WO2000075169A1 - Novel fabh enzyme, compositions capable of binding to said enzyme and methods of use thereof - Google Patents

Abstract

A novel E. coli FabH crystalline structure is identified. Also disclosed are methods of identifying inhibitors of these enzymes and/or active sites, and inhibitors identified by these methods.

Description

NOVEL FABH ENZYME, COMPOSITIONS CAPABLE OF BINDING TO SAID ENZYME AND METHODS OF USE THEREOF Technical Field of the Invention

The invention relates to the identification of a novel enzyme active site and methods enabling the design and selection of inhibitors of that active site. Background of the Invention

The pathway for the biosynthesis of saturated fatty acids is very similar in prokaryotes and eukaryotes. However, the organization of the biosynthetic apparatus is very different. Vertebrates possess a type I fatty acid synthase (FAS) in which all of the enzymatic activities are encoded on one multifunctional polypeptide, the mature protein being a homodimer. The acyl carrier protein (ACP) is an integral part of the complex. In contrast, in most bacterial and plant FASs (type II) each of the reactions are catalyzed by distinct monofunctional enzymes and the ACP is a discrete protein. Mycobacteria are unique in that they possess both type I and II FASs. There therefore appears to be considerable potential for selective inhibition of the bacterial systems by broad-spectrum antibacterial agents (Rock, C. & Cronan, J. 1996. Biochimica et Biophysica Acta 1302, 1- 16; Jackowski, S. 1992. In Emerging Targets in Antibacterial and Antifungal Chemotherapy. Ed. J. Sutcliffe & N. Georgopapadakou. Chapman & Hall, New York; Jackowski, S. et al (1989). J. Biol. Chem. 264, 7624-7629.) The first step in the biosynthetic cycle is the condensation of malonyl-ACP with acetyl-CoA by FabH. Prior to this, malonyl-ACP is synthesized from ACP and malonyl- CoA by FabD, malonyl CoA:ACP transacylase. In subsequent rounds malonyl-ACP is condensed with the growing-chain acyl-ACP (FabB and FabF, synthases I and II respectively). The second step in the elongation cycle is ketoester reduction by NADPH- dependent β-ketoacyl-ACP reductase (FabG). Subsequent dehydration by β-hydroxyacyl- ACP dehydrase (either FabA or FabZ) leads to trans-2-enoyl-ACP which is in turn converted to acyl-ACP by enoyl-ACP reductase (Fabl). Further rounds of this cycle, adding two carbon atoms per cycle, eventually lead to palmitoyl-ACP whereupon the cycle is stopped largely due to feedback inhibition of FabH and I by palmitoyl-ACP (Heath, et al, (1996), J.Biol.Chem. 271, 1833-1836).

Cerulenin and thiolactomycin are potent and selective inhibitors of bacterial fatty acid biosynthesis. Extensive work with these inhibitors has proved that this biosynthetic pathway is essential for bacterial viability. No marketed antibiotics are targeted against fatty acid biosynthesis, therefore it is unlikely that novel antibiotics would be rendered inactive by known antibiotic resistance mechanisms. There is an unmet need for developing new classes of antibiotic compounds, such as those that target FabH.

FabH enzymes are of interest as potential targets for antibacterial agents.

There is a need in the art for novel FabH enzyme active sites and catalytic sequences to enable identification and structure-based design of inhibitors, which are useful in the treatment or prophylaxis of diseases, particularly diseases caused by bacteria which may share catalytic domains with those of the invention. Summary of the Invention

In one aspect, the present invention provides a novel FabH enzyme active site crystalline form.

In another aspect, the present invention provides a novel FabH composition characterized by the catalytic residues Cysl 12, His244 and Asn274.

In still another aspect, the present invention provides a novel FabH composition characterized by the active site of 33 amino acid residues (including the catalytic residues). In yet another aspect, the invention provides a method for identifying inhibitors of the compositions described above which methods involve the steps of: providing the coordinates of the structure of the invention to a computerized modeling system; identifying compounds which will bind to the structure; and screening the compounds identified for FabH inhibitory bioactivity. In a further aspect, the present invention provides an inhibitor of the catalytic activity of any composition bearing the catalytic domain described above.

Another aspect of this invention includes machine readable media encoded with data representing the coordinates of the three-dimensional structure of the FabH crystal.

Other aspects and advantages of the present invention are described further in the following detailed description of the preferred embodiments thereof. Brief Description of the Drawings

Fig. 1 provides the atomic coordinates of the E. coli FabH di er.

Fig. 2 provides the atomic coordinates of the E. coli FabH monomer in complex with acetyl-CoA. Fig. 3 provides a projection of the ribbon diagram of the E. coli FabH dimer. The two monomers are drawn with a light or dark gray shading. The catalytic Cysl 12 is shown in dark ball-and-stick model.

Fig. 4 provides the ribbon diagram of the E. coli FabH monomer with the catalytic residue Cysl 12 is shown in dark ball-and-stick model. The N- and C-termini are labeled. Fig. 5 provides the stereoview of the oc-carbon superposition between the structures of FabH and FabF. FabH is drawn in a thin black line and FabF in a thick gray line.

Fig. 6 provides the ribbon diagram of the E. coli FabH monomer with acetylated Cysl 12 and the CoA molecule in black ball-and-stick model. The orientation of the view is the same as that of Fig. 4.

Fig. 7 provides the superposition of the E. coli FabH catalytic residues in comparison to those of FabF. FabH is drawn in thick gray lines and FabF in thin black lines. FabH residues are label Cysl 12, His244 and Asn274, which corresponds to Cys 163, His303 and His340, respectively. Detailed Description of the Invention

The present invention provides a novel E. coli FabH crystalline structure, a novel FabH active site, and methods of use of the crystalline form and active site to identify FabH inhibitor compounds (peptide, peptidomimetic or synthetic compositions) characterized by the ability to competitively inhibit binding to the active site of a FabH enzyme. Also provided herein is a novel FabH crystalline structure in complex with the substrate acetyl- CoA, and the identification of acetyl-CoA interacting residues in FabH.

I. The Novel FabH Crystalline Three-Dimensional Structure

The present invention provides a novel FabH crystalline structure based on the E. coli FabH. The amino acid sequences of the FabH are provided in Table 1 as SEQ ID NO:l.

TABLE 1

Met Tyr Thr Lys He He Gly Thr Gly Ser Tyr Leu Pro Glu Gin

1 5 10 15

Val Arg Thr Asn Ala Asp Leu Glu Lys Met Val Asp Thr Ser Asp

16 20 25 30

Glu Trp He Val Thr Arg Thr Gly He Arg Glu Arg His He Ala

31 35 40 45 Ala Pro Asn Glu Thr Val Ser Thr Met Gly Phe Glu Ala Ala Thr

46 50 55 60

Arg Ala He Glu Met Ala Gly He Glu Lys Asp Gin He Gly Leu

61 65 70 75

He Val Val Ala Thr Thr Ser Ala Thr His Ala Phe Pro Ser Ala 76 80 85 90

Ala Cys Gin He Gin Ser Met Leu Gly He Lys Gly Cys Pro Ala

91 95 100 105

Phe Asp Val Ala Ala Ala Cys Ala Gly Phe Thr Tyr Ala Leu Ser 106 110 115 120

Val Ala Asp Gin Tyr Val Lys Ser Gly Ala Val Lys Tyr Ala Leu

121 125 130 135

Val Val Gly Ser Asp Val Leu Ala Arg Thr Cys Asp Pro Thr Asp

136 140 145 150 Arg Gly Thr He He He Phe Gly Asp Gly Ala Gly Ala Ala Val

151 155 160 165

Leu Ala Ala Ser Glu Glu Pro Gly He He Ser Thr His Leu His

166 170 175 180

Ala Asp Gly Ser Tyr Gly Glu Leu Leu Thr Leu Pro Asn Ala Asp 181 185 190 195

Arg Val Asn Pro Glu Asn Ser He His Leu Thr Met Ala Gly Asn

196 200 205 210

Glu Val Phe Lys Val Ala Val Thr Glu Leu Ala His He Val Asp

211 215 220 225 Glu Thr Leu Ala Ala Asn Asn Leu Asp Arg Ser Gin Leu Asp Trp

226 230 235 240

Leu Val Pro His Gin Ala Asn Leu Arg He He Ser Ala Thr Ala

241 245 250 255

Lys Lys Leu Gly Met Ser Met Asp Asn Val Val Val Thr Leu Asp 256 260 265 270

Arg His Gly Asn Thr Ser Ala Ala Ser Val Pro Cys Ala Leu Asp

271 275 280 285

Glu Ala Val Arg Asp Gly Arg He Lys Pro Gly Gin Leu Val Leu

286 290 295 300 Leu Glu Ala Phe Gly Gly Gly Phe Thr Trp Gly Ser Ala Leu Val Arg Phe

301 305 310 317

As illustrated herein, the crystal structure is a tightly associated FabH dimer. Each monomer has two structural domains: the N-terminal domain (residues 1-170 of SEQ ID NO: l) and the C-terminal domain (residues 171-317 of SEQ ID NO: l). The two domains are similar in their overall fold: each contains a 5-stranded β-sheet sandwiched between α- helices and covered by other β-strands, α-helices and loops. The structural similarity between the two halves of the protein indicates that FabH is probably evolved from two genes of similar origin. The active site of FabH is at the center of the FabH monomer, formed at the junction of the N- and C-terminal domains. While the core architecture of the E. coli FabH bears some similarity to that of the FabF (Huang, et al, (1998), EMBO J. 17, 1183-1191), large differences exit in the atomic positions of the core β-strands, and the structures outside of the core β-strand are completely different. With amino acid sequence identity between FabH and FabF being below 20%, the large differences are well expected. Therefore, the crystalline structure ofE. coli FabH is novel.

As described above, the E. coli FabH is a dimer, each monomer contains an active site. The dimer formation is essential for the FabH activity because the active site of a monomer is comprised of at least Phe87 of the other monomer in the dimer. The present invention provides both a crystalline monomer and dimer structure of E. coli FabH. Inhibitors that perturb or interact with this dimer interface are another target for the design and selection of anti-bacterial agents.

According to the present invention, the crystal structure of E. coli FabH has been resolved at 2.0 A (crystal form 1), and its selenomethionine mutant protein in complex with acetyl-CoA has been determined at 1.9 A (crystal form 2). The structure was determined using the methods of MAD phasing and molecular replacement, and refined to R-factors of 18.9% and 27%, respectively.

Further refinement of the atomic coordinates will change the numbers in Figure 1-2 and Tables I - HI, refinement of the crystal structure from another crystal form will result in a new set of coordinates. However, distances and angles in Tables II will remain the same within experimental errors, and relative conformation of residues in the active site will remain the same within experimental error. For example, the two independently determined monomers in our crystal form 1 and the monomer in crystal form 2 do not have identical numerical coordinates, but the structures of these three monomers have very similar structures, and the spatial relationship between amino acid residues are considered the same within experimental error. In fact, we would consider any structure that can be superimposed onto that of FabH with an rms error of less than 1.5 A on α-carbon atoms being a close structural homologue and the same rms error but over all protein atoms being an identical structure. Figure 1 provides the atomic coordinates of the E. coli FabH dimer, which contains 634 amino acids. Figure 2 provides the atomic coordinates of the E. coli FabH monomer in complex with acetyl-CoA, which contains 317 amino acids. The FabH enzyme is characterized by an active site which preferably contains a binding site for the first substrate acetyl-CoA and the second substrate malonyl-ACP. The catalytic residues in FabH are Cysl 12, His244 and Asn274, compared to Cys 163, His303 and His340 in FabF. The difference in catalytic residues is not only limited to their amino acid identity (His340 to Asn274 change), but also their relative spatial arrangement. While FabH Cys 1 12 and Asn274 can be well superimposed onto FabF Cys 163 and His340, His244 of FabH occupies a very different position from that of His303 of FabF. This indicated the catalytic mechanisms of the two enzymes are very different. The crystal structure described herein was solved in the presence and absence of acetyl-CoA. We identified that the catalytic Cysl 12 has been covalently aceytlated, and the product CoA is still bound to the active site. The bound CoA enabled us to identify the active site cavity, which is long and narrow and shaped nicely to bind the β-mercaptoethylamine-patotheinate arm of CoA. The structure of the acetyl-CoA complex also revealed all the key residues that are interacting with CoA and lining the active site, which is identified as a set of 33 amino acid residues listed in Table I. For example, the adenine part of CoA is sandwiched between the side chains of Argl51 and Trp32. Our structures are determined in the absence of malonyl-ACP. However, the same acetyl-CoA binding cavity should bind malonyl-ACP as well because their active site binding regions are very similar and there is no apparent additional entrance to the active site. Moreover, while the FabH molecular surface in general negatively charged, a region just outside of the active site cavity is positively charge. This surface is mainly comprised of three α-helices (30-37, 209-231 and 248-258) and contains a number of positively charged amino acids (Arg36, Arg40, Lys214, His222, Arg235 Arg249, Lys256, Lys257). Since the acyl-carrier protein (ACP) is known to be very acidic or negatively charged, it is reasonable to assume this surface being the ACP binding surface.

Table I provides the the atomic coordinates of the apo E. coli FabH structure in the active site (in crystal form 1). Solvent molecules are omitted here for clarity, but can be found in Fig. 1. Residue 487 is Phe87 from the other monomer. TABLE I

