An antibody exo Diels-Alderase inhibitor complex at 1.95 angstrom resolution

Science

Volumn 279, Issue 5358, 1998, Pages 1934-1940

  Heine, Andreas ^a   Stura, Enrico A ^a   Yli Kauhaluoma, Jari T ^a   Gao, Changshou ^a   Deng, Qiaolin ^b   Beno, Brett R ^b   Houk, Kendall N ^b   Janda, Kim D ^a   Wilson, Ian A ^a  

^a SCRIPPS RESEARCH INSTITUTE   (United States)

^b UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CATALYTIC ANTIBODY; IMMUNOGLOBULIN F(AB) FRAGMENT;

ARTICLE; CATALYSIS; CRYSTAL STRUCTURE; HYDROGEN BOND; PRIORITY JOURNAL; REACTION ANALYSIS; STEREOCHEMISTRY;

ANTIBODIES, CATALYTIC; CATALYSIS; CHEMISTRY, ORGANIC; CRYSTALLOGRAPHY, X-RAY; FERROUS COMPOUNDS; HAPTENS; HYDROGEN BONDING; HYDROGEN-ION CONCENTRATION; IMMUNOGLOBULIN FAB FRAGMENTS; MODELS, MOLECULAR; STEREOISOMERISM; THERMODYNAMICS;

BETULACEAE;

EID: 0032549734     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5358.1934     Document Type: Article

References (71)

