which is responsible for the biosynthesis of the autoinducers of quorum sensing, can be a main target for the development of quorum sensing inhibitors. Particularly, recent information on the tertiary structures of two different AHL synthases has been expected to provide a starting point for protein structure-based drug design. In contrast to such an expectation, AHL synthases LasI (T. A. Gould, H. P. Schweizer, M. E. Churchill, Mol. Microbiol. 53, 1135 (2004)) and EsaI (W. T. Watson, T. D. Minogue, D. L. Val, S. B. von Bodman, M. E. Churchill, Mol. Cell 9, 685 (2002)), which are involved in the biosynthesis of the autoinducers 3-oxo-C12-HSL (3-oxo-C12-homoserine lactone) and 3-oxo-C6-HSL(3-oxo-C6-homoserine lactone) in Pseudomonas aeruginosa and Pantoeaste wartii, respectively, are difficult to utilize in the development of inhibitors useful for blocking quorum sensing in them, mainly because the absence of information on the tertiary structures of the synthases bound to their substrates or reaction by-products gives limitations to the investigation of the mechanism of the AHL synthases. Although the AHL synthases of pathogens which produce autoinducers may be the main targets for the development of inhibitors, the protein structure-based drug design of inhibitors against AHL synthases has currently not advanced by much throughout the world because there is no information on the tertiary structure of substrate- or reaction by-product-bound proteins, which is attributed to the fact that the solubilization of AHL synthases and protein crystallization required for a protein structure determination of AHL synthases are difficult to carry out.

There has been an increasing number of trials and efforts made into developing novel drugs by using NMR (Nuclear magnetic resonance spectroscopy) which allows a protein structure determination of AHL synthases (P. J. Hajduk, J. Greer, Nat. Rev. Drug Discovery 6, 211 (2007)), but the solubilization of proteins is a prerequisite for NMR analysis. Thus, the development of novel drugs of AHL synthase inhibitors using NMR analysis is impossible until the solubilization of AHL synthases has been achieved, but there are only a few reports on the solubilization of AHL synthases.

Leading to the present invention, intensive and thorough research into protein structure-based drug design of quorum sensing inhibitors, now arising as the next antibacterial agent, conducted by the present inventors, resulted in the finding that when mutated at specific positions using a protein engineering technique in full consideration of the properties of the target protein AHL synthease, TofI, an AHL synthase derived from the Gram-negative pathogen Burkholderia glumae, can be expressed in a soluble form and can form crystals which are effective for the X-ray analysis of the tertiary structure of the AHL synthase bound to a substrate analog and a reaction by-product.

It is an object of the present invention to provide a TofI variant protein which is devoid of the histidine (His) residue at position 91 and the proline residue (Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1 encoding TofI.

It is another object of the present invention to provide a nucleic acid coding for the ToFI variant protein, a vector carrying the nucleic acid, and a transformant anchoring the vector therein.

It is a further object of the present invention to provide a method for crystallizing a TofI variant protein which is devoid of histidine (His) at position 91 and proline (Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1 encoding a wild-type TofI, with methionine (Met) substituting for at least one of phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149 and leucine (Leu) at position 152.

It is still a further object of the present invention to provide a method for preparing a TofI variant protein which is devoid of the histidine (His) residue at position 91 and the proline residue (Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1 encoding TofI.

Provided with solubility and optionally crystallinity by the site-specific mutation resulting from protein engineering in accordance with the present invention, variants of the AHL synthase TofI may be expressed in a soluble form effective for the NMR analysis of drug design, and may find further use in the analysis of TofI tertiary structures when they bind to a substrate analog and a reaction by-product, thus guaranteeing a platform on the basis of which drug design of AHL synthase inhibitors can be developed.

FIG. 1 is a view showing the synthesis of AHL, an autoinducer, in the presence of an AHL synthase of Gram-negative bacteria. TofI, an AHL synthase derived from the Gram-negative plant pathogenic bacterium Burkholderia glumae, catalyzes the synthesis of the autoinducer C8-HSL (C8-homoserine lactone) from the substrates acyl-ACP and SAM.