ATOM RESIDUE X Y Z Occ B

1 N THR 28 -24, .151 18. .846 61. .990 1. .00 36. .45

2 CA THR 28 -23, .735 19. .054 60. .610 1. .00 36. .69

3 CB THR 28 -22, .196 19, .086 60. .565 1. .00 32. .66

4 OG1 THR 28 -21, .760 20, .076 59. .636 1. .00 33, .79

5 CG2 THR 28 -21, .645 17, .737 60. .183 1. .00 27. .40

6 C THR 28 -24. .238 17. .990 59. .627 1. .00 38. .85

7 O THR 28 -24. .732 16. .923 60. .023 1. ,00 42. .97

8 N TRP 32 -24. .091 20. .068 53. .681 1. .00 30. .06

9 CA TRP 32 -23. .725 21. .413 54. .092 1. .00 28. .93

10 CB TRP 32 -24. .277 21, .708 55. .486 1. .00 29. .27

11 CG TRP 32 -24. .036 23, .126 55. .939 1. .00 31. .13

12 CD2 TRP 32 -22. .895 23, .622 56. .644 1. .00 32, .44

13 CE2 TRP 32 -23. .118 25, .005 56. .890 1. .00 35, .25

14 CE3 TRP 32 -21. .707 23, .038 57. .096 1. .00 32, .45

15 CD1 TRP 32 -24. .880 24. .197 55. .779 1. .00 33, .86

16 NE1 TRP 32 -24, .333 25, .331 56. .351 1. .00 35, .49

17 CZ2 TRP 32 -22, .200 25, .800 57. .565 1. .00 35, .24

18 CZ3 TRP 32 -20, .793 23, .832 57, .765 1. .00 34. .43

19 CH2 TRP 32 -21, .046 25, .197 57, .994 1. .00 36, .72

20 C TRP 32 -22, .203 21, .582 54. .091 1. .00 27, .24

21 O TRP 32 -21, .675 22, .617 53, .674 1. .00 26. .75

22 N I E 33 -21. .503 20, .566 54, .581 1. .00 26, .32

23 CA ILE 33 -20, .042 20, .617 54. .642 1. .00 25, .89

24 CB ILE 33 -19, .459 19, .370 55, .333 1. .00 25 .18

25 CG2 ILE 33 -17, .925 19, .444 55, .366 1. .00 26, .64

26 CGI ILE 33 -20. .024 19 .253 56, .744 1. .00 18 .01

27 CD1 ILE 33 -19. .621 18 .008 57, .421 1. .00 19 .10

28 C ILE 33 -19, .432 20, .755 53, .258 1, .00 24 .76

29 O ILE 33 -18, .630 21, .650 53, .022 1. .00 23 .20

30 N ARG 36 -20, .198 24 .159 51, .621 1. .00 26 .35

31 CA ARG 36 -19, .545 25 .296 52, .237 1, .00 27 .73

32 CB ARG 36 -20, .083 25 .473 53, .649 1, .00 34 .96

33 CG ARG 36 -19, .562 26 .715 54, .326 1. .00 47 .48

34 CD ARG 36 -20, .581 27 .250 55, .290 1, .00 56 .04

35 NE ARG 36 -21, .775 27 .729 54, .600 1, .00 63 .48

36 CZ ARG 36 -22, .490 28 .780 54, .996 1, .00 67 .12

37 NHl ARG 36 -23, .564 29 .153 54, .303 1, .00 67 .75

38 NH2 ARG 36 -22. .127 29, .465 56. .082 1. .00 68, .27 TABLE I - Cont.

ATOM RESIDUE X Y Z Occ B

39 C ARG 36 -18. .014 25, .292 52, .233 1. .00 23. .26

40 0 ARG 36 -17. .386 26, .346 52, .208 1. .00 21. .26

41 N THR 37 -17, .423 24, .103 52, .214 1. .00 20. .79

42 CA THR 37 -15. .973 23, .969 52, .258 1. .00 19. .72

43 CB^° THR 37 -15. .549 23, .164 53, .509 1, .00 20. .01

44 OGl THR 37 -16, .014 21, .812 53, .384 1, .00 17. .59

45 CG2 THR 37 -16, .157 23, .752 54, .765 1. .00 18. .21

46 C THR 37 -15, .363 23, .272 51, .047 1. .00 20. .77

47 0 THR 37 -14, .234 23, .571 50 .657 1. .00 20. .98

48 N CYS 112 -0, .698 28, .695 58, .467 1, .00 12. .58

49 CA CYS 112 -0, .984 28, .096 57, .174 1, .00 11. .86

50 CB CYS 112 -2, .457 28, .264 56, .808 1. .00 10. .86

51 SG CYS 112 -3 .580 27 .460 57 .935 1 ..oc 1 22 :.06

52 C CYS 112 -0, .126 28, .620 56, .037 1. .00 10. .86

53 O CYS 112 -0, .003 27, .939 55, .025 1. .00 13. .89

54 N LEU 142 -3, .033 20, .066 62, .705 1. .00 16. .58

55 CA LEU 142 -4, .063 20, .954 63, .207 1. .00 17. .95

56 CB LEU 142 -4, .281 22, .159 62, .287 1. .00 15. .72

57 CG LEU 142 -3, .100 23, .125 62 .126 1. .00 18. .13

58 CD1 LEU 142 -3. .628 24, .499 61 .738 1. .00 14. .84

59 CD2 LEU 142 -2. .246 23, .204 63, .415 1. .00 12. .26

60 C LEU 142 -5. .396 20, .321 63, .598 1, .00 17. .45

61 O LEU 142 -6. .111 20, .883 64, .417 1. .00 17. .68

62 N ARG 151 -17. .927 23. .092 65, .249 1, .00 22. .20

63 CA ARG 151 -18. .230 22. .887 63, .841 1. .00 25. .49

64 CB ARG 151 -19. .699 23. .217 63, .534 1. .00 24. .14

65 CG ARG 151 -20. .051 22. .998 62, .052 1. .00 33. .87

66 CD ARG 151 -21. .530 23. .158 61, .748 1. .00 37. .44

67 NE ARG 151 -21. .991 24. .545 61, .780 1. .00 41. .79

68 CZ ARG 151 -23. .272 24. .897 61, .737 1. .00 44. .63

69 NH1 ARG 151 -23. .612 26. .173 61, .771 1. .00 46. .51

70 NH2 ARG 151 -24. .219 23. .970 61, .666 1. .00 47. .88

71 C ARG 151 -17. .304 23. .634 62, .868 1. .00 26. .00

72 O ARG 151 -16. .686 23. .018 61, .992 1. .00 26. .64

73 N GLY 152 -17. .164 24. .940 63. .077 1. .00 24. .63

74 CA GLY 152 -16. .353 25. .769 62. .201 1. .00 23. .08

75 C GLY 152 -14. .912 25. .371 61. .944 1. .00 22. .21

76 O GLY 152 -14. .366 25. .679 60. .880 1. .00 21. .32

77 N ILE 155 -14. ,484 20. .649 60. .878 1. .00 18. .82

78 CA ILE 155 -14. ,866 20. .149 59. .564 1. .00 18. .77

79 CB ILE 155 -16. ,223 20. .733 59. .071 1. .00 17. .77

80 CG2 ILE 155 -17. ,365 20. .321 60. .018 1. .00 12. .79 TABLE I - Cont.

ATOM RESIDUE X Y Z Occ B

81 CGI ILE 155 -16. .127 22. .249 58. .924 1. .00 15. ,46

82 CD1 ILE 155 -17. .339 22. .892 58. .331 1. .00 20. .95

83 C ILE 155 -13. .823 20. .489 58. .531 1. .00 18. .45

84 0 ILE 155 -13. .819 19. .909 57. .461 1. .00 21. .51

85 N ILE 156 -12. .958 21. .450 58. .819 1. .00 18. .70

86 CA ILE 156 -11. .985 21. .825 57. .812 1. .00 19. .10

87 CB ILE 156 -11. .999 23. .375 57, .499 1. .00 24. .79

88 CG2 ILE 156 -13. .391 23. .974 57. .563 1. .00 23. .59

89 CGI ILE 156 -11. .095 24. .139 58, .438 1. .00 24. .77

90 CD1 ILE 156 -9. .886 24. .631 57, .730 1. .00 27. .97

91 C ILE 156 -10. .544 21. .338 57. .935 1. .00 18. .32

92 O ILE 156 -9. .922 21. .071 56. .918 1. .00 18. .31

93 N PHE 157 -10. .005 21. .200 59, .142 1. .00 16. .26

94 CA PHE 157 -8. .611 20, .780 59, .280 1. .00 15. .33

95 CB PHE 157 -7. .984 21, .371 60, .551 1. .00 15, .71

96 CG PHE 157 -7. .868 22, .858 60, .523 1. .00 19. .05

97 CD1 PHE 157 -8. .814 23. .654 61, .158 1. .00 19. .74

98 CD2 PHE 157 -6. .844 23. .476 59, .814 1. .00 15. .77

99 CE1 PHE 157 -8. .737 25. .057 61, .076 1. .00 21. .28

100 CE2 PHE 157 -6. .761 24. .855 59, .727 1. .00 11. .63

101 CZ PHE 157 -7, .701 25. .650 60, .351 1. .00 17. .65

102 C PHE 157 -8. .278 19. .286 59, .190 1. .00 16. .07

103 0 PHE 157 -9. .045 18. .413 59, .622 1. .00 17. .36

104 N LEU 189 -7, .786 34. .391 64, .172 1, .00 19. .01

105 CA LEU 189 -7. .338 33, .021 63, .922 1. .00 19. .46

106 CB LEU 189 -6. .897 32. .907 62, .463 1. .00 23. .06

107 CG LEU 189 -6. .422 31. .587 61, .872 1. .00 23, .21

108 CD1 LEU 189 -7. .435 30. .493 62, .157 1. .00 24, .24

109 CD2 LEU 189 -6. .253 31. .811 60, .355 1. .00 25, .52

110 C LEU 189 -6. .164 32, .746 64, .850 1. .00 18, .26

111 O LEU 189 -5, .082 33. .338 64, .688 1, .00 15, .62

112 N LEU 205 -7. .765 25, .549 68, .834 1. .00 19, .58

113 CA LEU 205 -7, .699 26, .448 67, .677 1, .00 19, .69

114 CB LEU 205 -7. .475 25, .601 66, .398 1. .00 19, .66

115 CG LEU 205 -7. .104 26, .238 65, .052 1, .00 19, .36

116 CD1 LEU 205 -6. .309 25, .259 64, .201 1, .00 18, .01

117 CD2 LEU 205 -8, .366 26, .671 64, .321 1, .00 18, .66

118 C LEU 205 -8. .996 27, .273 67, .597 1, .00 17, .98

119 O LEU 205 -10. .088 26, .731 67. .804 1. .00 20, .78

120 N MET 207 -11. .189 30, .405 65, .330 1. .00 16, .50

121 CA MET 207 -11. .285 31, .040 64, .025 1, .00 18, .56

122 CB MET 207 -11. .105 30. .003 62, .931 1. .00 20, .76 TABLE I - Cont.

ATOM RESIDUE X Y Z Occ B

123 CG MET 207 -11. .293 30. .550 61. .542 1. .00 23. .66

124 SD MET 207 -10. .858 29. .292 60. .353 1. ,00 32. .43

125 CE MET 207 -12. .262 28. .166 60. .555 1. .00 31. .26

126 C MET 207 -12. .599 31. .742 63. .776 1. .00 18. .83

127 0 MET 207 -13. .666 31. .152 63. .934 1. .00 19. .82

128 N GLY 209 -14. .190 32. .425 61. .134 1. .00 20. .42

129 CA GLY 209 -14. .305 32. .056 59. .737 1. .00 23. .52

130 C GLY 209 -14. .623 33. .114 58. .701 1. .00 24. .44

131 0 GLY 209 -13. .771 33. .456 57. .884 1. .00 26. .52

132 N ASN 210 -15. .839 33. .640 58. .738 1. .00 23. .37

133 CA ASN 210 -16. .291 34. .615 57. .758 1. .00 25. .94

134 CB ASN 210 -17. .724 35. .006 58. .035 1. .00 24. .49

135 CG ASN 210 -18. .633 33. .818 58. .029 1. .00 25. .13

136 OD1 ASN 210 -18. .680 33. .068 57, .061 1. .00 25. .86

137 ND2 ASN 210 -19. .325 33. .603 59, .130 1. .00 25. .81

138 C ASN 210 -15. .426 35. .831 57, .639 1. .00 26. .62

139 0 ASN 210 -15. .214 36. .334 56, .545 1. .00 27. .59

140 N VAL 212 -12. .110 35. .950 58, .414 1. .00 25. .15

141 CA VAL 212 -10. .808 35. .645 57, .793 1. .00 25. .13

142 CB VAL 212 -10. .004 34, .469 58, .486 1. .00 23. .52

143 CGI VAL 212 -10. .492 34. .190 59, .896 1. .00 20. .23

144 CG2 VAL 212 -9, .958 33. .220 57, .653 1. .00 23. .20

145 C VAL 212 -10, .971 35. .405 56, .272 1. .00 23. .49

146 0 VAL 212 -10, .095 35. .769 55, .493 1. .00 20. .62

147 N PHE 213 -12, .115 34. .859 55, .853 1. .00 22, .05

148 CA PHE 213 -12, .371 34. .627 54, .431 1. .00 22. .19

149 CB PHE 213 -13, .718 33. .954 54, .244 1. .00 20. .95

150 CG PHE 213 -14, .116 33. .771 52. .794 1. .00 23. .47

151 CD1 PHE 213 -14. .758 34. .788 52. .101 1. .00 22. .38

152 CD2 PHE 213 -13 .833 32 .587 52 .132 1 .00 21 ..51

153 CE1 PHE 213 -15, .098 34. .634 50. .784 1. .00 23, .71

154 CE2 PHE 213 -14, .173 32. .423 50. .813 1. .00 26, .06

155 CZ PHE 213 -14. .805 33. .446 50, .133 1. .00 25, .34

156 C PHE 213 -12, .307 35. .935 53, .645 1. .00 22, .07

157 0 PHE 213 -11, .618 36. .045 52, .629 1. .00 22, .83

158 N ALA 216 -8, .801 37. .118 53, .586 1. .00 18, .14

159 CA ALA 216 -7. .964 36. .216 52, .808 1. .00 19, .00

160 CB ALA 216 -8, .183 34. .775 53, .218 1. .00 17, .94

161 C ALA 216 -8, .146 36. .371 51, .303 1. .00 17. .97

162 0 ALA 216 -7, .166 36. .285 50, .563 1. .00 16. .52

163 N LEU 220 -4. .879 37. .537 49, .135 1. .00 15. .55

164 CA LEU 220 -4, .379 36. .571 48, .174 1. .00 17. .76 TABLE I - Cont.

ATOM RESIDUE X Y Z Occ B

165 CB LEU 220 -5. .127 35. .233 48. .275 1. ,00 15. ,75

166 CG LEU 220 -4. .703 34. .362 49. .466 1. .00 13. ,65

167 CD1 LEU 220 -5. .621 33. .177 49. .608 1. .00 13. .89

168 CD2 LEU 220 -3. .278 33. .915 49. .310 1. .00 9. .45

169 C LEU 220 -4. .491 37. .186 46. .769 1. .00 17. .28

170 0 LEU 220 -3. .618 36. .957 45, .932 1. .00 20. .62

171 N HIS 244 -3. .012 27, .197 48, .689 1. .00 18. .90

172 CA HIS 244 -3. .165 26, .587 49, .988 1. .00 17. .18

173 CB HIS 244 -2, .914 27, .594 51, .111 1. .00 17. .15

174 CG HIS 244 -3. .178 27. .035 52. .465 1. .00 17. .42

175 CD2 HIS 244 -2. .579 26. .015 53. .138 1. .00 14. .15

176 ND1 HIS 244 -4. .285 27. .385 53. .212 1. .00 16. .36

177 CE1 HIS 244 -4. .370 26. .596 54, .264 1. .00 16. .12

178 NE2 HIS 244 -3. .354 25. .760 54, .244 1. .00 19. .14

179 C HIS 244 -4. .631 26. .151 49, .971 1. .00 15. .41

180 0 HIS 244 -5. .503 26. .936 49, .591 1. .00 14. .10

181 N ALA 246 -7. .440 26. .051 51, .721 1. .00 19. .76

182 CA ALA 246 -8. .240 26. .512 52, .864 1. .00 20. .02

183 CB ALA 246 -8. .166 28, .017 52, .975 1, .00 22. .28

184 C ALA 246 -9, .687 26, .075 52 .772 1, .00 23. .08

185 0 ALA 246 -10, .281 25, .620 53, .759 1, .00 23. .37

186 N ASN 247 -10, .280 26, .349 51 .615 1. .00 21, .20

187 CA ASN 247 -11, .645 25, .983 51 .311 1, .00 22, .48

188 CB ASN 247 -12, .653 26, .733 52 .190 1, .00 24, .84

189 CG ASN 247 -12, .700 28, .195 51 .888 1, .00 26, .54

190 OD1 ASN 247 -13, .343 28, .618 50 .942 1, .00 32, .63

191 ND2 ASN 247 -12, .016 28, .987 52 .686 1, .00 31, .62

192 C ASN 247 -11, .824 26. .292 49 .825 1, .00 23, .43

193 0 ASN 247 -11, .076 27, .097 49 .249 1, .00 23, .61

194 N ARG 249 -14, .126 27, .939 48 .119 1 .00 27, .79

195 CA ARG 249 -14, .566 29, .305 47 .811 1 .00 28 .98

196 CB ARG 249 -15 .376 29 .912 48 .966 1 .00 34 .43

197 CG ARG 249 -16 .577 29, .118 49 .433 1 .00 45 .16

198 CD ARG 249 -17 .307 29 .859 50 .557 1 .00 52 .72

199 NE ARG 249 -18 .235 30 .862 50 .037 1 .00 60 .09

200 CZ ARG 249 -18 .607 31 .976 50 .675 1 .00 62 .73

201 NH1 ARG 249 -19 .469 32 .803 50 .096 1 .00 64 .82

202 NH2 ARG 249 -18 .112 32 .290 51 .867 1 .00 60 .24

203 C ARG 249 -13 .369 30 .208 47 .562 1 .00 24 .80

204 0 ARG 249 -13 .358 31 .007 46 .629 1 .00 24 .09

205 N ILE 250 -12 .393 30 .135 48 .453 1 .00 24 .48

206 CA ILE 250 -11, .201 30, .951 48 .306 1 .00 24, .93 TABLE I - Cont.

ATOM RESIDUE X Y Z Occ B

207 CB ILE 250 -10. .365 30. .965 49. .621 1. ,00 26. ,91

208 CG2 ILE 250 -8. .880 31. .128 49. .350 1. ,00 22. .69

209 CGI ILE 250 -10. 902 32. 091 50. 506 1. 00 32. 57

210 CD1 ILE 250 -10. .216 32. .245 51. .828 1. ,00 38. .42

211 C ILE 250 -10. .391 30. .533 47. .076 1. ,00 23. .12

212 0 ILE 250 -10. .024 31. .380 46. .265 1. .00 20. .24

213 N ASN 274 -7. .884 20. .993 55. .104 1. .00 16. .04

214 CA ASN 274 -6. .887 22. .042 55. .213 1. .00 16. .17

215 CB ASN 274 -7. .524 23. .307 55. .790 1. .00 16. .71

216 CG ASN 274 -6. .524 24. .433 56. .031 1. .00 15. .26

217 OD1 ASN 274 -5. .290 24. .259 55. .970 1. .00 14. .69

218 ND2 ASN 274 -7. .058 25. .607 56. .319 1. .00 17. .12

219 C ASN 274 -5. .800 21. .538 56, .144 1. .00 18. .93

220 0 ASN 274 -6. .016 21. .456 57, .366 1. .00 18. .02

221 N SER 276 -2. .883 23. .010 56, .745 1. .00 14. .34

222 CA SER 276 -1. .996 24. .086 57, .152 1. .00 14. .51

223 CB SER 276 -1. .772 23. .993 58, .686 1. .00 16, .80

224 OG SER 276 -1. .051 25. .104 59, .218 1. .00 17, .07

225 C SER 276 -0. .675 24. .141 56, .352 1, .00 13, .90

226 O SER 276 -0. .719 24, .199 55, .132 1, .00 15, .64

227 N ALA 303 -0. .360 31, .072 49, .683 1. .00 15, .48

228 CA ALA 303 -0. .934 30, .617 50, .937 1. .00 13, .31

229 CB ALA 303 0. .045 29, .692 51, .624 1, .00 11, .10

230 C ALA 303 -1. .261 31, .801 51, .853 1, .00 12, .59

231 O ALA 303 -0. .614 32, .842 51, .789 1. .00 11, .22

232 N PHE 304 -2. .299 31, .642 52, .666 1, .00 14, .82

233 CA PHE 304 -2. .726 32, .650 53, .626 1, .00 15 .34

234 CB PHE 304 -4. .075 33. .248 53, .207 1. .00 17 .57

235 CG PHE 304 -4 .561 34 .355 54 .119 1 ..00i 23i.99

236 CD1 PHE 304 -5. .356 34. .060 55, .243 1, .00 22 .77

237 CD2 PHE 304 -4. .220 35. .687 53, .866 1, .00 22 .34

238 CE1 PHE 304 -5. .794 35. .064 56, .089 1, .00 19 .22

239 CE2 PHE 304 -4. .657 36, .705 54 .712 1, .00 24 .60

240 CZ PHE 304 -5. .447 36, .389 55 .826 1, .00 19 .92

241 C PHE 304 -2. .831 31, .946 54 .982 1, .00 15 .89

242 O PHE 304 -3. .176 30, .768 55 .041 1 .00 14 .69

243 N GLY 305 -2. .490 32, .637 56 .065 1 .00 14 .20

244 CA GLY 305 -2. .583 32, .002 57 .363 1 .00 13 .74

245 C GLY 305 -2, .765 32, .889 58 .578 1, .00 13 .32

246 O GLY 305 -2. .788 34, .115 58 .496 1, .00 12 .57

247 N GLY 306 -2. .856 32, .235 59, .727 1, .00 17 .80 TABLE I - Cont.

ATOM RESIDUE X Y Z Occ B

248 CA GLY 306 -3. .033 32, .929 60. .990 1. .00 17. .87

249 C GLY 306 -1. .928 33. .892 61, .357 1. .00 19. .45

250 0 GLY 306 -0. .758 33, .751 60, .965 1, .00 19 .00

251 N PHE 487 0. ,118 30. .521 66. .721 1. ,00 16. .05

252 CA PHE 487 -0, .254 31, .597 65 .800 1. .00 15 .49

253 CB PHE 487 -0. .539 31. .168 64 .330 1. .00 10 .60

254 CG PHE 487 -1. .559 30 .100 64 .167 1. .00 9 .77

255 CD1 PHE 487 -1. .169 28 .788 63 .944 1. .00 11 .44

256 CD2 PHE 487 -2, .916 30 .410 64 .157 1, .00 12 .27

257 CE1 PHE 487 -2, .109 27. .796 63. .715 1, .00 10 .46

258 CE2 PHE 487 -3, .878 29, .416 63. .925 1, .00 11 .90

259 CZ PHE 487 -3. .477 28, .111 63. .705 1, .00 9 .96

260 C PHE 487 -1. .381 32, .376 66 .460 1, .00 13 .45

261 0 PHE 487 -2, .233 31, .776 67 .132 1, .00 15 .58

Table II provides the distances between (D) atoms of the active site residues that are within 5.0 angstroms of one another as defined by Table I.

TABLE II Distance Atom 1 Atom 2 Between (D=) Atom 1 Atom 2 Between (D=)

28CA 151CD 4.796 32N 33CA 4.198

28CB 33CD1 4.204 32N 33C 4.728

28CB 151CD 4.292 32N 33CB 4.967

28CB 33CGI 4.398 45 32CA 33N 2.428

28CB 151CG 4.703 32CA 33CA 3.808

280G1 33CG1 3.472 32CA 33C 4.422

280G1 33CD1 3.709 32CA 33CB 4.890

280G1 151CD 3.743 32CB 33N 3.133

280G1 32CE3 3.902 50 32CB 33CA 4.454

280G1 151CG 4.159 32CG 33N 3.849

280G1 32CZ3 4.306 32CG 33CA 4.892

280G1 155CG2 4.418 32CD2 33N 3.941

280G1 32CD2 4.776 32CD2 36CB 4.506

280G1 33CB 4.930 55 32CD2 36CD 4.511

280G1 151NE 4.962 32CD2 33CA 4.602

28CG2 33CD1 3.435 32CD2 36NE 4.722

28CG2 33CG1 4.093 32CE2 36CD 3.747

32N 33N 2.785 32CE2 36NE 3.804 CE2 36CZ 4.270 32CZ3 33CG1 4..754 CE2 36CB 4.465 32CZ3 151CZ 4. .802 CE2 36NH2 4.640 45 32CH2 36CD 3. .427 CE2 36CG 4.706 32CH2 151NE 3. .956 CE2 151CZ 4.851 32CH2 36CG 4. .238 CE2 36NH1 4.909 32CH2 36NE 4. .297 CE3 33N 3.532 32CH2 151CD 4. .299 CE3 33CA 3.828 50 32CH2 151CZ 4. .365 CE3 33CG1 4.157 32CH2 155CD1 4. .378 CE3 36CB 4.522 32CH2 36CB 4. .459 CE3 155CD1 4.542 32CH2 151NH1 4. .669 CE3 33CB 4.649 32CH2 151CG 4. .722 CE3 151CD 4.657 55 32CH2 36NH2 4. .800 CE3 36CD 4.719 32CH2 36CZ 4. .890 CE3 151NE 4.929 32C 33CA 2, .430 CD1 36NE 4.848 32C 33C 3. .009 NE1 36NE 3.919 32C 330 3. .730 NE1 36CZ 4.139 60 32C 33CB 3, .737 NE1 36CD 4.346 32C 36N 4, .094 NE1 36NH1 4.404 32C 33CG1 4. .149 NE1 36NH2 4.693 32C 36CB 4, .453 CZ2 36CD 3.146 32C 36CA 4, .929 CZ2 36NE 3.563 65 32C 33CG2 4, .950 CZ2 36CZ 3.945 320 33N 2, .249 CZ2 36NH2 3.954 320 33CA 2, .757 CZ2 36CG 4.276 320 33C 2, .945 CZ2 151CZ 4.401 320 36N 2. .962 CZ2 151NE 4.403 70 320 330 3, .261 CZ2 151NH1 4.452 320 36CB 3, .270 CZ2 36CB 4.464 320 36CA 3, .712 CZ2 36NH1 4.873 320 33CB 4 .267 CZ2 151NH2 4.924 320 36CG 4, .657 CZ2 151CD 4.993 75 320 37N 4, .735 CZ3 155CD1 3.624 320 36C 4 .758 CZ3 151CD 4.106 320 33CG1 4, .844 CZ3 36CD 4.225 33N 36N 4 .835 CZ3 151NE 4.250 33CA 370G1 4 .386 CZ3 151CG 4.430 80 33CA 36N 4 .658 CZ3 36CB 4.488 33CA 36CB 4 .957 CZ3 33CA 4.545 33CA 37N 4 .991 CZ3 33N 4.616 33CA 37CG2 4 .994 CZ3 36CG 4.653 33CB 370G1 4 .651 33CG2 37OGl 3.631 36CB 37CG2 4.430