1. A. Streitwieser and C. H. Heathcock, Introduction to Organic Chemistry (Macmillan, New York, 1981).
2. J. Sauer and R. Sustmann, Angew. Chem. Int. Ed. Engl. 19, 779 (1980); Y. Li and K. N. Houk, J. Am. Chem. Soc. 115, 7478 (1993).
3. J. Sauer and R. Sustmann, Angew. Chem. Int. Ed. Engl. 19, 779 (1980); Y. Li and K. N. Houk, J. Am. Chem. Soc. 115, 7478 (1993).
4. D. C. Rideout and R. Breslow, J. Am. Chem. Soc. 102, 7816 (1980); P. A. Grieco, P. Garner, Z. He, Tetrahedron Lett. 24, 1897 (1983); J. F. Blake and W. L. Jorgensen, J. Am. Chem. Soc. 113, 7430 (1991); W. L. Jorgensen, J. F. Blake, D. Lim, D. L. Severance, J. Chem. Soc. Faraday Trans. 90, 1727 (1994); J. F. Blake, D. Lim, W. L. Jorgensen, J. Org. Chem. 59, 803 (1994).
5. D. C. Rideout and R. Breslow, J. Am. Chem. Soc. 102, 7816 (1980); P. A. Grieco, P. Garner, Z. He, Tetrahedron Lett. 24, 1897 (1983); J. F. Blake and W. L. Jorgensen, J. Am. Chem. Soc. 113, 7430 (1991); W. L. Jorgensen, J. F. Blake, D. Lim, D. L. Severance, J. Chem. Soc. Faraday Trans. 90, 1727 (1994); J. F. Blake, D. Lim, W. L. Jorgensen, J. Org. Chem. 59, 803 (1994).
6. D. C. Rideout and R. Breslow, J. Am. Chem. Soc. 102, 7816 (1980); P. A. Grieco, P. Garner, Z. He, Tetrahedron Lett. 24, 1897 (1983); J. F. Blake and W. L. Jorgensen, J. Am. Chem. Soc. 113, 7430 (1991); W. L. Jorgensen, J. F. Blake, D. Lim, D. L. Severance, J. Chem. Soc. Faraday Trans. 90, 1727 (1994); J. F. Blake, D. Lim, W. L. Jorgensen, J. Org. Chem. 59, 803 (1994).
7. D. C. Rideout and R. Breslow, J. Am. Chem. Soc. 102, 7816 (1980); P. A. Grieco, P. Garner, Z. He, Tetrahedron Lett. 24, 1897 (1983); J. F. Blake and W. L. Jorgensen, J. Am. Chem. Soc. 113, 7430 (1991); W. L. Jorgensen, J. F. Blake, D. Lim, D. L. Severance, J. Chem. Soc. Faraday Trans. 90, 1727 (1994); J. F. Blake, D. Lim, W. L. Jorgensen, J. Org. Chem. 59, 803 (1994).
8. D. C. Rideout and R. Breslow, J. Am. Chem. Soc. 102, 7816 (1980); P. A. Grieco, P. Garner, Z. He, Tetrahedron Lett. 24, 1897 (1983); J. F. Blake and W. L. Jorgensen, J. Am. Chem. Soc. 113, 7430 (1991); W. L. Jorgensen, J. F. Blake, D. Lim, D. L. Severance, J. Chem. Soc. Faraday Trans. 90, 1727 (1994); J. F. Blake, D. Lim, W. L. Jorgensen, J. Org. Chem. 59, 803 (1994).
9. P. G. Schultz and R. A. Lerner, Science 269, 1835 (1995); B. Lavey and K. D. Janda, in Antibody Engineering, H. Y. Wang and T. Imanaka, Eds. (American Chemical Society, Washington, DC, 1995), pp. 123-138; K. D. Janda, C. G. Shevlin, C.-H. L. Lo, in Comprehensive Supramolecular Chemistry, J. M. Lehn et al., Eds. (Pergamon, New York, 1996), pp. 43-73; E. Keinan and R. A. Lerner, Isr. J. Chem. 36, 133 (1996).
10. P. G. Schultz and R. A. Lerner, Science 269, 1835 (1995); B. Lavey and K. D. Janda, in Antibody Engineering, H. Y. Wang and T. Imanaka, Eds. (American Chemical Society, Washington, DC, 1995), pp. 123-138; K. D. Janda, C. G. Shevlin, C.-H. L. Lo, in Comprehensive Supramolecular Chemistry, J. M. Lehn et al., Eds. (Pergamon, New York, 1996), pp. 43-73; E. Keinan and R. A. Lerner, Isr. J. Chem. 36, 133 (1996).
11. P. G. Schultz and R. A. Lerner, Science 269, 1835 (1995); B. Lavey and K. D. Janda, in Antibody Engineering, H. Y. Wang and T. Imanaka, Eds. (American Chemical Society, Washington, DC, 1995), pp. 123-138; K. D. Janda, C. G. Shevlin, C.-H. L. Lo, in Comprehensive Supramolecular Chemistry, J. M. Lehn et al., Eds. (Pergamon, New York, 1996), pp. 43-73; E. Keinan and R. A. Lerner, Isr. J. Chem. 36, 133 (1996).