FIG. 2 shows amino acid sequences (a) of wild-type TofI (SEQ ID NO: 1), and TofI variants TofI(Δ) (SEQ ID NO: 2), TofI(3MΔ) (SEQ ID NO: 3) and TofI(F42MΔ) (SEQ ID NO: 4) wherein mutated and deleted portions are marked with asterisks and triangles, respectively, and nucleotide sequences (b) of SEQ ID NOS: 5, 6 and 7 respectively corresponding to SEQ ID NOS: 2, 3 and 4 wherein portions of different bases have been marked with asterisks.

FIG. 3 is of views illustrating the process flow of X-ray protein crystallography. According to the process flow, DNA coding for a target protein is cloned after which the target protein is expressed, separated and purified. The purified proteins are allowed to crystallize using various techniques. The protein crystals thus obtained are irradiated with X-rays to give data on the basis of which the tertiary structure of the protein is analyzed. The TofI variant TofI(3MΔ) prepared according to the present invention acts as a milestone for the crystallization of TofI proteins, which is the most difficult step in X-ray protein crystallography.

FIG. 4 is a thin-layer chromatogram showing the biosynthesis of an autoinducer by TofI variants. Thin-layer chromatography was used to examine the ability of the TofI variants and the wild-type to catalyze the biosynthesis of the autoinducer C8-HSL (C8-homoserine lactone). The autoinducers synthesized in the presence of the TofI variants or the wild-type or in the absence of TofI (control) were run together with chemically synthesized C8-HSL (standard). As shown in the chromatogram, all of the TofI variants catalyzed the biosynthesis of the autoinducer C8-HSL although its levels were different to some degree.

FIG. 5 is a photograph showing a protein crystal of the TofI(3MΔ) complexed with the autoinducer analog C8J8 and the reaction by-product MTA.

FIG. 6 is a schematic diagram showing a tertiary structure of the TofI(3MΔ) complexed with the autoinducer analog C8J8 and the reaction by-product MTA. The TofI(3MΔ) protein prepared according to the method of the present invention succeeded in crystallization, which is the most difficult problem of X-ray protein crystallography. Thus, the tertiary structure of a TofI(3MΔ) complex bound to the autoinducer analog C8J8 and the reaction by-product MTA can be determined.

In accordance with an aspect thereof, the present invention provide a TofI variant protein which is devoid of the histidine (His) residue at position 91 and the proline residue (Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1 encoding TofI.

As used herein, the term “TofI” refers to an AHL (N-acylhomoserine lactone) synthase derived from the Gram-negative plant pathogen Burkholderia glumae, which catalyzes the biosynthesis of C8-HSL (C8-homoserine lactone) as an autoinducer. Burkholderia glumae is a plant pathogen that produces a toxoflavin responsible for causative of grain and seedling rot in rice and of bacterial wilt in many field crops. A recent report has it that the biosynthesis and extracellular transport of toxoflavin is correlated with quorum sensing-dependent gene expression (J. Kim et al., J. Bacteriol. 191, 4870 (2009)). AHL refers to an autoinducer which acts as a signaling molecule in quorum sensing in Gram-negative bacteria. AHL-mediated quorum sensing is found in a wide range of pathogenic bacteria, and similarity amongst the mechanisms of quorum sensing in pathogenic bacteria is making AHL into a new target of anti-infection therapy.

The term “AHL”, as used herein, refers to a class of compounds based on a homoserine lactone ring which plays a pivotal role in the quorum sensing of Gram-negative bacteria. Examples of AHL include N-β-oxo-hexanoyl-L-homoserine lactone, N-β-oxo-octanoyl-L-homoserine lactone, N-β-oxo-decanoyl-L-homoserine lactone, N-hexanoyl-L-homoserine lactone, N-butanoyl-homoserine lactone, and N-β-oxo-dodecanoyl-L-homoserine lactone, but are not limited thereto. Preferably, the AHL that is synthesized by the TofI of the present invention is an N-β-oxo-octanoyl-L-homoserine lactone such as C8-HSL (C8-homoserine lactone).

The term “TofI variant protein” may be interchangeably used with “TofI variant” and, as used herein, is intended to include any TofI variant protein so long as it can be solubilized in buffer solutions whether crystallizable or not. Preferably, the TofI variant is devoid of the histidine (His) residue at position 91 and the proline residue (Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1 coding for a wild-type TofI protein. More preferably, the TofI variant has an amino acid sequence of SEQ ID NO: 2. In addition, the TofI variant may be further modified to have methionine residues instead of phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149 and leucine (Leu) at position 152 in the amino acid sequence of SEQ ID NO: 1, respectively. More preferably, the TofI variant has an amino acid sequence of SEQ ID NO: 3 or 4.