33CG2 155CB 4.276 36CB 37CA 4 .592

33CG2 155CD1 4.585 45 36CG 37N 3 .982

33CG2 1550 4.633 36CG 37CG2 4, .535

33CG2 37CG2 4.695 36CG 37CA 4, .970

33CG2 155CG2 4.767 36C 37CA 2, .432

33CG2 37CB 4.789 36C 37CG2 3 .497

33CG2 155CG1 4.874 50 36C 37CB 3 .498

33CG1 155CG2 4.351 36C 37C 3 .538

33CG1 155CB 4.696 36C 370G1 4 .176

33CG1 155CD1 4.793 36C 370 4 .442

33CD1 155CG2 4.145 36C 249CD 4 .916

33CD1 155CB 4.658 55 36C 249CG 4 .954

33C 370G1 3.580 360 37N 2 .243

33C 36N 3.854 360 37CA 2 .766

33C 37N 4.042 360 37CG2 3 .844

33C 37CB 4.576 360 37C 3 .859

33C 36CA 4.656 60 360 249CD 3 .882

33C 37CG2 4.688 360 37CB 3, .898

33C 36CB 4.779 360 249CG 4, .005

33C 37CA 4.826 360 370 4, .477

33C 36C 4.863 360 247CB 4, .749

330 37OGl 2.646 65 360 2470D1 4, .807

330 37N 2.851 360 370G1 4, .881

330 36N 3.274 37CA 247CB 4, .321

330 37CB 3.467 37CA 247CA 4 .867

330 37CA 3.608 37CB 156CG2 4 .663

330 37CG2 3.684 70 37CB 247CB 4 .782

330 36C 3.777 37CG2 155CD1 3 .854

330 36CA 3.840 37CG2 156CG2 3 .941

330 36CB 4.138 37CG2 155CG1 4 .422

330 37C 4.148 37CG2 156CB 4 .991

330 360 4.926 75 37C 247CB .542

36N 37N 2.838 37C 247CA 4. .609

36N 37CA 4.277 37C 247C 4, .810

36N 37C 4.949 370 247CA 3, .598

36CA 37N 2.434 370 247C 3. .729

36CA 37CA 3.811 80 370 247CB 3, .853

36CA 37CG2 4.500 370 247N 4, .926

36CA 37CB 4.704 370 2470 4, .938

36CA 37C 4.796 112N 2760G 3, .686

36CB 37N 3.318 112N 305CA 3, .963 112N 306N 4.333 112SG 304O 4..414

112N 305C 4.677 112SG 157CE2 4. .485

112N 3040 4.709 45 112SG 274CG 4. .632

112N 276CB 4.828 112SG 276N 4. .659

112N 305N 4.952 112SG 305CA 4. .685

112N 276CA 4.966 112SG 276C 4. .686

112CA 2760G 3.624 112SG 244ND1 4. .776

112CA 276C 4.051 50 112SG 142CD1 4. .820

112CA 3040 4.061 112C 3040 3. .861

112CA 276CA 4.136 112C 305CA 4. .386

112CA 305CA 4.225 112C 304C 4. .415

112CA 2760 4.408 112C 276C 4. .524

112CA 244NE2 4.434 55 112C 303CB 4, .545

112CA 276CB 4.443 112C 2760 4, .551

112CA 244CE1 4.710 112C 244CD2 4, .605

112CA 304C 4.800 112C 305N 4, .661

112CA 244CD2 4.813 112C 244NE2 4, .671

112CA 305N 4.911 60 112C 2760G 4, .831

112CB 304O 3.148 112C 244CG 4, .958

112CB 244CE1 3.594 1120 244CD2 3, .728

112CB 244NE2 3.694 1120 2760 3, .809

112CB 305CA 3.781 1120 303CB 3, .827

112CB 304C 4.127 65 1120 244NE2 4, .073

112CB 244ND1 4.129 1120 276C 4, .079

112CB 2760G 4.216 1120 244CG 4, .177

112CB 276CA 4.217 1120 3040 4, .251

112CB 244CD2 4.306 1120 244CE1 4, .632

112CB 305N 4.436 70 1120 244ND1 4, .683

112CB 276C 4.515 1120 276CA 4, .831

112CB 244CG 4.571 1120 244CB 4, .890

112CB 276CB 4.716 1120 304C 4, .905

112CB 2760 4.728 1120 304N 4. .955

112CB 306N 4.945 75 1120 303CA 4, .975

112CB 305C 4.962 142CA 157CB 4, .754

112CB 2740D1 4.977 142CA 205CD1 4, .956

112SG 2760G 3.687 142CB 157CD2 3, .797

112SG 276CA 3.809 142CB 157CG 4, .058

112SG 244CE1 3.853 80 142CB 157CB 4, .165

112SG 276CB 3.982 142CB 205CD1 4, .170

112SG 244NE2 4.070 142CB 157CE2 4. .469

112SG 2740D1 4.127 142CB 276CB 4, .757

112SG 274ND2 4.259 142CB 157CD1 4, .905 142CG 276CB 3.788 151C 155N 4..563

142CG 2760G 4.071 151C 155CD1 4, .597

142CG 205CD1 4.377 45 151C 155CB 4, .899

142CG 157CD2 4.414 1510 152N 2, .258

142CG 157CE2 4.706 1510 152CA 2, .779

142CD1 276CB 3.608 1510 152C 2, .947

142CD1 2760G 3.655 1510 155CG1 3, .212

142CD1 205CD1 3.719 50 1510 155CG2 3, .411

142CD1 157CE2 3.740 1510 155N 3, .421

142CD1 157CD2 3.885 1510 1520 3, .701

142CD1 487CZ 4.116 1510 155CD1 3, .721

142CD1 487CE1 4.134 1510 155CB 3 .737

142CD1 157CZ 4.454 55 1510 155CA 4 .176

142CD1 157CG 4.706 152CA 155CG1 4 .815

142CD1 276CA 4.885 152CA 155CD1 4 .922

142CD2 487CE1 4.604 152C 207CE 4 .094

142CD2 205CD1 4.620 152C 155CG1 4 .510

142CD2 2760G 4.759 60 152C 156CG2 4 .843

142CD2 276CB 4.818 152C 155N 4 .860

142C 157CB 4.133 1520 207CE 3 .274

142C 157CG 4.691 1520 156CG2 3 .855

1420 157CB 4.323 1520 155CG1 4 .323

1420 205CD1 4.386 65 1520 156CG1 4, .363

1420 157CG 4.706 1520 156CB 4, .727

151N 152N 2.952 1520 155CD1 4, .807

151N 152CA 4.351 1520 156N 4, .911

151CA 152N 2.436 155N 156N 2, .685

151CA 152CA 3.810 70 155N 156CA 4, .127

151CA 152C 4.558 155N 156CG2 4, .821

151CA 155CG2 4.685 155N 157N 4, .835

151CB 152N 3.099 155N 156C 4, .966

151CB 152CA 4.414 155CA 156N 2, .427

15ICG 152N 3.627 75 155CA 156CA 3, .765

15ICG 155CG2 4.303 155CA 156CG2 4, .562

15ICG 155CD1 4.606 155CA 156C 4, .769

15ICG 152CA 4.623 155CA 156CB 4, .784

151CD 152N 4.899 155CA 157N 4, .991

151C 152CA 2.431 80 155CB 156N 3. .352

151C 152C 3.097 155CB 156CA 4. .554

151C 1520 4.095 155CB 156CG2 4. .561

151C 155CG1 4.343 155CG2 156N 4. .705

151C 155CG2 4.371 155CG1 156N 3. .270 155CG1 156CG2 3.509 156CG1 157CE2 4..578

155CG1 156CA 4.310 156CG1 157CB 4. .670

155CG1 156CB 4.510 45 156CG1 207CE 4, .697

155CD1 156CG2 4.165 156CG1 274ND2 4 .790

155CD1 156N 4.638 156CG1 2460 4 .975

155C 156CA 2.383 156CD1 274ND2 3 .308

155C 156C 3.439 156CD1 274CB 3 .331

155C 156CB 3.567 50 156CD1 157CZ 3 .561

155C 156CG2 3.643 156CD1 157CE1 3 .563

155C 157N 3.931 156CD1 157N 3 .712

155C 1560 4.261 156CD1 157CE2 3 .715

155C 156CG1 4.558 156CD1 157CD1 3, .722

1550 156N 2.227 55 156CD1 274CG 3, .772

1550 156CA 2.675 156CD1 157CD2 3, .864

1550 156C 3.604 156CD1 157CG 3, .875

1550 156CB 3.915 156CD1 2460 4 .111

1550 156CG2 4.089 156CD1 157CA 4, .343

1550 1560 4.103 60 156CD1 274CA 4 .694

1550 157N 4.363 156CD1 157CB 4 .712

156N 157N 2.981 156CD1 274N 4 .913

156N 157CA 4.422 156CD1 2740D1 4 .936

156CA 157N 2.466 156C 157N 1 .329

156CA 157CA 3.825 65 156C 157CA 2 .420

156CA 157C 4.700 156C 157C 3 .305

156CA 1570 4.854 156C 157CB 3 .660

156CA 157CB 4.870 156C 1570 3 .694

156CA 157CD1 4.959 156C 274N 3 .900

156CA 274N 4.980 70 156C 157CG 4 .021

156CB 157N 3.377 156C 274CB 4 .195

156CB 157CA 4.624 156C 157CD1 4 .329

156CB 2460 4.688 156C 2740 4 .565

156CB 274CB 4.791 156C 274CA 4 .613

156CB 157CD1 4.859 75 156C 157CD2 4 .668

156CG2 157N 4.653 1560 157N 2 .229

156CG1 157N 3.213 1560 157CA 2 .717

156CG1 157CD1 3.583 1560 274N 2 .729

156CG1 157CE1 3.655 1560 157C 3 .324

156CG1 157CG 4.050 80 1560 274CB 3 .467

156CG1 157CZ 4.179 1560 274CA 3 .614

156CG1 157CA 4.262 1560 1570 3 .892

156CG1 157CD2 4.517 1560 2740 3 .950

156CG1 274CB 4.523 1560 157CB 4, .129 1560 274C 4.220 157CZ 205CD1 4,.113

1560 157CG 4.518 157CZ 205CD2 4, .153

1560 274CG 4.862 45 157CZ 274CG 4, .640

1560 157CD2 4.863 157CZ 205CG 4, .775

157N 2740 4.374 157CZ 207SD 4, .820

157N 274N 4.566 157C 2740 3, .627

157N 274CB 4.672 157C 274N 4, .446

157CA 2740 3.295 50 157C 274C 4, .527

157CA 274N 4.244 1570 2740 4, .850

157CA 274C 4.279 189N 207CA 4, .847

157CA 274CB 4.444 189CA 207CA 4, .417

157CA 274CA 4.594 189CA 207N 4, .864

157CB 2740 3.745 55 189CA 207CB 4, .928

157CB 274C 4.921 189CA 487CE2 4, .997

157CG 2740 3.919 189CB 306CA 4, .135

157CG 205CD1 4.661 189CB 212CG1 4, .600

157CG 274CB 4.767 189CB 487CE2 4, .841

157CG 274CG 4.946 60 189CB 306N 4, .926

157CD1 205CD1 4.256 189CB 487CD2 4, .995

157CD1 205CD2 4.394 189CG 306CA 3, .750

157CD1 205CG 4.976 189CG 487CE2 3, .924

157CD2 2740 3.280 189CG 306N 4. .212

157CD2 274CG 3.915 65 189CG 487CD2 4, .347

157CD2 274CB 4.085 189CG 487CZ 4, .911

157CD2 274ND2 4.099 189CG 207CG 4, .991

157CD2 2740D1 4.220 189CD1 207CB 3, .783

157CD2 274C 4.280 189CD1 207CG 3 , .907

157CD2 205CD1 4.766 70 189CD1 207SD 4, .051

157CD2 274CA 4.819 189CD1 487CE2 4. .116

157CE1 205CD2 3.643 189CD1 207CA 4. .314

157CE1 205CD1 3.963 189CD1 205CD2 4. .490

157CE1 205CG 4.458 189CD1 487CZ 4. .872

157CE1 207CE 4.729 75 189CD1 207N 4. .916

157CE1 207SD 4.791 189CD1 487CD2 4. .942

157CE2 274ND2 3.503 189CD2 306CA 3. .467

157CE2 274CG 3.728 189CD2 306N 3. .480

157CE2 2740D1 4.078 189CD2 305C 4. .060

157CE2 2740 4.205 80 189CD2 305O 4. .557

157CE2 274CB 4.299 189CD2 305CA 4. .739

157CE2 205CD1 4.515 189CD2 212CG2 4. .797

157CE2 274C 4.976 189CD2 212CG1 4. .883

157CZ 274ND2 4.083 189CD2 306C 4. .903 189CD2 487CE2 4.911 209N 212N 4.,914 189CD2 212CB 4.963 209CA 210N 2. ,421 189C 487CD2 4.060 45 209CA 21OCA 3. ,796 189C 487CE2 4.144 209CA 212CG1 4. .372 189C 4870 4.648 209CA 210C 4. .462 189C 306CA 4.974 209CA 212N 4. .662 1890 487CD2 3.681 209CA 210CB 4. .826 1890 4870 4.066 50 209CA 212CG2 4. .959 1890 487CE2 4.173 209CA 210CG 4. .975 1890 487C 4.215 209C 21OCA 2. .434 1890 306CA 4.247 209C 210C 3. .026 1890 306C 4.621 209C 210CB 3. .693 1890 487CG 4.813 55 209C 212N 3. .800 205CG 487CZ 4.299 209C 210O 3. .920 205CG 487CE2 4.667 209C 210CG 4. .126 205CD1 487CZ 4.050 209C 213N 4. .177 205CD1 487CE2 4.824 209C 210OD1 4. .376 205CD1 487CE1 4.931 60 209C 212CG1 4. .433 205CD2 207CB 4.532 209C 213CB 4. .625 205CD2 207N 4.789 209C 212CA 4. .667 205C 207N 4.445 209C 210ND2 4. .747 205O 207N 4.564 209C 212CG2 4, .782 207CA 209N 4.326 65 209C 212CB 4, .818 207CB 209N 4.314 209C 212C 4, .948 207CB 209CA 4.966 209O 210N 2, .245 207CG 209N 3.475 209O 21OCA 2, .777 207CG 209CA 3.821 209O 210C 2, .905 207CG 212CG1 4.074 70 209O 213N 2, .972 207CG 212CG2 4.903 209O 212N 3, .043 207SD 209CA 4.461 209O 210O 3, .487 207SD 209N 4.640 209O 213CB 3, .674 207SD 212CG2 4.851 209O 212CA 3, .685 207SD 212CG1 4.933 75 209O 212C 3. .773 207CE 209CA 4.469 2090 212CG2 3 .827 207CE 209N 4.711 2090 213CA 3. .906 207C 209N 3.159 209O 212CG1 3 .916 207C 209CA 4.396 209O 212CB 3 .947 207O 209N 3.120 80 209O 210CB 4 .249 207O 209CA 4.341 209O 210CG 4 .878 209N 210N 3.152 209O 2120 4 .958 209N 212CG1 4.281 209O 210OD1 4 .993 209N 210CA 4.540 210N 212N 4 .398 210N 213N 4.866 212C 216CA 4..658

210N 213CB 4.979 212C 213CG 4. .966

21OCA 213CB 4.405 45 2120 213N 2. .245

21OCA 212N 4.438 2120 216N 2. .670

21OCA 213N 4.596 2120 213CA 2. .759

210C 213N 3.886 2120 213C 2. .887

210C 213CB 4.239 2120 216CB 3. .134

210C 213CA 4.591 50 2120 2130 3. .255

210C 212CA 4.624 2120 216CA 3. .457

210C 212C 4.679 2120 213CB 4. .240

210O 213N 3.501 2120 304CE1 4. .399

210O 213CB 3.633 2120 216C 4. .660

210O 212N 3.644 55 2120 304CZ 4. .701

210O 213CA 3.933 213N 216N 4. .607

210O 213C 4.126 213N 216CB 4. .734

210O 212C 4.352 213CA 250CD1 4, .134

210O 212CA 4.631 213CA 216CB 4. .363

210O 213CG 4.674 60 213CA 216N 4. .434

210O 213CD1 4.727 213CA 250CG1 4, .898

212N 213N 2.784 213CA 216CA 4. .958

212N 213CA 4.205 213CB 250CD1 4, .585

212N 213C 4.773 213CG 250CG1 4. .288

212N 213CB 4.895 65 213CG 250CD1 4. .298

212CA 213N 2.468 213CG 249NH2 4. .361

212CA 213CA 3.845 213CD1 249NH2 4. .189

212CA 213C 4.420 213CD1 250CG1 4. .969

212CA 216N 4.888 213CD1 249CZ 4, .976

212CA 213CB 4.891 70 213CD2 250CG1 3, .388

212CB 213N 3.397 213CD2 250CD1 3. .646

212CB 213CA 4.698 213CD2 247ND2 4. .070

212CB 304CE1 4.881 213CD2 2470D1 4. .172

212CG1 213N 4.408 213CD2 249NH2 4. .297

212CG2 213N 3.253 75 213CD2 249CB 4. .423

212CG2 213CA 4.264 213CD2 247CG 4, .542

212CG2 304CE1 4.815 213CD2 250CB 4, .579

212C 213N 1.335 213CD2 250N 4, .650

212C 213CA 2.440 213CD2 249CD 4. .689

212C 213C 2.994 80 213CD2 250CA 4. .924

212C 213CB 3.710 213CE1 249NH2 3. .969

212C 2130 3.755 213CE1 249CZ 4. .403

212C 216N 3.855 213CE1 249NH1 4. .789

212C 216CB 4.183 213CE1 250CG1 4. .914 213CE1 249NE 4.963 2130 213N 3..471 213CE2 250CG1 3.302 2130 216CA 3. .662 213CE2 249CB 3.341 45 2130 216CB 3. .709 213CE2 250N 3.738 2130 216C 3, .731 213CE2 213CB 3.785 2130 250CD1 4, .129 213CE2 2470D1 3.897 2130 250CG1 4. .545 213CE2 249C 4.015 2130 2160 4. .914 213CE2 249CD 4.057 50 216N 304CZ 4. .099 213CE2 249NH2 4.080 216N 304CE2 4. .314 213CE2 250CD1 4.089 216N 304CE1 4. .419 213CE2 25OCA 4.157 216N 304CD2 4. .807 213CE2 250CB 4.248 216N 304CD1 4, .895 213CE2 249CG 4.314 55 216CA 304CE2 3. .847 213CE2 249CA 4.346 216CA 304CD2 3. .926 213CE2 249NE 4.420 216CA 304CZ 3. .934 213CE2 249CZ 4.459 216CA 304CG 4. .094 213CE2 247ND2 4.468 216CA 304CE1 4. .099 213CE2 2490 4.492 60 216CA 304CD1 4, .169 213CE2 247CG 4.604 216CA 250CD1 4. .669 213CZ 249CB 3.765 216CA 304CB 4 .908 213CZ 249NH2 3.909 216CA 220N 4, .975 213CZ 249CZ 4.112 216CA 220CD1 4, .997 213CZ 250CG1 4.148 65 216CB 250CD1 3, .531 213CZ 249NE 4.295 216CB 304CD1 3, .550 213CZ 249C 4.377 216CB 304CE1 3, .746 213CZ 249CD 4.394 216CB 304CG 3, .756 213CZ 250N 4.428 216CB 304CZ 4. .110 213CZ 2490 4.508 70 216CB 304CD2 4, .118 213CZ 249NH1 4.708 216CB 304CE2 4. .288 213CZ 249CG 4.729 216CB 304CB 4, .383 213CZ 25OCA 4.749 216CB 250CG1 4, .685 213CZ 249CA 4.754 216CB 220CD1 4, .706 213C 216N 3.701 75 216C 220N 4, .091 213C 216CB 4.305 216C 220CG 4 .389 213C 216CA 4.432 216C 220CD1 4 .410 213C 250CD1 4.614 216C 220CB 4 .425 213C 216C 4.795 216C 250CD1 4 .646 2130 216N 3.163 80 216C 304CD2 4, .738 2130 2120 3.255 216C 304CE2 4, .889 2130 213CB 3.375 216C 22 OCA 4, .901 2130 213CG 3.382 216C 304CG 4, .985 2130 213CD1 3.423 2160 220N 2, .973 2160 220CB 3.240 244CG 303CB 4..261