12. P. G. Schultz and R. A. Lerner, Science 269, 1835 (1995); B. Lavey and K. D. Janda, in Antibody Engineering, H. Y. Wang and T. Imanaka, Eds. (American Chemical Society, Washington, DC, 1995), pp. 123-138; K. D. Janda, C. G. Shevlin, C.-H. L. Lo, in Comprehensive Supramolecular Chemistry, J. M. Lehn et al., Eds. (Pergamon, New York, 1996), pp. 43-73; E. Keinan and R. A. Lerner, Isr. J. Chem. 36, 133 (1996).
13. W. P. Jencks, Catalysis in Chemistry and Enzymology (McGraw-Hill, New York, 1969); Adv. Enzymol. 43, 219 (1975).
14. W. P. Jencks, Catalysis in Chemistry and Enzymology (McGraw-Hill, New York, 1969); Adv. Enzymol. 43, 219 (1975).
15. A. Tramontano, K. D. Janda, R. A. Lerner, Science 234, 1566 (1986); K. D. Janda, S. J. Benkovic, R. A. Lerner, ibid. 244, 437 (1989); G. W. Zhou, J. Guo, W. Huang, R. J. Fletterick, T. S. Scanlan, ibid. 265, 1059 (1994); J. Guo, W. Huang, G. W. Zhou, R. J. Fletterick, T. S. Scanlan, Proc. Natl. Acad. Sci. U.S.A. 92, 1694 (1995); P. A. Patten et al., Science 271, 1086 (1996); B. Golinelli-Pimpaneau et al., Structure 2, 175 (1994); J.-B. Charbonnier et al., Science 275, 1140 (1997).
16. A. Tramontano, K. D. Janda, R. A. Lerner, Science 234, 1566 (1986); K. D. Janda, S. J. Benkovic, R. A. Lerner, ibid. 244, 437 (1989); G. W. Zhou, J. Guo, W. Huang, R. J. Fletterick, T. S. Scanlan, ibid. 265, 1059 (1994); J. Guo, W. Huang, G. W. Zhou, R. J. Fletterick, T. S. Scanlan, Proc. Natl. Acad. Sci. U.S.A. 92, 1694 (1995); P. A. Patten et al., Science 271, 1086 (1996); B. Golinelli-Pimpaneau et al., Structure 2, 175 (1994); J.-B. Charbonnier et al., Science 275, 1140 (1997).
17. A. Tramontano, K. D. Janda, R. A. Lerner, Science 234, 1566 (1986); K. D. Janda, S. J. Benkovic, R. A. Lerner, ibid. 244, 437 (1989); G. W. Zhou, J. Guo, W. Huang, R. J. Fletterick, T. S. Scanlan, ibid. 265, 1059 (1994); J. Guo, W. Huang, G. W. Zhou, R. J. Fletterick, T. S. Scanlan, Proc. Natl. Acad. Sci. U.S.A. 92, 1694 (1995); P. A. Patten et al., Science 271, 1086 (1996); B. Golinelli-Pimpaneau et al., Structure 2, 175 (1994); J.-B. Charbonnier et al., Science 275, 1140 (1997).
18. A. Tramontano, K. D. Janda, R. A. Lerner, Science 234, 1566 (1986); K. D. Janda, S. J. Benkovic, R. A. Lerner, ibid. 244, 437 (1989); G. W. Zhou, J. Guo, W. Huang, R. J. Fletterick, T. S. Scanlan, ibid. 265, 1059 (1994); J. Guo, W. Huang, G. W. Zhou, R. J. Fletterick, T. S. Scanlan, Proc. Natl. Acad. Sci. U.S.A. 92, 1694 (1995); P. A. Patten et al., Science 271, 1086 (1996); B. Golinelli-Pimpaneau et al., Structure 2, 175 (1994); J.-B. Charbonnier et al., Science 275, 1140 (1997).
19. A. Tramontano, K. D. Janda, R. A. Lerner, Science 234, 1566 (1986); K. D. Janda, S. J. Benkovic, R. A. Lerner, ibid. 244, 437 (1989); G. W. Zhou, J. Guo, W. Huang, R. J. Fletterick, T. S. Scanlan, ibid. 265, 1059 (1994); J. Guo, W. Huang, G. W. Zhou, R. J. Fletterick, T. S. Scanlan, Proc. Natl. Acad. Sci. U.S.A. 92, 1694 (1995); P. A. Patten et al., Science 271, 1086 (1996); B. Golinelli-Pimpaneau et al., Structure 2, 175 (1994); J.-B. Charbonnier et al., Science 275, 1140 (1997).
20. A. Tramontano, K. D. Janda, R. A. Lerner, Science 234, 1566 (1986); K. D. Janda, S. J. Benkovic, R. A. Lerner, ibid. 244, 437 (1989); G. W. Zhou, J. Guo, W. Huang, R. J. Fletterick, T. S. Scanlan, ibid. 265, 1059 (1994); J. Guo, W. Huang, G. W. Zhou, R. J. Fletterick, T. S. Scanlan, Proc. Natl. Acad. Sci. U.S.A. 92, 1694 (1995); P. A. Patten et al., Science 271, 1086 (1996); B. Golinelli-Pimpaneau et al., Structure 2, 175 (1994); J.-B. Charbonnier et al., Science 275, 1140 (1997).