Preferably, the TofI variant protein is expressed in a soluble form.

The phrase “expressed in a soluble form”, as used herein, means the alteration of the water-solubility of the protein from insoluble to soluble. In one embodiment of the present invention, the TofI variant protein devoid of histidine (His) at position 91 and proline (Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1 was observed to be expressed in a soluble form without negatively impacting its enzymatic activity.

A soluble form of a protein is a prerequisite condition for the NMR analysis step of drug design. Further, because proteins cannot be structurally determined unless they are soluble, a soluble form of the protein is also a prerequisite for crystallization. In one embodiment of the present invention, a TofI variant protein (TofI(3M)) which has three methionine residues instead of phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149 and leucine (Leu) at position 152 in the amino acid sequence of SEQ ID NO: 1 without being devoid of the histidine (His) at position 91 and the proline at position 92 was not crystallized unlike the TofI variants (TofI(Δ), TofI(3MΔ), TofI(F42MΔ)) which are devoid of both the histidine (His) at position 91 and the proline at position 92. Since crystallization requires solubilization in advance, the result indicates that the deletion of amino acids at positions 91 and 92 in the amino acid sequence of SEQ ID NO: 1 is important to the expression of TofI proteins in a soluble form.

Preferably, a TofI variant protein which is devoid of the histidine (His) at position 91 and the proline (Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1 with a methionine residue substituting for at least one selected from among phenylalanine (Phe) at position 42, isoleucine at position 149 and leucine at position 152 can be crystallized. More preferably, the TofI variant protein useful in the present invention has the amino acid sequence of SEQ ID NO: 3 or 4.

The term “crystallization” or “crystallinity” in all its grammatical forms and spelling variations, as used herein, is intended to be used in conjunction with a protein state suitable for X-ray crystallography which is obtained either by introducing a mutation into the protein to allow it to form a homogeneous solution from which solid particles of constant morphology and size are formed or by improving the crystalline state of the protein. The tertiary structure of a protein is very important in understanding the in vivo function of the protein and developing the protein structure-based drug. Knowledge of the three-dimensional structure of an AHL synthase and the arrangement of its atoms makes it possible to analyze the tertiary structure of the AHL synthase complexed with a substrate analog and a reaction by-product, which provides a basis for the development of novel drugs inhibitive of the enzymatic activity of the AHL synthase. Thus, such knowledge is a common subject in both the biological and medical fields. However, there has been no information about the structure of AHL synthase that can give a clue about the tertiary structure of AHL bound to a substrate- or a reaction by-product. The analysis of a protein for tertiary structure is conducted on the basis of crystallization which in turn requires a soluble form of the protein. In accordance with the present invention, TofI is made to be soluble and crystallizable by mutation.

In one embodiment, a TofI variant having an amino acid sequence of SEQ ID NO: 2, devoid of histidine (his) at position 91 and proline (Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1, was designated “TofI(Δ)”. A TofI variant having an amino acid sequence of SEQ ID NO: 3 which is devoid of histidine (His) at position 91 and proline (Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1 with respective methionine residues substituting for phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149 and leucine at position 152 was designated “TofI(3MΔ)”, and a TofI variant having an amino acid sequence of SEQ ID NO: 4 which is devoid of histidine (His) at position 91 and proline (Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1 with methionine substituting for phenylalanine (Phe) at position 42 was designated “TofI(F42MΔ)”. All of the TofI variants were observed to retain their original enzymatic activity (FIG. 4). It was also observed that TofI(Δ), TofI(3MΔ) and TofI(F42MΔ) were expressed in soluble forms suitable for the NMR analysis step of drug design and that TofI(3MΔ) and TofI(F42MΔ) formed protein crystals suitable for X-ray crystallography. Thus, the histidine (His) and proline (Pro) residues at respective positions 91 and 92 play a critical role in the maintenance of the TofI protein in an insoluble form, but play a minor role in the enzymatic function of the TofI protein.

In addition, a TofI variant which was derived from the TofI variant of SEQ ID NO: 2 by substituting methionine for one or more of the amino acid residues at positions 42, 149 and 152, for example, TofI(3MΔ) or TofI(F42MΔ) was found to be crystallized under certain conditions (FIG. 5).