2160 220CG 3.312 244CG 246N 4. .437

2160 220CD1 3.600 45 244CG 303CA 4. .495

2160 22 OCA 3.682 244CG 304O 4. .536

2160 304CD2 4.466 244CG 2760 4. .605

2160 220CD2 4.723 244CG 304N 4. .694

2160 220C 4.729 244CG 2740D1 4. .945

2160 304CG 4.812 50 244CD2 2760 3. .276

2160 304CE2 4.867 244CD2 276C 4. .179

220CG 304CB 3.954 244CD2 2740D1 4. .296

220CG 304CD2 4.620 244CD2 276CA 4. .491

220CG 304CG 4.655 244CD2 276N 4. .705

220CG 304N 4.839 55 244CD2 303CB 4 .764

220CG 303C 4.910 244ND1 246N 3 .736

220CG 304CA 4.914 244ND1 246CB 3 .939

220CG 303O 4.942 244ND1 304O 4 .002

220CD1 250CG2 3.858 244ND1 246CA 4 .065

220CD1 304CB 3.918 60 244ND1 2740D1 4 .288

220CD1 304N 4.769 244ND1 274ND2 4 .528

220CD1 304CG 4.781 244ND1 274CG 4 .656

220CD1 304CA 4.980 244ND1 304N 4 .729

220CD2 303O 3.794 244CE1 2740D1 3 .036

220CD2 303C 3.874 65 244CE1 274ND2 3, .525

220CD2 304CB 4.033 244CE1 274CG 3, .527

220CD2 303N 4.091 244CE1 246N 4 .024

220CD2 304N 4.170 244CE1 246CA 4, .116

220CD2 303CA 4.361 244CE1 246CB 4, .253

220CD2 304CA 4.531 70 244CE1 304O 4, .409

220CD2 304CD2 4.978 244CE1 2760 4. .453

220CD2 304CG 4.997 244CE1 276CA 4, .503

244N 303CA 4.590 244CE1 276N 4, .607

244N 303N 4.800 244CE1 274CB 4, .806

244N 303CB 4.918 75 244CE1 276C 4, .903

244CA 246N 4.644 244NE2 2740D1 2, .997

244CA 303CA 4.703 244NE2 2760 3, .189

244CA 303CB 4.756 244NE2 276CA 3, .620

244CB 303CA 3.618 244NE2 276N 3, .747

244CB 303CB 3.663 80 244NE2 276C 3, .774

244CB 304N 4.380 244NE2 274CG 3, .873

244CB 303N 4.545 244NE2 274ND2 4, .248

244CB 303C 4.581 244NE2 246N 4, .811

244CB 246N 4.821 244C 246N 3 .311 244C 246CA 4.639 246C 274CG 4..829

2440 246N 3.012 2460 247N 2. .265

2440 246CA 4.288 45 2460 246CA 2. .400

2440 246CB 4.440 2460 247CA 2. .826

246N 247N 2.858 2460 247CB 3. .054

246N 247CA 4.225 2460 247ND2 3. .937

246N 2470 4.519 2460 247CG 3. .998

246N 274ND2 4.635 50 2460 274ND2 4. .116

246N 274CG 4.694 2460 274CB 4. .132

246N 247C 4.783 2460 247C 4. .279

246N 274CB 4.908 2460 274CG 4. .548

246CA 247N 2.398 2460 2470 4. .812

246CA 274ND2 3.762 55 247CA 249N 4. .491

246CA 247CA 3.780 247CB 249N 4. .494

246CA 274CG 4.159 247CG 250N 3, .957

246CA 274CB 4.398 247CG 249N 4, .038

246CA 247CB 4.470 247CG 250CB 4. .274

246CA 247ND2 4.518 60 247CG 249CB 4, .318

246CA 2470 4.632 247CG 250CG1 4. .508

246CA 247C 4.704 247CG 249CA 4, .619

246CA 2740D1 4.840 247CG 249CG 4, .681

246CA 247CG 4.866 247CG 250CD1 4, .751

246CB 247N 3.017 65 247CG 25OCA 4, .762

246CB 247ND2 3.981 247CG 249C 4, .818

246CB 274ND2 4.268 2470D1 249N 3, .007

246CB 247CA 4.360 2470D1 250N 3, .066

246CB 247CG 4.666 2470D1 249CB 3, .116

246CB 247CB 4.733 70 2470D1 249CA 3, .431

246CB 2470 4.816 2470D1 249CG 3, .604

246CB 250CG2 4.830 2470D1 249C 3, .735

246CB 250CD1 4.837 2470D1 250CB 4, .015

246CB 250CB 4.978 2470D1 25OCA 4, .121

246CB 274CG 4.988 75 2470D1 249CD 4, .172

246C 247CA 2.445 2470D1 250CG1 4, .267

246C 247CB 3.093 2470D1 250CD1 4, .870

246C 247C 3.647 2470D1 2490 4, .930

246C 247ND2 3.730 247ND2 250CD1 3 .820

246C 247CG 3.789 80 247ND2 250CG1 3, .953

246C 2470 3.922 247ND2 250CB 4, .004

246C 274ND2 4.440 247ND2 250N 4, .402

246C 274CB 4.631 247ND2 25 OCA 4, .869

246C 2470D1 4.815 247C 249N 3, .305 247C 250N 4.120 2740 276CA 4..809

247C 249CA 4.545 303N 304CA 4, .862

247C 249C 4.779 45 303CA 304N 2, .430

247C 250CB 4.900 303CA 304CA 3, .818

247C 25OCA 4.940 303CA 304C 4, .661

2470 249N 3.360 303CA 304O 4, .679

2470 250N 3.406 303CA 304CB 4, .684

2470 250CB 3.950 50 303CB 304N 3, .222

2470 25OCA 3.970 303CB 304CA 4, .521

2470 250C 4.123 303CB 304O 4, .818

2470 249C 4.217 303CB 304C 4. .963

2470 249CA 4.373 303C 304CA 2. .452

2470 250CG2 4.591 55 303C 304CB 3, .442

249N 250N 2.817 303C 304C 3, .504

249N 25OCA 4.203 303C 304O 3, .860

249N 250C 4.666 303C 305N 4, .467

249CA 250N 2.413 303C 304CG 4, .748

249CA 25OCA 3.779 60 303O 304N 2, .247

249CA 250C 4.413 303O 304CA 2. .806

249CA 250CB 4.866 303O 304CB 3, .762

249CB 250N 3.035 303O 304C 3. .989

249CB 25OCA 4.353 303O 304O 4, .630

249CG 250N 4.416 65 303O 305N 4. .674

249C 25OCA 2.410 303O 304CG 4. .827

249C 250C 3.035 304N 305N 3. .547

249C 250CB 3.720 304N 305CA 4, .719

249C 250O 3.774 304CA 305N 2. .450

249C 250CG1 4.278 70 304CA 305CA 3. .795

249C 250CG2 4.919 304CA 305C 4. .958

2490 250N 2.240 304CB 305N 3. .325

2490 25OCA 2.733 304CG 305N 3. .321

2490 250C 3.038 304CG 305CA 4 .469

2490 250O 3.374 75 304CG 305O 4. .729

2490 250CB 4.232 304CD1 305N 3. .304

2490 250CG1 4.716 304CD1 305CA 4. .052

2740D1 276CB 4.452 304CD1 305O 4. .145

2740D1 276C 4.632 304CD1 305C 4. .383

2740D1 2760 4.648 80 304CD2 305N 4. .139

274ND2 276N 4.935 304CE1 305O 3. .966

274C 276N 3.322 304CE1 305N 4. .100

274C 276CA 4.688 304CE1 305C 4. .483

2740 276N 3.552 304CE1 305CA 4. .616 ωn ωo r ro

Ol o UI O cn

(-0 OJ OJ OJ OJ o o o o o en cn cn en σi

O O O O

OJ OJ 0J OJ O o o o o

LΠ LΠ LΠ LΠ L

2 2 O O

*. tf^**. rf-. *- OJ t to to OJ J to OJ t OJ to to *> OJ NJ ι-> #»

UJ to ^1 on t cn LΠ LΠ o o to o t t o *-. ^1 J so 1 so 00 K^* cn to ] l-> 1 OJ 0J o UJ 0 to #*. OJ ^1 cn LΠ to o so \-> so OJ -J to cn o O so cn o to to UJ OJ LΠ 0J H^* UJ to oo O LΠ to CO *- en LΠ LΠ LΠ to 00 to σs to 00 cn l o H¹ 0J to cn LΠ σs UJ *- o CO LΠ UJ OJ H^* to #*

Table III provides the the atomic coordinates of the acetyl-CoA complex structure in the active site. Solvent molecules are omitted here for clarity, but can be found in Fig. 2. Residue 487 is Phe87 from the other monomer. Residue CAC is acetylated cysteine, and COA is the bound CoA molecule.

TABLE III

ATOM RESIDUE X Y Z Occ B

1 N THR 28 32. .909 0. .319 26. .935 1. .00 14. .64

2 CA THR 28 31. .524 0, .759 27. .053 1. .00 16. .73

3 CB THR 28 31. .399 2, .311 26. .861 1. .00 18. .66

4 OGl THR 28 30. .140 2, .771 27. .368 1. .00 21, .07

5 CG2 THR 28 31. .523 2. .702 25. .394 1. .00 14. .87

6 C THR 28 30. .671 -0. .021 26. .041 1. .00 15. .95

7 O THR 28 31. .196 -0. .755 25. .190 1. .00 14. .39

8 N TRP 32 24. .685 1. .112 27. .156 1. .00 18. .61

9 CA TRP 32 24. .896 1. .996 28. .316 1. .00 17. .67

10 CB TRP 32 26. .253 1. .657 28. .999 1. .00 18. .46

11 CG TRP 32 26. .543 2. .508 30. .252 1. .00 14. .22

12 CD2 TRP 32 26. .947 3. .865 30. .325 1. .00 16. .45

13 CE2 TRP 32 27. .044 4, .089 31. .715 1. .00 13. .95

14 CE3 TRP 32 27. .232 4. .916 29. .444 1. .00 14. .91

15 CD1 TRP 32 26. .405 1, .970 31. .509 1. .00 19. .11

16 NE1 TRP 32 26. .722 2, .948 32. .369 1. .00 17. .55

17 CZ2 TRP 32 27. .417 5, .348 32. .222 1. .00 16. .49

18 CZ3 TRP 32 27. .602 6, .164 29. .953 1. .00 8, .45

19 CH2 TRP 32 27, .698 6, .373 31. .321 1, .00 11. .56

20 C TRP 32 24, .917 3, .414 27. .781 1, .00 16, .08

21 O TRP 32 24. .363 4, .325 28. .378 1, .00 17, .69

22 N ILE 33 25. .536 3. .534 26. .593 1. .00 16, .72

23 CA ILE 33 25. .591 4. .911 26. .052 1. .00 17, .89

24 CB ILE 33 26, .670 5, .169 24. .944 1. .00 20, .24

25 CG2 ILE 33 26, .790 6, .671 24, .704 1, .00 18, .87

26 CGI ILE 33 28. .038 4, .571 25, .295 1. .00 16, .21

27 CD1 ILE 33 28. .930 4, .480 24. .013 1. .00 24, .09

28 C ILE 33 24. .196 5, .403 25. .732 1, .00 18, .98

29 O ILE 33 23. .877 6, .540 26. .194 1, .00 18, .61

30 N ARG 36 22, .046 6 .096 28. .836 1, .00 20, .61

31 CA ARG 36 22, .587 7 .077 29, .780 1, .00 20 .93

32 CB ARG 36 23, .940 6 .602 30, .339 1, .00 19, .27

33 CG ARG 36 23, .882 5 .328 31, .146 1, .00 20 .40

34 CD ARG 36 23, .627 5 .619 32, .605 1, .00 22 .27

35 NE ARG 36 23, .511 4 .396 33, .393 1, .00 27 .02

36 CZ ARG 36 23, .867 4 .298 34, .670 1, .00 25 .93

37 NH1 ARG 36 23. .734 3, .152 35. .315 1, .00 26, .63 Table III Cont

ATOM ] RESIDUE Occ

38 NH2 ARG 36 24. .330 5. .355 35. .318 1. .00 23. ,35

39 C ARG 36 22. .702 8. .517 29. .247 1. ,00 18. ,28

40 0 ARG 36 22. .703 9. .462 30. .029 1. ,00 17. ,41

41 N THR 37 22, .798 8. .697 27. .936 1. ,00 18. .97

42 CA THR 37 22, .932 10. .050 27. .405 1. ,00 21. .02

43 CB THR 37 24, .388 10. .371 26. .949 1. ,00 18. .78

44 OGl THR 37 24, .793 9, .461 25. .925 1. ,00 17. .72

45 CG2 THR 37 25, .347 10, .293 28. .084 1. ,00 21. .35

46 C THR 37 22, .048 10. .362 26. .222 1. .00 20. .16

47 0 THR 37 21, .914 11. .534 25. .839 1. ,00 25. .43

48 N CAC 112 30, .456 25. .709 28. .104 1. ,00 10. .38

49 CA CAC 112 29, .270 25, .229 27. .412 1. .00 14. .44

50 CB CAC 112 28, .799 23, .888 27, .980 1. .00 17. .69

51 SG CAC 112 29, .712 22, .439 27, .254 1. .00 17. .65

52 CD CAC 112 32, .183 21, .508 28, .594 1. .00 24. .17

53 CE CAC 112 30, .937 22, .403 28, .616 1. .00 21. .28

54 OE CAC 112 30, .752 23, .125 29, .602 1. .00 25. .29

55 C CAC 112 28, .167 26, .294 27, .295 1. .00 11. .81

56 O CAC 112 27, .369 26, .232 26, .368 1. .00 10. .19

57 N LEU 142 35. .611 19, .985 21, .261 1. .00 10. .22

58 CA LEU 142 35, .860 19. .347 22, .539 1. .00 13. .06

59 CB LEU 142 34, .735 19, .597 23, .555 1. .00 12, .36

60 CG LEU 142 34. .583 20, .999 24, .171 1. .00 11. .62

61 CD1 LEU 142 33, .937 20, .919 25, .543 1. .00 5. .06

62 CD2 LEU 142 35, .940 21, .651 24. .300 1. .00 10. .88

63 C LEU 142 36, .175 17, .851 22, .433 1. .00 13. .55

64 0 LEU 142 36, .786 17, .299 23, .322 1. .00 19, .07

65 N ARG 151 36, .295 6, .724 29 .164 1. .00 23, .03

66 CA ARG 151 34, .919 6, .417 28, .730 1. .00 23, .11

67 CB ARG 151 34. .470 5, .004 29 .175 1. .00 16, .86

68 CG ARG 151 34, .348 4, .774 30 .666 1. .00 15, .32

69 CD ARG 151 33, .926 3, .335 30 .928 1. .00 7, .13

70 NE ARG 151 33, .779 3, .086 32 .349 1. .00 10, .71

71 CZ ARG 151 33 .378 1, .927 32 .869 1. .00 3, .91

72 NH1 ARG 151 33 .268 1, .783 34 .179 1. .00 4, .61

73 NH2 ARG 151 33 .078 0. .930 32 .071 1. .00 10. .10

74 C ARG 151 33 .873 7, .478 29 .120 1. .00 17, .49

75 O ARG 151 33 .012 7, .828 28 .317 1. .00 17, .71

76 N GLY 152 34 .016 8, .044 30 .309 1, .00 17, .52

77 CA GLY 152 33 .070 9, .045 30, .776 1. .00 16, .37

78 C GLY 152 33, .062 10, .401 30 .082 1. .00 15, .84

79 O GLY 152 32 .246 11, .248 30, .439 1. .00 21, .56 Table III Cont

ATOM RESIDUE X Occ

80 N ILE 155 32. .443 9. .844 25. .187 1. .00 7. .71

81 CA ILE 155 31. .083 9. .426 24. .707 1. .00 12. .55

82 CB ILE 155 30. .385 8. .425 25. .708 1. .00 11. .77

83 CG2 ILE 155 31. .197 7. .148 25. .866 1. .00 11. .90

84 CGI ILE 155 30. .158 9, .085 27. .088 1. .00 12. .15

85 CD1 ILE 155 29, .158 8, .276 27. .966 1. .00 11, .79

86 C ILE 155 30, .193 10. .622 24. .373 1. .00 10. .55

87 0 ILE 155 29, .530 10, .593 23. .314 1. .00 14. .21

88 N ILE 156 30, .115 11, .601 25. .228 1. .00 15, .15

89 CA ILE 156 29, .284 12, .781 24. .971 1. .00 13, .87

90 CB ILE 156 28, .912 13, .460 26, .383 1. .00 18, .45

91 CG2 ILE 156 27, .632 12, .860 26. .931 1. .00 23, .09

92 CGI ILE 156 30, .082 13, .252 27, .370 1. .00 15, .34

93 CD1 ILE 156 29, .617 12, .611 28, .714 1. .00 19, .30

94 C ILE 156 29, .845 13, .826 24. .026 1. .00 13, .98

95 0 ILE 156 29, .049 14, .365 23. .211 1. .00 9, .76

96 N PHE 157 31, .114 14, .104 24. .000 1. .00 10, .77

97 CA PHE 157 31, .656 15, .152 23. .157 1. .00 7, .33

98 CB PHE 157 32. .859 15, .790 23. .759 1. .00 4, .54

99 CG PHE 157 32. .560 16, .451 25, .090 1. .00 7, .66

100 CD1 PHE 157 32, .946 15, .788 26. .255 1. .00 3, .98

101 CD2 PHE 157 31, .915 17, .650 25. .184 1. .00 5, .65

102 CE1 PHE 157 32, .660 16, .349 27, .491 1. .00 9, .88

103 CE2 PHE 157 31, .630 18, .205 26. .422 1. .00 4, .05

104 CZ PHE 157 32, .018 17, .536 27. .588 1. .00 6, .80

105 C PHE 157 31, .810 14, .851 21, .690 1. .00 10, .70

106 0 PHE 157 32, .380 13. .859 21, .257 1. .00 13, .03

107 N LEU 189 34, .231 20, .663 36, .441 1. .00 15, .69

108 CA LEU 189 34, .309 20, .542 34. .989 1. .00 15, .11

109 CB LEU 189 32. .983 20, .982 34. .350 1. .00 10, .07

110 CG LEU 189 32, .807 20, .922 32. .844 1. .00 7, .51

111 CD1 LEU 189 33, .311 19, .593 32. .263 1. .00 10, .35

112 CD2 LEU 189 31, .343 21, .142 32. .523 1. .00 7, .61

113 C LEU 189 35, .464 21, .418 34, .538 1. .00 15, .40

114 0 LEU 189 35, .452 22, .612 34, .812 1. .00 16, .51

115 N LEU 205 40, .306 17, .390 29, .143 1. .00 13, .16

116 CA LEU 205 39, .050 17, .802 29, .770 1, .00 15, .27

117 CB LEU 205 37, .963 17, .874 28. .694 1. .00 12, .62

118 CG LEU 205 36, .505 18, .215 29. .034 1. .00 14, .99

119 CD1 LEU 205 35, .817 18, .527 27. .706 1. .00 15, .12

120 CD2 LEU 205 35, .773 17, .085 29. .762 1. .00 11, .51

121 C LEU 205 38, .658 16, .793 30. .846 1. .00 15, .81 Table III Cont

ATOM ] RESIDUE X Occ

122 0 LEU 205 38. .675 15. .588 30. .594 1. .00 20. .20

123 N MET 207 35. .792 15. .888 34. .121 1. .00 18. .42

124 CA MET 207 34. .419 16. .232 34. .463 1. .00 16. .18

125 CB MET 207 33. .555 16. .227 33. .174 1, .00 17. .87

126 CG MET 207 32. .024 16. .237 33. .467 1. .00 17. .17

127 SD MET 207 30. .990 16, .464 32. .044 2. .00 17. .60

128 CE MET 207 31, .340 14, .797 31. .582 1. .00 22. .99

129 C MET 207 33. .790 15, .238 35. .466 1. .00 16. .62

130 0 MET 207 33. .726 14, .046 35. .222 1. .00 18. .22

131 N GLY 209 30. .811 14. .103 36. .169 1. .00 12. .42

132 CA GLY 209 29. .492 14. .040 35. .588 1. .00 16. .72

133 C GLY 209 28. .358 14. .011 36. .516 1. .00 19. .06

134 0 GLY 209 27. .487 14. .883 36. .423 1. .00 20. .59

135 N ASN 210 28. .284 13. .037 37. .418 1. .00 21. .24

136 CA ASN 210 27. .150 13. .010 38. .362 1. .00 24. .44

137 CB ASN 210 27. .198 11. .753 39. .171 1. .00 25, .49

138 CG ASN 210 27. .160 11. .958 40. .631 1. .00 33. .50

139 OD1 ASN 210 26. .177 11. .619 41. .309 1, .00 34. .80

140 ND2 ASN 210 28. .217 12. .429 41. .247 1. .00 32, .41

141 C ASN 210 26. .970 14, .201 39. .196 1. .00 25. .55

142 0 ASN 210 25. .858 14, .799 39. .270 1. .00 27. .11

143 N VAL 212 27. .967 17, .255 38. .323 1. .00 18. .86

144 CA VAL 212 27. .657 18, .365 37. .397 1. .00 19. .45

145 CB VAL 212 28, .483 18, .363 36. .115 1, .00 13. .66

146 CGI VAL 212 28, .142 19, .417 35. .091 1. .00 10. .49

147 CG2 VAL 212 29, .921 18, .642 36. .480 1. .00 11. .31

148 C VAL 212 26. .176 18, .359 36. .977 1, .00 25, .20

149 O VAL 212 25. .455 19 .359 36. .929 1, .00 27, .15

150 N PHE 213 25, .738 17, .114 36, .763 1, .00 24, .63

151 CA PHE 213 24, .361 16, .813 36, .336 1, .00 25, .87

152 CB PHE 213 24, .203 15 .287 36, .398 1, .00 23, .74

153 CG PHE 213 22, .788 14 .958 36, .099 1, .00 23, .97

154 CD1 PHE 213 22, .533 14 .398 34, .810 1, .00 27, .08

155 CD2 PHE 213 21, .752 14 .909 36, .974 1. .00 22, .61

156 CE1 PHE 213 21. .275 13 .964 34. .464 1, .00 23, .26

157 CE2 PHE 213 20 .480 14 .482 36, .625 1, .00 23, .27

158 CZ PHE 213 20 .223 13 .976 35, .335 1, .00 21 .74

159 C PHE 213 23 .356 17 .458 37, .319 1, .00 26 .46

160 O PHE 213 22 .395 18 .091 36, .945 1, .00 28 .12

161 N ALA 216 23 .435 21 .215 37, .390 1, .00 21, .80

162 CA ALA 216 22 .949 21 .675 36, .100 1, .00 19 .74

163 CB ALA 216 23, .464 20 .861 34. .933 1. .00 18, .25 Table III Cont

ATOM ] RESIDUE Y Z Occ B

164 C ALA 216 21. .440 21. .811 36. ,028 1. ,00 20. ,86

165 0 ALA 216 20. .936 22. .882 35. .612 1. .00 14. .72 i 166 N HIS 244 21. .005 23. .509 25. .764 1. .00 14. .90

167 CA HIS 244 22. .348 23. .098 25. .390 1. .00 17. .43

168 CB HIS 244 23. .328 23. .551 26. .478 1. .00 17. .97

169 CG HIS 244 24. .644 22. .836 26. .459 1. .00 18. .58

170 CD2 HIS 244 25. .582 22. .714 25. .488 1. .00 18. .43

1 171 ND1 HIS 244 25. .123 22. .136 27. .546 1. .00 18. .75

172 CE1 HIS 244 26. .295 21. .608 27. .243 1. .00 21. .88

173 NE2 HIS 244 26. .597 21. .944 26. .000 1. .00 17. .34

174 C HIS 244 22. .190 21. .563 25. .366 1. .00 17. .94

175 0 HIS 244 21. .579 20. .979 26. .286 1. .00 18. .08

176 N ALA 246 23. .569 18. .461 26. .118 1. .00 19. .92

177 CA ALA 246 24. .594 17. .753 26. .886 1. .00 22. .75

178 CB ALA 246 24. .851 18. .474 28. .207 1. .00 20. .40

179 C ALA 246 24. .197 16. .301 27. .174 1. .00 25. .65

180 0 ALA 246 24. .941 15. .364 26. .869 1. .00 27. .18

I 181 N ASN 247 23, .035 16. .122 27, .793 1. .00 26. .14

182 CA ASN 247 22, .545 14, .795 28, .146 1. .00 26. .38

183 CB ASN 247 22, .964 14, .464 29, .587 1. .00 28, .11

184 CG ASN 247 22, .574 13, .044 30, .019 1. .00 32. .46

185 OD1 ASN 247 21, .552 12, .486 29, .583 1. .00 30. .09

1 186 ND2 ASN 247 23, .371 12, .470 30, .912 1, .00 31, .19