In accordance with another aspect thereof, the present invention pertains to nucleic acids coding for the TofI variant proteins.

As long as it encodes one of the TofI variant proteins, any nucleic acid may be used in the present invention. Preferred is a nucleic acid encoding the TofI variant protein devoid of histidine (His) at position 91 and proline (Pro) at position 92 in the TofI amino acid sequence of SEQ ID NO: 1. More preferably, the nucleic acid may encode a TofI variant protein selected from among TofI(Δ) having the amino acid sequence of SEQ ID NO: 2, TofI(3MΔ) having the amino acid sequence of SEQ ID NO: 3 and TofI(F42MΔ) having the amino acid sequence of SEQ ID NO: 4, or may be selected from nucleic acids of SEQ ID NOS: 5 to 7 coding respectively for TofI(Δ), TofI(3MΔ) and TofI(F42MΔ).

In accordance with a further aspect thereof, the present invention pertains to a recombinant vector carrying one of the nucleic acids.

The term “vector”, as used herein, refers to any vehicle for the cloning or and/or transfer of a nucleic acid of interest into a host cell. As a vector, a genetic construct in which a gene of interest is operably linked to regulatory elements necessary for the expression of the gene and that permits the expression of the gene within a suitable host cell is called an “expression vector”. A functional linkage in a recombinant vector can be achieved using typical genetic engineering techniques. For site-specific DNA cleavage and ligation, enzymes known in the art may be employed. A suitable expression vector may be constructed in such a way to encompass a signal sequence for membrane targeting or secretion or a leader sequence as well as regulatory sequences such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, an enhancer, etc., depending on the purpose. In an expression vector, a promoter may be constitutive or inducible. In addition, an expression vector may comprise a selection marker for selecting host cells transformed with the expression vector, and a replicable expression vector comprises a replication origin. The vector can replicate by itself or as a factor incorporated into the chromosome of the host cell. Examples of the expression vector useful in the present invention include plasmid vectors, cosmid vectors, viral vectors, etc. pET-28 may be preferably employed as a vector in the present invention, but these are not limited thereto.

In accordance with still a further aspect thereof, the present invention pertains to a transformant transformed with the recombinant vector.

As used herein, the term “transformation” in all its grammatical forms and spelling variations refers to the artificial genetic alteration of a cell resulting from the introduction of foreign DNA into the host cell. Transformation may be conducted using any method if it is known in the art, typical examples of which include CaCl₂ precipitation, a Hanahan method in which the effect of CaCl₂ precipitation is improved by using DMSO (dimethyl sulfoxide) as a reducing material, electroporation, calcium phosphate transfection, protoplast fusion, silicon carbide fiber-mediated transformation, agrobacterium-mediated transformation, PEG-, dextran sulfate-, and lipofectamine-mediated transformation. In addition, because the expression level of a protein varies depending on the host cell, it is necessary to choose a host cell suitable to the purpose. Examples of the host cells useful in the present invention include, but are not limited to, prokaryotes such as Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas, Proteus mirabilis, and Staphylococcus, lower eukaryotes such as fungi (e.g., Aspergillus), yeasts (e.g., Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Neurospora crassa, etc.), and higher eukaryotes such as insect cells, plant cells and mammal cells, with preference for E. coli.

The term “transformant”, as used herein, is intended to refer to a host cell transformed with the vector which can produce a TofI variant protein having very high solubility and optionally very high crystallinity. Also, the transformant can be used for screening drug candidates by NMR after the TofI variant protein is introduced thereinto, or for screening drug candidates inhibitory of AHL synthase after the TofI variant protein is allowed to bind to a substrate analog or a reaction by-product.

In accordance with still another aspect thereof, the present invention pertains to a method for crystallizing a TofI variant protein, comprising (a) preparing a TofI variant protein having the same amino acid sequence as that of SEQ ID NO: 1 encoding a TofI protein, with the exception that both the histidine (His) at position 91 and the proline (Pro) at position 92 are deleted and an amino acid selected from among phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149, leucine (Leu) at position 152 and a combination thereof is substituted with methione; and (b) crystallizing the TofI variant protein of step (a). In step (b), preferably, the crystallization may be performed using a hanging-drop vapor diffusion method.