187 C ASN 247 21, .021 14, .827 28, .020 1. .00 26. .38

188 0 ASN 247 20, .381 15. .783 28, .497 1, .00 24. .78

189 N ARG 249 18, .806 13. .418 29. .619 1. .00 26. .17

190 CA ARG 249 18. .082 13. .368 30, .918 1. .00 30. .19

I 191 CB ARG 249 18. .684 12. .450 31. .892 1. .00 35. .11

192 CG ARG 249 20. .149 12. .514 32. .084 1. .00 40. .00

193 CD ARG 249 20. .737 11. .377 33. .040 1. .00 40. .00

194 NE ARG 249 19. .770 10. .270 32. .981 1. .00 40. .00

195 CZ ARG 249 20. .131 8. .991 32. .720 1. .00 40. .00

196 NH1 ARG 249 19. .206 8. .005 32. .728 1. .00 40. .00

197 NH2 ARG 249 21. .400 8. .728 32. .469 1. .00 40. .00

198 C ARG 249 17, .883 14, .720 31, .505 1. .00 30, .27

199 0 ARG 249 16, .848 15, .128 31, .999 1, .00 29, .21

200 N ILE 250 19, .022 15, .485 31, .317 1, .00 31, .13

I 201 CA ILE 250 18, .989 16, .891 31, .777 1, .00 30, .26

202 CB ILE 250 20, .417 17, .557 31, .646 1, .00 31, .49

203 CG2 ILE 250 20, .269 19, .060 31. .848 1, .00 27. .57

204 CGI ILE 250 21, .391 16, .967 32. .703 1, .00 27. .31

205 CD1 ILE 250 22, .804 17. .587 32, .626 1, .00 29. .25 Table III Cont

ATOM ] RESIDUE Y Z Occ B

206 C ILE 250 17, .878 17. .652 31. .051 1. .00 29. .32

207 0 ILE 250 17. .014 18. .274 31. .667 1. .00 30. .29

208 N ASN 274 27. .325 16. .399 22. .555 1. .00 13. .47

209 CA ASN 274 27, .474 17. .720 23. .155 1. .00 13. .39

210 CB ASN 274 27, .818 17. .530 24. .622 1. .00 15. .56

211 CG ASN 274 27, .960 18. .816 25, .366 1. .00 17. .87

212 OD1 ASN 274 28, .135 19. .881 24. .780 1. .00 24. .67

213 ND2 ASN 274 27, .890 18. .729 26, .689 1. .00 19. .64

214 C ASN 274 28, .638 18. .414 22, .458 1. .00 14. .33

215 0 ASN 274 29 .770 17. .971 22 .613 1, .00 12. .94

216 N SER 276 29 .549 21. .633 22 .863 1. .00 7. .51

217 CA SER 276 29 .823 22, .861 23 .613 1, .00 13, .37

218 CB SER 276 31 .354 23, .045 23 .758 1, .00 16, .08

219 OG SER 276 31 .709 24, .178 24 .552 1. .00 13, .44

220 C SER 276 29 .132 24, .114 23 .029 1. .00 13, .89

221 O SER 276 27 .945 24, .062 22 .700 1. .00 11, .72

222 N PHE 304 24 .334 25, .567 30 .088 1, .00 14, .66

223 CA PHE 304 25 .107 25, .471 31 .332 1, .00 17, .36

224 CB PHE 304 24 .396 24, .476 32 .274 1, .00 14, .19

225 CG PHE 304 25 .035 24, .321 33 .630 1, .00 14, .80

226 CD1 PHE 304 26 .179 23, .562 33 .795 1, .00 13, .55

227 CD2 PHE 304 24 .464 24, .909 34 .748 1. .00 18, .11

228 CE1 PHE 304 26 .751 23. .388 35, .053 1, .00 13, .17

229 CE2 PHE 304 25 .024 24. .744 36, .014 1, .00 19, .56

230 CZ PHE 304 26 .175 23 , .977 36, .166 1, .00 18, .61

231 C PHE 304 26 .495 24. .936 30, .934 1, .00 18, .48

232 O PHE 304 26, .597 24, .072 30, .048 1. .00 19, .82

233 N GLY 305 27, .546 25, .411 31, .603 1. .00 20, .16

234 CA GLY 305 28. .889 24, .966 31, .272 1. .00 18, .15

235 C GLY 305 29, .950 25, .008 32, .367 1, .00 15, .06

236 O GLY 305 29, .701 25, .407 33, .507 1. .00 11, .78

237 N GLY 306 31, .145 24. .556 31, .988 1. .00 16, .84

238 CA GLY 306 32. .290 24, .514 32, .875 1, .00 16, .87

239 C GLY 306 32. .529 25. .856 33, .525 1. .00 19, .36

240 O GLY 306 32, .236 26. .899 32, .934 1, .00 17, .63

241 N PHE 487 38, .425 26. .469 30, .807 1. .00 13, .64

242 CA PHE 487 37, .277 26. .474 31, .704 1. .00 12. .94

243 CB PHE 487 35, .953 26. .064 31, .031 1. .00 16. .12

244 CG PHE 487 35, .967 24. .728 30, .332 1, .00 10. .50

245 CD1 PHE 487 36, .055 24. .668 28, .952 1. .00 11. .96

246 CD2 PHE 487 35, .776 23. .548 31, .043 1. .00 14. .59

247 CE1 PHE 487 35 .943 23, .450 28 .275 1, .00 11. .67 Table III Cont

ATOM RESIDUE X Occ

248 CE2 PHE 487 35. .665 22. .321 30. .373 1. ,00 10. .83

249 CZ PHE 487 35. .748 22, .283 28. .989 1. .00 9. .41

250 C PHE 487 37. .606 25, .574 32. .861 1. .00 13. .75

251 0 PHE 487 38. .217 24, .529 32, .661 1. .00 18. .53

252 A06 COA 350 25. .886 9. .541 33, .559 1. .00 40, .00

253 AP2 COA 350 25. .938 8, .466 34, .779 1. .00 40, .00

254 A04 COA 350 25. .984 7, .033 34, .193 1. .00 40. .00

255 A05 COA 350 24. .688 8. .689 35, .674 1. .00 40, .00

256 A03 COA 350 27, .383 8, .800 35, .491 1. .00 40, .00

257 API COA 350 27. .959 7, .998 36, .780 1. .00 40, .00

258 AOl COA 350 26. .887 7. .993 37, .879 1. .00 40, .00

259 A02 COA 350 29. .237 8, .653 37, .296 1. .00 40, .00

260 A05* COA 350 28. .201 6. .460 36, .164 1. .00 40. .00

261 AC5* COA 350 27. .718 5. .279 36, .817 1. .00 39. .18

262 AC4* COA 350 28. .472 4. .019 36, .378 1. .00 37. .65

263 A04* COA 350 28. .702 4, .012 34, .931 1. .00 35. .45

264 AC3* COA 350 29. .898 3. .856 36, .965 1. .00 37. .54

265 A03* COA 350 30. .205 2 .474 37 .178 1. .00 40 .00

266 AP3* COA 350 31, .518 2. .029 38 .160 1. .00 40. .00

267 A07 COA 350 32, .888 2, .220 37 .337 1, .00 40 .00

268 A08 COA 350 31. .503 3, .018 39 .420 1. .00 40 .00

269 A09 COA 350 31. .296 0, .500 38 .522 1. .00 40, .00

270 AC2* COA 350 30, .688 4, .469 35 .850 1. .00 32, .65

271 A02* COA 350 32. .112 4, .433 35 .932 1. .00 24, .96

272 AC1* COA 350 30. .098 3, .815 34 .584 1. .00 27, .72

273 AN9 COA 350 30, .429 4, .564 33, .382 1. .00 20. .99

274 AC8 COA 350 30, .840 5, .878 33, .186 1. .00 21, .31

275 AN7 COA 350 30. .992 6. .002 31, .788 1. .00 18, .53

276 AC5 COA 350 30. .700 4, .873 31, .234 1. .00 12, .67

277 AC6 COA 350 30. .698 4, .501 29. .898 1. .00 12, .21

278 AN6 COA 350 31. .039 5, .381 28. .963 1. .00 15, .81

279 AN1 COA 350 30. .338 3. .249 29, .672 1. .00 17, .72

280 AC2 COA 350 30. .014 2, .442 30, .654 1. .00 11, .38

281 AN3 COA 350 29 .997 2 .743 31 .973 1, .00 15 .08

282 AC4 COA 350 30. .341 3 .964 32 .268 1, .00 15 .56

283 PS1 COA 350 27 .926 20 .647 30 .314 1, .00 40 .00

284 PC2 COA 350 26 .439 19 .897 31 .045 1. .00 40 .00

285 PC3 COA 350 26 .760 18, .654 31 .835 1. .00 40 .00

286 PN4 COA 350 26, .965 17, .518 30 .873 1. .00 40 .00

287 PC5 COA 350 27, .350 16, .338 31 .273 1. .00 40 .00

288 P05 COA 350 27, .542 15. .370 30 .476 1. .00 40, .00

289 PC6 COA 350 27, .580 16. .199 32 .745 1. .00 40, .00 Table III Cont.

ATOM RESIDUE X Y Z Occ

290 PC7 COA 350 26 .255 15. .801 33 .363 1. .00 40. .00

291 PN8 COA 350 26 .292 14, .370 33 .634 1. .00 40. .00

292 PC9 COA 350 26 .176 13, .440 32 .669 1. .00 37, .37

293 P09 COA 350 25 .948 13, .691 31 .437 1. .00 31, .87

294 PC10 COA 350 26 .320 11, .982 33 .151 1. .00 38, .48

295 PO10 COA 350 26 .849 11, .940 34 .496 1. .00 37, .07

296 PC11 COA 350 27 .172 11, .057 32 .178 1. .00 40, .00

297 PC13 COA 350 28 .667 11, .476 32 .189 1. .00 40, .00

298 PC14 COA 350 26 .632 11, .101 30 .745 1. .00 40, .00

299 PC12 COA 350 26 .933 9 .588 32 .579 1. .00 40 .00

Mutants and Derivatives The invention further provides homologues, co-complexes and mutants of the E. coli FabH crystal structure of the invention.

The term "homologue" means a protein having at least 30% amino acid sequence identity with E. coli FabH or any of its functional domains.

The term "co-complex" means FabH or a mutant or homologue of FabH in covalent or non-covalent association with a chemical entity or compound.

The term "mutant" refers to a FabH polypeptide, i.e., a polypeptide displaying the biological activity of wild-type FabH activity, characterized by the replacement of at least one amino acid from the wild-type FabH sequence. Such a mutant may be prepared, for example, by expression of E. coli FabH cDNA previously altered in its coding sequence by oligonucleotide-directed mutagenesis.

E. coli FabH mutants may also be generated by site-specific incorporation of unnatural amino acids into the FabH proteins using the general biosynthetic method of C. J. Noren et al, Science. 244:182-188 (1989). In this method, the codon encoding the amino acid of interest in wild-type FabH enzyme is replaced by a "blank" nonsense codon, TAG, using oligonucleotide-directed mutagenesis. A suppressor tRNA directed against this codon is then chemically aminoacylated in vitro with the desired unnatural amino acid. The aminoacylated tRNA is then added to an in vitro translation system to yield a mutant FabH enzyme with the site-specific incorporated unnatural amino acid.

Selenomethionine may be incorporated into wild-type or mutant FabH by expression of E. coli FabH -encoding cDNAs in auxotrophic or non- auxotrophic E. coli strains [W. A. Hendrickson et al, EMBO J.. 9(5): 1665-1672 (1990)]. In this method, the wild-type or mutagenized FabH cDNA may be expressed in a host organism on a growth medium depleted of either natural methionine but enriched in selenomethionine. The location(s) of the Se atom(s) can be determined by X-ray diffraction analysis at three or four different wavelengths. This information, in turn, is used to generate the phase information used to construct three-dimensional structure of the enzyme. II. Methods of Identifying Inhibitors of the Novel FabH Crystalline Structure

Another aspect of this invention involves a method for identifying inhibitors of a FabH enzyme characterized by the crystal structure and novel active site described herein, and the inhibitors themselves. The novel E. coli FabH crystalline structure of the invention, or the structure of E. coli FabH homologue, permits the identification of inhibitors of FabH activity. Such inhibitors may be competitive, binding to all or a portion of the active site of FabH; or non-competitive and bind to and inhibit FabH whether or not it is bound to another chemical entity.

One design approach is to probe the FabH crystal of the invention with molecules composed of a variety of different chemical entities to determine optimal sites for interaction between candidate inhibitors and the enzyme. For example, high resolution X- ray diffraction data collected from crystals saturated with solvent allows the determination of where each type of solvent molecule sticks. Small molecules that bind tightly to those sites can then be designed and synthesized and tested for their FabH inhibitor activity [J. Travis, Science. 262:1374 (1993)]. This invention also enables the development of compounds that can isomerize to short-lived reaction intermediates in the chemical reaction of a substrate or other compound that binds to or with FabH. Thus, the time-dependent analysis of structural changes in FabH during its interaction with other molecules is permitted. The reaction intermediates of FabH can also be deduced from the reaction product in co-complex with FabH. Such information is useful to design improved analogues of known FabH inhibitors or to design novel classes of inhibitors based on the reaction intermediates of the enzyme and enzyme- inhibitor co-complex. This provides a novel route for designing FabH inhibitors with both high specificity and stability.

Another approach made possible by this invention, is to screen computationally small molecule data bases for chemical entities or compounds that can bind in whole, or in part, to the FabH enzyme. In this screening, the quality of fit of such entities or compounds to the binding site may be judged either by shape complementarity or by estimated interaction energy [E. C. Meng et al, J. Comp. Chem.. J3:505-524 (1992)]. Because FabH may crystallize in more than one crystal form, the structure coordinates of FabH, or portions thereof, as provided by this invention are particularly useful to solve the structure of those other crystal forms of FabH. They may also be used to solve the structure of FabH mutant co-complexes, or of the crystalline form of any other protein with significant amino acid sequence homology to any functional domain of FabH. One method that may be employed for this purpose is molecular replacement. In this method, the unknown crystal structure, whether it is another crystal form of FabH, a FabH mutant, a FabH co-complex, a FabH from a different bacterial species, or the crystal of some other protein with significant amino acid sequence homology to any domain of FabH, may be determined using the FabH structure coordinates of this invention as provided in Figure 1 and Tables I - IH. This method will provide an accurate structural form for the unknown crystal more quickly and efficiently than attempting to determine such information ab initio.

Thus, the FabH structure provided herein permits the screening of known molecules and/or the designing of new molecules which bind to the structure, particularly at the active site or substrate binding site, via the use of computerized evaluation systems. For example, computer modeling systems are available in which the sequence of the FabH, and the FabH structure (i.e., the atomic coordinates, bond distances between atoms in the active site region, etc. as provided by Figures 1-2 and Tables I - HI herein) may be input. Thus, a machine readable medium may be encoded with data representing the coordinates of Figures 1-2 and Tables I - III. The computer then generates structural details of the site into which a test compound should bind, thereby enabling the determination of the complementary structural details of said test compound.

More particularly, the design of compounds that bind to or inhibit FabH according to this invention generally involves consideration of two factors. First, the compound must be capable of physically and structurally associating with FabH. Non-covalent molecular interactions important in the association of FabH with its substrate include hydrogen bonding, van der Waals and hydrophobic interactions.

Second, the compound must be able to assume a conformation that allows it to associate with FabH. Although certain portions of the compound will not directly participate in this association with FabH, those portions may still influence the overall conformation of the molecule. This, in turn, may have a significant impact on potency. Such conformational requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the binding site, e.g., active site or substrate binding sites of FabH, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with FabH. The potential inhibitory or binding effect of a chemical compound on FabH may be analyzed prior to its actual synthesis and testing by the use of computer modelling techniques. If the theoretical structure of the given compound suggests insufficient interaction and association between it and FabH, synthesis and testing of the compound is obviated. However, if computer modelling indicates a strong interaction, the molecule may then be synthesized and tested for its ability to bind to FabH and inhibit using a suitable assay. In this manner, synthesis of inoperative compounds may be avoided. An inhibitory or other binding compound of FabH may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the individual binding pockets or other areas of FabH.

One skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with FabH and more particularly with the individual binding pockets of the FabH active site or accessory binding sites. This process may begin by visual inspection of, for example, the active site on the computer screen based on the FabH coordinates in Figures 1 -2 and Tables I - HI. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within a binding pocket of FabH. Docking may be accomplished using software such as Quanta and Sybyl, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM and AMBER.

Specialized computer programs may also assist in the process of selecting fragments or chemical entities. These include: 1. GRID [P. J. Goodford, "A Computational Procedure for Determining

Energetically Favorable Binding Sites on Biologically Important Macromolecules", J. Med. Chem.. 28:849-857 (1985)]. GRID is available from Oxford University, Oxford, UK.

2. MCSS [A. Miranker and M. Karplus, "Functionality Maps of Binding Sites: A Multiple Copy Simultaneous Search Method", Proteins: Structure. Function and Genetics. 11:29-34 (1991)]. MCSS is available from Molecular Simulations, Burlington, MA.

3. AUTODOCK [D. S. Goodsell and A. J. Olsen, "Automated Docking of Substrates to Proteins by Simulated Annealing", Proteins: Structure. Function, and Genetics. 8: 195-202 (1990)]. AUTODOCK is available from Scripps Research Institute, La Jolla, CA.

4. DOCK [I. D. Kuntz et al, "A Geometric Approach to Macromolecule-

Ligand Interactions", J. Mol. Biol.. 161:269-288 (1982)]. DOCK is available from University of California, San Francisco, CA.

Once suitable chemical entities or fragments have been selected, they can be assembled into a single compound or inhibitor. Assembly may be proceed by visual inspection of the relationship of the fragments to each other on the three-dimensional image displayed on a computer screen in relation to the structure coordinates of FabH. This would be followed by manual model building using software such as Quanta or Sybyl.

Useful programs to aid one of skill in the art in connecting the individual chemical entities or fragments include:

1. CAVEAT [P. A. Bartlett et al, "CAVEAT: A Program to Facilitate the Structure-Derived Design of Biologically Active Molecules", in Molecular Recognition in Chemical and Biological Problems". Special Pub., Royal Chem. Soc. 78, pp. 182-196 (1989)]. CAVEAT is available from the University of California, Berkeley, CA.

2. 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, CA). This area is reviewed in Y. C. Martin, "3D Database Searching in Drug Design", J. Med. Chem.. 35:2145-2154 (1992). 3. HOOK (available from Molecular Simulations, Burlington, MA).

Instead of proceeding to build a FabH inhibitor in a step-wise fashion one fragment or chemical entity at a time as described above, inhibitory or other FabH binding compounds may be designed as a whole or "de novo" using either an empty active site or optionally including some portion(s) of a known inhibitor(s). These methods include: 1. LUDI [H.-J. Bohm, "The Computer Program LUDI: A New Method for the

De Novo Design of Enzyme Inhibitors", J. Comp. Aid. Molec. Design, 6:61-78 (1992)]. LUDI is available from Biosym Technologies, San Diego, CA.

2. LEGEND [Y. Nishibata and A. Itai, Tetrahedron, 47:8985 (1991)].

LEGEND is available from Molecular Simulations, Burlington, MA. 3. LeapFrog (available from Tripos Associates, St. Louis, MO).

Other molecular modelling techniques may also be employed in accordance with this invention. See, e.g., N. C. Cohen et al, "Molecular Modeling Software and Methods for Medicinal Chemistry", J. Med. Chem.. 33:883-894 (1990). See also, M. A. Navia and M. A. Murcko, "The Use of Structural Information in Drug Design", Current Opinions in Structural Biology, 2:202-210 (1992). For example, where the structures of test compounds are known, a model of the test compound may be superimposed over the model of the structure of the invention. Numerous methods and techniques are known in the art for performing this step, any of which may be used. See, e.g., P.S. Farmer, Drug Design, Aliens, E.J., ed., Vol. 10, pp 1 19-143 (Academic Press, New York, 1980); U.S. Patent No. 5,331,573; U.S. Patent No. 5,500,807; C. Verlinde, Curr. Biol., 2:577-587 (1994); and I. D. Kuntz, Science, 257:1078-1082 (1992). The model building techniques and computer evaluation systems described herein are not a limitation on the present invention.

Thus, using these computer evaluation systems, a large number of compounds may be quickly and easily examined and expensive and lengthy testing avoided. Moreover, the need for actual synthesis of many compounds is effectively eliminated.

Once identified by the modelling techniques, the FabH inhibitor may be tested for bioactivity using standard techniques. For example, structure of the invention may be used in binding assays using conventional formats to screen inhibitors. One particularly suitable assay format includes the enzyme-linked immunosorbent assay (ELISA). Other assay formats may be used; these assay formats are not a limitation on the present invention.

In another aspect, the FabH structure of the invention permit the design and identification of synthetic compounds and/or other molecules which are characterized by the conformation of FabH of the invention. Using known computer systems, the coordinates of the FabH structure of the invention may be provided in machine readable form, the test compounds designed and/or screened and their conformations superimposed on the structure of the FabH of the invention. Subsequently, suitable candidates identified as above may be screened for the desired FabH inhibitory bioactivity, stability, and the like.

Once identified and screened for biological activity, these inhibitors may be used therapeutically or prophylactically to block FabH activity, and thus, bacterial replication.

The following examples illustrate various aspects of this invention. These examples do not limit the scope of this invention which is defined by the appended claims.

Example 1 - The Expression of FabH from Escherichia coli in Escherichia coli. The strategy for the expression of the FabH from Escherichia coli, using

Escherichia coli as a host was based on the PCR amplification of the fabH gene and the introduction of suitable restriction sites that allowed the cloning of the /αb-H-containing DNA fragment in the pET29 expression vector. After the PCR amplification the insert of the resultant recombinant plasmid, (pET29c hereafter), was sequenced to verify the absence of artifacts introduced by the Taq polymerase reaction. Expression was monitored by SDS- polyacrylamide gel analysis.

A. Bacterial strains, Plasmids and Medium

The Escherichia coli strains used were: MAXEfficiency DH10B

Competent Cells Genotype: F^" mcrA A(mrr-hsdRMS-mcrBC) φ80d/αcZΔM15 Δ/αcX74 deoR rec Al αraD139 A(ara, leu)1691 galX) galK λ" rpsL nupG. E. coli cells were grown at 37°C in Luria Bertani broth (LB). These strains may all be obtained from commercial sources. BL21 (DE3) competent cells for protein expression purchased by Novagen. The protocol used to make them electroporation-competent was the one provided by Invitrogen. The plasmid used was pET29 [Novagen]. A detailed description of pET29 is provided in Figure 2. Briefly, pET29 is an expression vector of E.coli which is based on the T7 promoter-driven system and allows the selection of the recombinant clones by kanamycin resistance.

LB Medium. Per liter:

For plasmid propagation 0.1 mg/ml of kanamycin was added to the medium.

B. DNA manipulations Plasmid DNA was prepared by the rapid alkaline method (Sambrook et al,

1989). Transformations of E. coli cells were carried out according to the protocol provided with the DH10B or the electroporation method. The plasmids for sequencing were purified using QIAGEN mini-prep kit [QIAGEN]. DNA sequencing was carried out on supercoiled plasmid DNA by the dideoxy chain-termination method using the Thermo Sequenase cycle sequencing kit [ABI, BigDye, Applied BioSystems, USA]. Synthetic oligonucleotides

[ordered in-house] were used as primers. Restriction enzymes and T4 DNA ligase were obtained from Gibco BRL (Life Technologies) and the experiments were carried out following the instructions provided by the suppliers.

The fabH gene from E.coli cloned in the pET29 vector was amplified by PCR using the primers:

(5'-TATACATATGTATACGAAGATTATTGGT-3'; SEQ ID NO:2) and: (5'-ATATGGATCCCTAGAAACGAACCAGCGCGG-3'; SEQ ID NO:3).

Ndel and BamHI restriction sites were incorporated at the 5' and 3' ends respectively of each primer to facilitate ligation of the amplified DNA into vectors. Plasmid DNA (480 ng) was amplified in 100 ul of PCR mixture containing 200 uM deoxynucleotide triphosphates (dNTPs), 0.20 M oligonucleotide primers, the manufacturer's buffer and 2.5 U of pfu (Stratagene). The following cycling parameters were used:

94°C 4 min

94°C 1 min, 55°C 40 sec, 72°C 1 min (30 cycles) 72°C 2 min

4°C Polymerase chain reaction (PCR) was performed using the GeneAmp, PCR System 2400 [Perkin Elmer Cetus Co]. PCR-amplified DNA fragments were purified using Qiaquick PCR Purification kit for Rapid Purification of DNA Fragments [Qiagen]. C. Cloning of the fabH gene of E. coli in the expression vector pET29 of E. coli.

The cloning strategy is shown in Figure 2. PCR amplification of the fabH gene from E. coli using the primers AKK2 and AKK3 resulted in a DNA fragment of about 960 bp. This fragment was purified with Qiaquick PCR purification kit protocol (Qiagen) digested with Ndel and BamHI, purified, ligated overnight to the Ndel and BamHI sites of already digested vector pET29 to obtain the recombinant plasmid pET29c. The ligation mix was used to transform E. coli DH10B competent cells. The construction of pET29c was initially confirmed by restriction analysis of the plasmid purified from the transformants. The amplification with Taq DNA polymerase made the sequencing of the fabH of pET29c an obligatory step to confirm that no changes were introduced due to the low fidelity of this enzyme. Sequence analysis was accomplished by using T7 promoter and terminator primers. The sequencing of both strands showed that no artifacts had been introduced during the amplification process.

D. Small-scale production of FabH from E. coli in E. coli

The plasmid pET29c and the negative control pET29 (vector without insert) were used to transform the E. coli BL21(DE3) host strain. Single clones of BL21(DE3): pET29c cells were grown overnight at 37°C in 2 ml of LB medium in the presence of 0.1 mg/ml kanamycin. The cells were then diluted 100-fold in 10 ml LB with kanamycin. When the cultures reached a value of 0.5 at OD₆₀₀ the fabH expression was induced by addition of isopropyl-thio-galactoside (IPTG) at 0.5 mM of final concentration. After this induction 2 ml samples were taken at different times (1 and 2 hours). The cells were harvested in a microfuge for 3 min, the pellets were washed with 20 mM Tris-HCl pH 8, lmM PMSF and resuspended in 100 ul of SDS-PAGE gel-loading buffer. The cells were broken by sonication (15 seconds). The samples were then boiled for 10 minutes and after one spin, 10 ml fractions were analyzed by SDS-PAGE according to the methods of Laemmli [U. K. Laemmli, Nature 227, 680-685 (1970)]. The 4-12% polyacrylamide gels [NOVEX] were stained with Coomassie blue. As shown in Figure 3 good expression levels were detected from the early stages after induction with IPTG. The evidence was the presence of a prominent band (lanes 2, 4 and 6 in Figure 3) that was in good agreement with the M_r predicted from the primary sequence. The FabH protein has a theoretical molecular weight of about 33,514 Da. Example 2 - Large Scale Growth and Purification of FabH

A. Large Scale Growth

A 4 liter fermentation of E.coli BL21(DE3): pET29c was carried out in Luria Bertani medium (LB), containing 100 ug/ml kanamycin. The 8x500 ml flasks were inoculated at 1% (v/v) from an overnight secondary seed culture in single strength LB medium, containing 100 mg/ml kanamycin. The flasks were incubated at 37°C, agitated at 250 rpm in a benchtop shaker. The OD at 600 nm was monitored, and at 0.5 absorbance units, FabH expression was induced with the addition of isopropyl-thiogalactosidase to 0.5 mM and the cells harvested by centrifugation in a Sorval CSA rotor, 2 hours post induction. A total of 20 grams of cell paste was recovered.

LB Medium, per liter, contains the following components. The medium was supplied by the in-house laboratory. Single strength Bacto Tryptone 10 g

Bacto Yeast Extract 5 g Sodium Chloride 5 g

B. Purification 1) Lysis 12.5 g of cells of E. coli overexpressing E. coli FabH obtained as described above, were resuspended in 140 ml of 20 mM Tris pH 7.9, 50 M NaCl, lmM EDTA, lmM DTT, 10% glycerol, 1 mM PMSF (buffer A). Lysozyme ( Sigma Chemicals: hen egg) was added to a final concentration of 1 mg/ml. Cells were incubated at 37°C for 20min. The cells were then lysed by sonication in an ice water bath (4x30sec). DNAse (Sigma; bovine pancreas type 1) was added along with MgCl₂ and held at 37°C for 5 minutes. The solution was centrifuged in a Beckman JA-HS centrifuge at 14,000 g for 60 minutes using a Beckman JA-14 rotor.

2) Anion exchange All chromatography was performed on a Pharmacia chromatography system, fitted with a UV detector (Pharmacia, Monitor UV-1). UV (at 280 nm) was monitored during all operations. All operations were performed at 4°C.

The supernatant from 1) was loaded onto a Q-Sepharose high performance (Pharmacia) column of 50 ml packed into a Pharmacia XK-26 column. The column was equilibrated with buffer A prior to loading. The column was then washed with buffer A (250 ml) at 4 ml/min, and eluted with a linear gradient of buffer A to 1M NaCl in buffer A over 80 minutes at 4 ml/min. The eluate was fractionated into 8 ml fractions using a Pharmacia FRAC 200.

The eluted fractions were assayed for FabH activity by measurement of incorporation of [¹ C]Acetyl-CoA to Malonyl-ACP and , and for protein by the Bradford method. Active fractions were analyzed by reducing SDS-PAGE

(NOVEX, NuPAGE Bis-Tris 4-12 % gradient gel). Active fractions pooled together and dialyzed against Buffer A.