The hanging-drop vapor diffusion method is the most popular method for the crystallization of macromolecules such as proteins by which crystalline proteins can be provided in sizes large enough that the structure of the protein can be analyzed using X-ray crystallography. In the hanging-drop vapor diffusion method, a drop composed of a mixture of sample and reagent is placed in vapor equilibration with a liquid reservoir of reagent. Typically the drop contains a lower reagent concentration than does the reservoir. To achieve equilibrium, water vapor leaves the drop and eventually ends up in the reservoir. As water leaves the drop, the sample undergoes an increase in relative supersaturation. Both the sample and reagent increase in concentration as water leaves the drop for the reservoir. Equilibration is reached when the reagent concentration in the drop is approximately the same as that in the reservoir. The hanging-drop vapor crystallization method enjoys the advantage of being cost effective, giving relatively easy access to crystals, and that multiple drops can be made using a single reservoir.

Any TofI variant protein having solubility and crystallinity that is obtained by the crystallization method falls within the scope of the present invention without limitation. Preferred is a TofI variant protein that is devoid of histidine (His) at position 91 and proline (Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1, optionally with a methionine residue substituting for phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149 and leucine (Leu) at position 152. More preferably, the TofI variant protein may have the amino acid sequence of SEQ ID NO: 3 or 4.

In accordance with yet a further aspect thereof, the present invention pertains to a method for producing a TofI variant protein, comprising (a) preparing a TofI variant protein devoid of histidine (His) at position 91 and proline (Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1 encoding a wild-type TofI protein; and (b) developing the TofI variant protein of step (a) into a soluble form.

The preparation step (a) comprises deleting histidine (His) at position 91 and proline (Pro) at position 92 out of the amino acid sequence of SEQ ID NO: 1 encoding the wild-type TofI protein, and further substituting methionine (Met) for phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149 and leucine (Leu) at position 152.

The preparation step (a) comprises i) preparing a nucleic acid molecule coding for a soluble TofI variant protein which is devoid of histidine (His) at position 91 and proline (Pro) at position 92 and has a methionine (Met) residue instead of at least one of phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149 and leucine (Leu) at position 159; ii) constructing a vector carrying the nucleic acid molecule; iii) forming a transformant transformed with the vector; and iv) expressing the nucleic acid molecule from the transformant.

If necessary, a sequence for facilitating the purification of the TofI variant protein may be employed in such a way that the sequence is fused to a gene of interest when constructing the vector. The sequence may be a tag for protein purification, examples of which include glutathione-5-transferase, maltose-binding protein, FLGA and 6x His (hexahistidine), but are not limited thereto. The fusion protein expressed by the vector may be purified using affinity chromatography. For example, the TofI variant proteins may be easily recovered using the substrate glutathione when they are fused to the enzyme glutathione-5-transferase or using Ni-NTA His-binding resin columns when they are fused with 6x His.

In one embodiment of the present invention, the TofI variant proteins, when expressed without a purification tag, were separated using ion-exchange chromatography and size exclusion chromatography sequentially.

After being separated, the TofI variant proteins expressed in soluble forms may be provided as enzymes for use in the NMR analysis step of drug design. Alternatively, the separated TofI variant proteins, if crystallizable, may be used to analyze their tertiary structure formed when they bind to substrate analogs or reaction by-products and may be provided as enzymes for use in the drug design of AHL synthase inhibitors.

According to a concrete embodiment, TofI(3MΔ), which is devoid of the histidine (His) at position 91 and the proline (Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1 encoding a wild-type TofI protein, with methionine (Met) substituting for all of the phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149 and leucine (Leu) at position 152, and TofI (F42MΔ), which is devoid of histidine (His) at position 91 and proline (Pro) at position 92 in the amino acid sequence of SEQ ID NO: 1 encoding a wild-type TofI protein, with methionine (Met) substituting for phenylalanine (Phe) at position 42, were prepared (FIGs. 2a and 2b). These variant proteins were found to retain their enzymatic activity because they catalyzed the biosynthesis of C8-HSL (FIG. 4). In addition, the crystallization of both TofI(3MΔ) and TofI(F42MΔ) were achieved by the hanging-drop vapor diffusion method (FIG. 5).

In another concrete embodiment, the autoinducer analog C8J8 (molecular weight 237.34) and the by-product MTA were added to a TofI(3MΔ) solution to form a TofI(3MΔ)-C8J8-MTA complex which was then identified to form protein crystals upon hanging-drop vapor diffusion. Hence, the TofI variant proteins with solubility and crystallinity in accordance with the present invention can be used to analyze the tertiary structures they form when they complex with a substrate analog and a reaction by-product.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention.

EXAMPLE 1: Design of TofI Variants

A TofI gene (GenBank Accession No. NC_012721) was amplified by PCR (Polymerase Chain Reaction) using nucleotide sequence-specific primers. The PCR product thus obtained and the vector pET-28 (Novagen) were treated with the restriction enzymes NdeI and XhoI, followed by ligation using T4 DNA ligase (Promega). The recombinant plasmid thus constructed was introduced into E. coli DH10b (Novagen) which was then grown to single colonies. From the colonies, the recombinant plasmid was isolated with the aid of a plasmid mini-prep kit (Qiagen).

After base sequencing, the TofI gene was mutated to change the codons encoding Phe42, Ile149, and Leu152 into the codon of methionine (Met) using a QuikChange Site-Directed Mutagenesis kit (Stratagene) on the plasmid. DNA base sequencing identified the mutations on the plasmid, and the protein expressed by the mutant plasmid was named TofI(3M). The mutant protein TofI(3M) was greatly increased in stability, compared to the wild-type, but did not succeed in crystallizing, which is essential for protein structure-based drug design of AHL inhibitors.

To achieve the crystallization of TofI proteins, the codons encoding His91 and Pro92 were eliminated using the QuikChange Site-Directed Mutagenesis kit because when comparing the amino acid sequence of TofI with that of Pseudomonas aeruginosa AHL synthase LasI (GeneBank Accession No. AM778435) or Pantoeaste wartii AHL synthase EsaI (GeneBank Accession No. L32183), the amino acid residues were thought to be located on the surface of the protein and thus to be in charge of the stabilization of the protein but to not participate to the formation of the secondary structure of the protein. The resulting mutant protein which was devoid of the histidine (His) at position 91 and proline (Pro) at position 92, with methionine substituting for phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149 and leucine (Leu) at position 152, was named TofI(3MΔ). TofI(3MΔ) was found to have high stability and to crystallize, thus making it possible to analyze the tertiary structure for protein structure-based drug design of AHL inhibitors. The amino acid sequence of TofI(3MΔ) is given in SEQ ID NO: 3.

In addition to the variants TofI(3M) and TofI(3MΔ), TofI(Δ), which is devoid of the histidine (His) at position 91 and proline (Pro) at position 92, and TofI(F42MΔ), which is devoid of histidine (His) at position 91 and proline (Pro) at position 92, with methionine substituting for phenylalanine (Phe) at position 42, were constructed. Amino acid sequences of TofI(Δ) and TofI(F42MΔ) are given as SEQ ID NOS: 2 and 4, respectively.

In FIGs. 2a and 2b, amino acid sequences of wild-type TofI and its variants (TofI(Δ), TofI(3MΔ) and TofI(F42MΔ)) are given as SEQ ID NOS: 1, 2, 3 and 4, respectively, where mutated and deleted portions are marked with asterisks and triangles, respectively.

EXAMPLE 2: Expression, Purification and Activity of TofI Variants

<2-1> Expression, Isolation and Purification of TofI Variants

A plasmid carrying a purification tag-free TofI(3MΔ) gene was introduced into the expression host E. coli BL21(DE3)pLysS(Novagen), which was then grown to form single coloinies. One of them was cultured at 37℃ for approximately 6 hours in Luria-Bertani broth. When the optical density of the cells reached 0.7 at 600 nm, the culture temperature was reduced to 28℃ at which the overexpression of the target protein TofI(3MΔ) was induced for 4 hours in the presence of 1mM IPTG(isopropyl-1-thio-β-D-galactopyranoside). After being harvested by centrifugation, the cells were suspended in buffer, pH 8.0, containing 50 mM Tris and 5% (v/v) glycerol (buffer A) and disrupted by sonication. Centrifugation gave a supernatant containing the target protein.