3) Anion Exchange chromatography The dialyzed material was loaded onto a MonoQ column equilibrated with buffer A (Pharmacia, 5/5). The column was washed with 20 ml of the equilibration buffer until 280 nm absorbance returned to base line and then eluted with a linear gradient of equilibration buffer to buffer A over 90 minutes at 0.5 ml/min. Fractions were pooled together, collected, assayed for FabH activity. 4) Hydroxyapatite/ buffer exchange

Eluted fractions are collected ( 1 ml fraction) and assayed for FabH activity and protein. Active fractions are pooled and the volume was doubled with Buffer B [20 mM Tris-HCl pH 7.4, 50 mM NaCl, 1 mM DTT and 10% glycerol] to reduce the salt concentration in half. The active eluate was loaded in a hydroxyapatite column and eluted with 0.5 M Potassium Phosphate pH 7.4. The active enzyme was buffer exchanged with 20 mM Tris pH 7.4, 50 mM NaCl 2 mM DTT. This product was greater than 97% purity by SDS PAGE and the activity showed an overall process yield of 60 % from 1). N*-terminal amino acid analysis confirmed identity. Example 3 - Fermentation. Purification and Characterization of seleno-methionine derivative of Escherichia coli FabH A. Fermentation

To obtain soluble selmet-FabH for purification and crystallization studies, E. coli strain BL21 (DE3) was transformed with pET29c FabH. 50 ul of the seed culture expressing FabH gene product was inoculated into 100 ml of Luria broth, containing kanamycin (50 ug/ml) and glucose (0.6%). On reaching target density of 2 OD, the cells from the seed culture were isolated by centrifugation, resuspended in 100 ml of M9 minimal medium containing 1 mM CaCl2, 1 mM MgSO kanamycin ( 50 ug/ml) and glucose (0.6% w/v). The resuspended pellets were then added to 900 ml of the same medium and the cells were grown at 37 C to mid-log phase, AgrjO °f 1 -5. ^at which point lysine, phenylalanine, threonine at 100 mg/1 each, and selenomethionine, isoleucine, leucine, and valine at 50 mg/1 were added. The culture was shaken for 15 minutes, and then induced with 0.5mM isopropyl b-D-thiogalactopyranoside (IPTG). The culture was grown for 13 hours, and harvested by centrifugation (speed). 5 ml aliquots were taken prior to and during induction to monitor the expression of selenomethionine FabH. 12g of cell paste (wet wt) was recovered from 5L. In addition, to compare the expression of selenomethionine derivative to that of wt FabH, a one 1 culture was prepared under identical conditions except that the cells were grown in LB media. B. Purification

All lysis and purification steps were carried out using degassed buffers in a cold room or on ice. 4.5 g of E. coli cells over expressing Fab H were resuspended in 50 ml of 20 mM Tris, 50 mM sodium chloride, 10% glycerol, 0.2 M PMSF, 2 mM DTT, pH 7.9 (buffer A). Cells were lysed twice at 10,000 psi using Microfluidizer (Microfluidics Corporation, MA). Cell debris was removed by centrifugation (Sorvall RC-5B) at 35,000 g for 30 min. The supernatant was applied to a 2.6 x 4 cm Source Q column (Pharmacia) equilibrated in buffer A. The column was washed with 10 column volumes of buffer A, and eluted with a 10 column volume gradient of 0 to 1.0 M NaCl in buffer A. Eluted fractions were analyzed by 10% SDS-PAGE under reducing conditions. Fab H eluted at 0.2 - 0.25 M NaCl. Fab H containing fractions were pooled and applied to a 2.6 x 6 cm Hydroxyapatite column (Bio- Rad, Type I, 40u) equilibrated in buffer A. The column was eluted with a 30 column volume linear gradient of buffer A to 400 mM potassium phosphate, 10% glycerol, 2 mM DTT. Fab H, which eluted at 80-180 mM potassium phosphate, was diluted 1 :2 with 50 mM Tris, 200 mM NaCl, 10% glycerol, 2 mM DTT, pH 7.5 (buffer B) and applied to a 1.6 X 7.5 cm Blue Sepharose column (Pharmacia) equilibrated in buffer B. The column was eluted with buffer B containing 1 M NaCl. Blue Sepharose eluted Fab H fractions were next applied to a 2.6 x 60 cm Superdex 200 size exclusion column (Pharmacia) equilibrated in 20 mM Tris, 50 mM NaCl, 2 M DTT, pH 7.4. A total of 23 mg of Fab H was recovered which was concentrated tol3 mg/ml using Amicon 3 filtration device.

C. Characterization i). Mass Spectoscopy

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) data were obtained on a PerSeptive Biosystems Voyager RP laser desorption time-of-flight mass spectrometer. Protein samples were prepared for analysis by diluting analyte 1:5 with 3,5- Dimethoxy-4-hydroxy-cinnamic acid (lOmg/ml in 2: 1 0.1% trifluoroacetic acid/ acetonitrile) for a final concentration of 1-10 pmol/ul. Bovine Beta lactoglobulin A (Sigma) was included as an internal calibrant (MH⁺ 18364 Da). Desorption/ionization was accomplished using photon irradiation from a 337-nm pulsed nitrogen laser and 30-keV accelerating energy. Spectra were averaged over ca. 100 laser scans.

The predicted molecular mass for native FabH is 33516 Da. MALDI-MS analysis of the selenomethionyl incoporated FabH protein construct provided a mass of 33,889 Da. This is in close agreement with the predicted +375Da shift in mass expected for the sulfur to selenium side-chain substitution of eight methionine residues within the protein (33,891 Da theoretical). ii). N-terminal sequence analysis

Sequence analysis was performed on a Hewlett-Packard G1000A N-terminal Protein

Sequencer with on-line PTH identification using an HP1100 HPLC. Samples were applied directly to biphasic sequencing cartridges and standard 3.1 sequencing method cycles were used.

N-terminal sequencing results showed negligible native methionine in the first residue.

Instead, a unique PTH (phenylthiohydantoin) derivative was observed which eluted 1.6 minutes later than PTH-methionine, and did not coelute with any natural PTH-amino acid derivatives. The increase in hydrophobicity is consistent with the direct detection of the

PTH-selenomethionyl amino acid derivative.

D. Measurement of β-ketoacyl-ACP synthase III activity.

The enzyme catalyses the condensation of acetyl-CoA with malonyl-ACP to form acetoacetyl-ACP. The reaction can be described by three distinct steps: (a) the acyl group of acetyl-CoA is transferred to the active site cysteine resulting in a acyl-enzyme thioester; (b) carbanion formation. by the decarboxylation of malonyl-ACP; and (c) carbon-carbon bond formation by nucleophillic attack of the carbanion onto the carbonyl carbon atom of the acyl-enzyme thioester to yield the acetoacetyl-ACP product. This reaction can be assayed in order to characterize the enzyme or identify specific inhibitors of its activity in two ways:

(1) Radiolabeled acetoacetyl-ACP formation can be specifically determined using [3H]-acetyl-CoA and malonyl-ACP. The [3H]-acetyl-CoA substrate is soluble in 10%TCA while the resulting [3H]-acetoacetyl-ACP is not. A reaction mixture containing lOOmM sodium phosphate buffer pH7.0, lmM DTT, 34uM acetyl-CoA, 0.15uM [3H]-acetyl-CoA (specific activity 25Ci/mmol), and 7uM malonyl-ACP is prepared and transfered to a microtiter plate with or without inhibitors already added. The enzyme is added last to start the reaction and the plate is incubated at 37 degrees C. Ten percent TCA is added to stop the reaction, and then 50ug of BSA as a carrier. Stopped reactions are filtered and washed 2 times with 10% TCA on Wallac GF/A filtermats using a TomTec harvester. The filtermats are dried at 60 degrees C and the radioactivity quantified using Wallac Betaplate scintillation cocktail and a Wallac Microbeta 1450 liquid scintillation counter. IC50s are generated using Grafit 4.0 software and solved using the equation v = Vmax/(1 + I/IC50).

(2) FabG coupling can also be used to measure FabH production of acetoacetyl-ACP by following NADPH consumption at 340nm. FabG (β-ketoacyl-ACP reductase) will specifically reduce the C3 carbonyl of acetoacetyl-ACP to form β-hydroxybutyryl-ACP. This reduction requires the conversion of NADPH to form NADP+ which can be monitored by following the optical density at wavelength 340nm. (3) FabD coupling is an available assay option in the absence of purified malonyl-ACP. FabD (Malonyl-CoA:ACP transacylase) is responsible for malonic acid transfer from malonyl-CoA to ACP to form malonyl-ACP. This activity can be exploited by applying the techniques described in (1) above together with de novo malonyl-ACP from the FabD reaction.

E. Ligand binding to FabH. It is also possible to define ligand interactions with FabH in experiments that are not dependent upon enzyme catalyzed turnover of substrates. This type of experiment can be done in a number of ways:

(1) Effects of ligand binding upon enzvme intrinsic fluorescence (e.g. of tryptophan). Binding of either natural ligands or inhibitors may result in enzyme conformational changes which alter enzyme fluorescence. Using stopped-flow fluorescence equipment, this can be used to define the microscopic rate constants that describe binding. Alternatively, steady-state fluorescence titration methods can yield the overall dissociation constant for binding in the same way that these are accessed through enzyme inhibition experiments.

(2) Spectral effects of ligands. Where the ligands themselves are either fluorescent or possess chromophores that overlap with enzyme tryptophan fluorescence, binding can be detected either via changes in the ligand fluorescence properties (e.g. intensity, lifetime or polarization) or fluorescence resonance energy transfer with enzyme tryptophans. The ligands could either be inhibitors or variants of the natural ligands.

(3) Thermal analysis of the enzyme:ligand complex. Using calorimetric techniques (e.g. Isothermal Calorimetry, Differential Scanning Calorimetry) it is possible to detect thermal changes, or shifts in the stability of FabH which reports and therefore allows the characterization of ligand binding.

Example 3 - Crystallization of E. coli wild-type and selenomethionine mutant of FabH

A. Crystallization

All crystals were grown at room temperature using the sitting-drop vapor diffusion method. The drop solution was always a 1:1 mixture of the protein sample and the well solutions. For the crystal form 1 of the wild-type protein, the well solution contained 0.1 M HEPES buffer at pH 7.5 and 20% PEG8000. For the crystal form 2 of the selenomethionine mutant protein in complex with acetyl-CoA, the well solution contained 0.05 M Bis-Tris propane buffer at pH 7.0, 0.1 M MgCl₂ and 14% PEG6000. Crystals grew overnight and are approximately 0.1 to 0.2 mm in sizes.

B. X-ray Diffraction Characterization

All crystals were frozen in liquid nitrogen streams before their characterization using synchrotron X-ray radiation. Diffraction data for the apo form 1 crystal was collected to 2.0 A resolution. The data is 97.1 % complete and 6 fold redundant with a merging R- factor of 7.7 %. The crystal belongs to the orthorhombic spacegroup P2-2-2-, with cell dimensions a = 63.1, b = 65.1 and c = 166.5 A. For the Se-Met protein in complex with acetyl-CoA, data were collected at three different wavelengths: 0.9789, 0.9785 and 0.9414 A. The three data set were of nearly identical quality, with about 80% completion, 6-fold redundancy, 8.5 % merging R-factor, and 1.9 A resolution. The form 2 crystal belongs to the tetragonal spacegroup P4-2-2, with a = b = 72. 4 and c = 102.8 A.

C. Structure Solution

The crystal structure of the Se-Met E. coli FabH mutant in complex with acetyl- CoA was solved to 1.9 A resolution using the MAD phasing technique with the data sets collected at three different wavelengths and the program SOLVE (Terwilliger & Berendzen, 1999, Acta Cryst. D55, 849-861). All eight Se-Met were located by SOLVE. The overall MAD phasing figure of merit was 0.6 to 1.9 A resolution, and the overall Z score was as high as 148. The resulting electron density map was of very high quality. The structure of the apo enzyme (crystal form 1) was solved with the molecular replacement method using the acetyl-CoA complex structure as the search model. This crystal form had a FabH dimer in the asymmetric unit, and the R-factor of the solution was only 33%. Twofold averaged map was then calculated and used for model building.

D. Model Building and Refinement The electron density for the acetyl-CoA complex was very clear and a structure model for the whole FabH protein, the bound acetyl group and CoA, as well as 98 solvent molecules were built in the first round. Standard structural refinement protocols and manual model building led to the current model, which has an R-factor of 27 % to 1.9 A resolution. The model for the apo FabH structure was also built readily, and refined to an R-factor of 18.9 % (R_free of 24.4%) to 2.0 A resolution. Both models have excellent geometry and do not have any outliers in the Ramanchandran plot, indicating high quality of the atomic coordinates, which contain an estimated error of less than 0.3 A.

This invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. The disclosures of the patents, patent applications and publications cited herein are incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising a E. coli FabH in crystalline form.

2. The composition according to claim 1 wherein said FabH is a dimer.

3. The composition according to claim 1 wherein said FabH comprises an active site cavity formed by amino acids comprising Cys 112, His244 and Asn274

4. The composition of claim 1 wherein said FabH is a E. coli FabH.

5. The composition of claim 3 wherein said FabH is characterized by the coordinates selected from the group consisting of the coordinates of Figures 1-2 and Tables I, π, and m.

6. A E. coli FabH crystal.

7. A selenomethionine mutant crystal of a E. coli FabH.

8. An isolated, properly folded FabH molecule or fragment thereof having a conformation comprising the protein coordinates of Figures 1 -2 and Tables I, π, and -XT.

9. The molecule according to claim 8 wherein said molecule is a dimer, wherein each monomer is characterized by two similar domains having core of five β- strands, each containing flanking helices, strands and loops, as illustrated in Fig. 3.

10. The molecule according to claim 8 wherein said molecule is a dimer characterized by the dimer interface of Fig. 3.

1 1. The molecule according to claim 10 which is E. coli FabH.

12. A peptide, peptidomimetic or synthetic molecule which interacts competitively or non-competitively with the active site of a FabH of claim 1.

13. A method of identifying an inhibitor compound capable of binding to, and inhibiting the enzymatic activity of, a E. coli FabH, said method comprising: introducing into a suitable computer program information defining an active site conformation of a E. coli FabH molecule comprising a conformation defined by the coordinates of Figures 1 -2 and Tables I, II, and HI, wherein said program displays the three-dimensional structure thereof; creating a three dimensional structure of a test compound in said computer program; displaying and superimposing the model of said test compound on the model of said active site; assessing whether said test compound model fits spatially into the active site; incorporating said test compound in a biological activity assay for a FabH characterized by said active site; and determining whether said test compound inhibits enzymatic activity in said assay.

14. The method according to claim 13 wherein said FabH molecule is a dimer, wherein each monomer is characterized by two similar domains having core of five β- strands, each containing flanking helices, strands and loops, as illustrated in Fig. 3.

15. A method of identifying an inhibitor compound capable of binding to, and inhibiting the enzymatic activity of, a E. coli FabH, said method comprising: introducing into a suitable computer program information defining an active site conformation of a FabH molecule comprising a conformation defined by the coordinates of Figures 1-2 and Tables I, II, and III, wherein said program displays the three-dimensional structure thereof; creating a three dimensional structure of a test compound in said computer program; displaying and superimposing the model of said test compound on the model of said active site; assessing whether said test compound model fits spatially into the active site; incorporating said test compound in a biological activity assay for a FabH characterized by said active site; and determining whether said test compound inhibits enzymatic activity in said assay.

16. The method according to claim 15 wherein said FabH molecule is a dimer, wherein each monomer is characterized by two similar domains having core of five β- strands, each containing flanking helices, strands and loops, as illustrated in Fig. 3.

17. A peptide, peptidomimetic or synthetic molecule identified by the method of claim 13 or 15.

18. A method for solving a crystal form comprising using the structural coordinates of a E. coli FabH crystal or portions thereof, to solve a crystal form of a mutant, homologue or co-complex of said FabH by molecular rearrangement.

19. A method of drug design comprising the step of using the structural coordinates of a E. coli FabH crystal to computationally evaluate a chemical entity for associating with the active site and substrate binding sites of E. coli FabH.

20. The method of drug design according to claim 19 comprising the step of using the structure coordinates of E. coli FabH to identify an intermediate in a chemical reaction between said FabH and a compound with is a substrate or inhibitor of said enzyme.

21. The method according to claim 20, wherein said entity is a competitive or non-competitive inhibitor of a E. coli FabH.

22. The method of drug design according to claim 19, using the structure of a FabH homologue that has similar amino acid identities as well as spacial arrangements as those of E. coli FabH listed in Tables I-m.

23. The method of drug design according to claim 20 using the structure of a FabH homologue that has similar amino acid identities as well as spacial arrangements as those of E. coli FabH listed in Tables I--H.

24. The method of drug design according to claim 21 using the structure of a FabH homologue that has similar amino acid identities as well as spacial arrangements as those of E. coli FabH listed in Tables I-HI.

25. The method according to claim 19 wherein said structure coordinates comprise the coordinates of Figures 1-2 and Tables I, II, and HI.

26. The method according to claim 20 wherein said structure coordinates comprise the coordinates of Figures 1-2 and Tables I, II, and DX

27. The method according to claim 21 wherein said structure coordinates comprise the coordinates of Figures 1-2 and Tables I, π, and HI.