For the purification of the target protein TofI(3MΔ), first, HiTrap Q HP ion exchange column chromatography (GE Healthcare) was conducted. In this regard, a linear concentration gradient of buffer B, prepared by adding up to 1 M NaCl to buffer A, was loaded onto the column and protein fractions eluted at 150~250mM NaCl were collected. Then, these TofI(3MΔ) fractions were subjected to size exclusion chromatography using Superdex 200 columns (GE Healthcare) to purify the target protein. For eluting the protein, 50 mM Tris buffer, pH 8.0, was used as an eluent.

In addition to TofI(3MΔ), the other variants prepared in Example 1, that is, TofI(3M), TofI(Δ), and TofI(F42MΔ) were expressed and purified in the same manner as described above.

<2-2> Enzymatic Activity of the TofI Variants

The TofI variants prepared in <2-1> were analyzed for the enzymatic activity of catalyzing the synthesis of the autoinducer. In this context, thin layer chromatography was used to examine the biosynthesis of the autoinducer C8-HSL (N-octanoyl-homoserine lactone) by the TofI variant proteins and the wild-type.

The autoinducer was biosynthesized by reacting 100 μL (6.9 μg μL1) cell lysate of the Burkholderia glumae mutant BGS2(tofI::Ω) which lacks C8-HSL biosynthesis (J. Kim et al., Mol. Microbiol. 54, 921(2004)) with 100μM SAM in the presence of 1μM of the purified TofI variants. The biosynthesis reaction was performed at 37℃ for one hour and terminated by two volumes of ethyl acetate which was then evaporated in a vacuum.

To the reaction mixture was added 20 μL ethanol, and 1-4 μL of the ethanol solutions was sampled and dropwise added to a thin layer chromatography plate. The samples were developed with a mixture of methanol and water (60:40, v/v) on the thin plate. Following evaporating the developing solution, agar containing an Agrobacterium indicator was poured onto the thin chromatography plate in the plastic container and incubated overnight at 28℃. The autoinducer, if present, turned blue, indicating the enzymatic activity of the TofI variants. For comparison, chemically synthesized C8-HSL was used as a positive control (Standard) while the reaction mixture in absence of TofI was used as a control.

As can be seen in FIG. 4, all of the TofI variants, although different in capacity, catalyzed the biosynthesis of the autoinducer C8-HSL, indicating that the variants retained their enzymatic activity and that the mutation sites of the present invention have no significant effect on enzymatic activity.

EXAMPLE 3: Crystallization of TofI Variants and Determination of Tertiary Structure

The four TofI variants, identified to have the enzymatic activity in Example 2, were allowed to crystallize for X-ray analysis of protein structures.

Using Centriprep (Millipore), the purified TofI(3MΔ) protein was concentrated to 7~10 mg/mL as calculated from the absorbance at 280 nm.

Because the TofI(3MΔ) protein is unlikely to crystallize when stored at 4℃ or higher, it was aliquoted and rapidly frozen at -70℃ in liquid nitrogen immediately after purification, and stored at that temperature until use. If necessary, it was completely thawed at 4℃.

The crystallization of the proteins was performed using a hanging-drop vapor diffusion method. The autoinducer analog C8J8 (Mw. 237.34) was dissolved in 100 % methanol of HPLC grade to give a 20 mM solution while the reaction by-product MTA (5'-methylthioadenosine, Mw 313.3) was dissolved in DMF (N,N-dimethylformamide) of molecular biology grade to give a 100 mM solution. To the TofI(3MΔ) protein solution was added 20 mM C8J8 to form a final concentration of 2 mM C8J8 while MTA was added in the amount necessary to form a final concentration of 4 mM. Next, 2 μl of the mixture of TofI(3MΔ), C8J8 and MTA was placed on a coverglass and dropwise overlaid with 2 μl of a crystallization solution, pH 7.5, containing 0.1M HEPES, 20mM MgCl₂, and 21-24 % (w/v) polyacrylic acid. The coverglass was turned upside down and placed on a reservoir containing 300 μl of the crystallization solution so that the drop on the coverglass faced the reservoir. The reservoir was made airtight by sealing the contact faces between the coverglass and the reservoir with Vaseline gel. After storing the reservoir for one to two days at 22℃, it was observed that TofI(3MΔ)-C8J8-MTA complex protein crystals had appeared (FIG. 5).

Single-wavelength data of the TofI(3MΔ)-C8J8-MTA complex protein crystals was collected at beamline 4A and 6C of the Pohang Light Source, South Korea. Before being used to collect data, the crystals were dipped for 1 min or longer in a crystallization solution containing 20% glycerol lest they should be damaged. The collected data was processed using the HKL2000 program and the protein structure was determined by molecular replacement in the CNS program.

The structure analysis revealed that the TofI(3MΔ) protein binds both the autoinducer analog C8J8 and the reaction by-product MTA (FIG. 6).

FIG. 6 shows a tertiary structure of the TofI(3MΔ) complexed with the autoinducer analog C8J8 and the reaction by-product MTA. The TofI(3MΔ) protein prepared according to the method of the present invention was successfully crystallized, which is the most difficult problem in X-ray protein crystallography. Thus, the tertiary structure of a TofI(3MΔ) complex bound to the autoinducer analog C8J8 and the reaction by-product MTA was determined.

Separately, the other variants prepared in Example 1, that is, TofI(3M), TofI(Δ), and TofI(F42MΔ), were subjected to protein crystallization for X-ray analysis of tertiary protein structures. Of them, the TofI(F42MΔ) variant was found to form protein crystals suitable for use in X-ray crystallography. Therefore, when it was modified by substituting methionine for at least one of the phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149 and leucine (Leu) at position 152, the variants of TofI of SEQ ID NO: 1 were observed to crystallize.

It is expected that the structures of the TofI variants complexed with the autoinducer analog C8J8 and the reaction by-product MTA, determined according to the present invention, will make a great contribution to the development of anti-bacterial agents which have inhibitory activity against AHL synthases, eventually disrupting quorum sensing. In detail, C8J8 mimetics with an acyl chain of various lengths may be anti-bacterial agents specific for the bacteria containing AHL synthase.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

A TofI variant protein, devoid of both histidine (His) at position 91 and proline (Pro) at position 92 in an amino acid sequence of SEQ ID NO: 1.
The TofI variant protein of claim 1, having an amino acid sequence of SEQ ID NO: 2.
The TofI variant protein of claim 1 wherein methionine (Met) is substituted for at least one of phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149, and leucine (Leu) at position 152.
The TofI variant protein of claim 3, having an amino acid sequence of SEQ ID NO: 3 or 4.
The TofI variant protein of any one of claims 1 to 4, being expressed in a soluble form.
The TofI variant protein of any one of claims 2 to 4, being able to crystallize.
A nucleic acid, encoding the TofI variant protein of any one of claims 1 to 4.
The nucleic acid of claim 7, having a nucleotide sequence of SEQ ID NO: 5, 6 or 7.
A vector, carrying the nucleic acid of claim 7.
A transformant transformed with the vector of claim 9.
The transformant of claim 10, being an E. coli species.
A method for crystallizing a TofI variant protein, comprising:

(a) preparing a TofI variant protein having a same amino acid sequence as that of SEQ ID NO: 1 encoding a TofI protein, with exception that both histidine (His) at position 91 and proline (Pro) at position 92 are deleted and an amino acid selected from among phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149, leucine (Leu) at position 152 and a combination thereof is substituted with methione; and

(b) crystallizing the TofI variant protein of step (a).
The method of claim 12, wherein the step (b) is carried out using a hanging-drop vapor diffusion method.
The method of claim 12, wherein the TofI variant protein has an amino acid sequence of SEQ ID NO: 3 or 4.
A method for producing a TofI variant protein, comprising:

(a) preparing a TofI variant protein devoid of histidine (His) at position 91 and proline (Pro) at position 92 in an amino acid sequence of SEQ ID NO: 1 encoding a wild-type TofI protein; and

(b) developing the TofI variant protein of step (a) into a soluble form.
The method of claim 15, wherein the preparation step (a) comprises further substituting methionine (Met) for at least one of phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149 and leucine (Leu) at position 152.
The method of claim 16 wherein the preparation step (a) comprises:

i) preparing a nucleic acid molecule coding for a soluble TofI variant protein which is devoid of histidine (His) at position 91 and proline (Pro) at position 92 and has a methionine (Met) residue substituted for at least one of phenylalanine (Phe) at position 42, isoleucine (Ile) at position 149 and leucine (Leu) at position 152;

ii) constructing a vector carrying the nucleic acid molecule;

iii) forming a transformant transformed with the vector; and

iv) expressing the nucleic acid molecule from the transformant.