21. A. Tramontano, K. D. Janda, R. A. Lerner, Science 234, 1566 (1986); K. D. Janda, S. J. Benkovic, R. A. Lerner, ibid. 244, 437 (1989); G. W. Zhou, J. Guo, W. Huang, R. J. Fletterick, T. S. Scanlan, ibid. 265, 1059 (1994); J. Guo, W. Huang, G. W. Zhou, R. J. Fletterick, T. S. Scanlan, Proc. Natl. Acad. Sci. U.S.A. 92, 1694 (1995); P. A. Patten et al., Science 271, 1086 (1996); B. Golinelli-Pimpaneau et al., Structure 2, 175 (1994); J.-B. Charbonnier et al., Science 275, 1140 (1997).
22. P. Wirsching, J. A. Ashley, C-H. L. Lo, K. D. Janda, R. A. Lerner, Science 270, 1775 (1995); J. Wagner, R. A. Lerner, C. F. Barbas III, ibid., p. 1797; R. Björnestedt, G. Zhong, R. A. Lerner, C. F. Barbas, J. Am. Chem. Soc. 118, 11720 (1996).
23. P. Wirsching, J. A. Ashley, C-H. L. Lo, K. D. Janda, R. A. Lerner, Science 270, 1775 (1995); J. Wagner, R. A. Lerner, C. F. Barbas III, ibid., p. 1797; R. Björnestedt, G. Zhong, R. A. Lerner, C. F. Barbas, J. Am. Chem. Soc. 118, 11720 (1996).
24. P. Wirsching, J. A. Ashley, C-H. L. Lo, K. D. Janda, R. A. Lerner, Science 270, 1775 (1995); J. Wagner, R. A. Lerner, C. F. Barbas III, ibid., p. 1797; R. Björnestedt, G. Zhong, R. A. Lerner, C. F. Barbas, J. Am. Chem. Soc. 118, 11720 (1996).
25. K. D. Janda, C. G. Shevlin, R. A. Lerner, Science 259, 490 (1993); B. F. Cravatt, J. A. Ashley, K. D. Janda, D. L. Boger, R. A. Lerner, J. Am. Chem. Soc. 116, 6013 (1994).
26. K. D. Janda, C. G. Shevlin, R. A. Lerner, Science 259, 490 (1993); B. F. Cravatt, J. A. Ashley, K. D. Janda, D. L. Boger, R. A. Lerner, J. Am. Chem. Soc. 116, 6013 (1994).
27. H. Oikawa, K. Katayama, Y. Suzuki, A. Ichihara, J. Chem. Soc. Chem. Commun. 1995, 1321 (1995).
28. M. R. Haynes, E. A. Stura, D. Hilvert, I. A. Wilson, Science 263, 646 (1994); M. R. Haynes, A. Heine, I. A. Wilson, Isr. J. Chem. 36, 133 (1996).
29. M. R. Haynes, E. A. Stura, D. Hilvert, I. A. Wilson, Science 263, 646 (1994); M. R. Haynes, A. Heine, I. A. Wilson, Isr. J. Chem. 36, 133 (1996).
30. D. Hilvert, K. W. Hill, K. D. Nared, M.-T. M. Auditor, J. Am. Chem. Soc. 111, 9261 (1989); A. C. Braisted and P. G. Schultz, ibid. 112, 7430 (1990).
31. D. Hilvert, K. W. Hill, K. D. Nared, M.-T. M. Auditor, J. Am. Chem. Soc. 111, 9261 (1989); A. C. Braisted and P. G. Schultz, ibid. 112, 7430 (1990).
32. V. E. Gouverneur et al., Science 262, 204 (1993).
33. J. T. Yli-Kauhaluoma et al., J. Am. Chem. Soc. 117, 7041 (1995).
34. H1 domains and was calculated with the programs OVRLAP [M. G. Rossman and P. Argos, J. Biol. Chem. 250, 7525 (1975)] and ELBOW (D. H. Fremont and T. O. Yeates, personal communication). All molecular surface areas buried by interaction were calculated with the program MS [M. L. Connolly, J. Appl. Crystallogr. 16, 439 (1983)] with a 1.7 Å probe sphere and standard atomic radii [S. Sheriff, Immunomethods 3, 191 (1993)].
35. H1 domains and was calculated with the programs OVRLAP [M. G. Rossman and P. Argos, J. Biol. Chem. 250, 7525 (1975)] and ELBOW (D. H. Fremont and T. O. Yeates, personal communication). All molecular surface areas buried by interaction were calculated with the program MS [M. L. Connolly, J. Appl. Crystallogr. 16, 439 (1983)] with a 1.7 Å probe sphere and standard atomic radii [S. Sheriff, Immunomethods 3, 191 (1993)].
36. H1 domains and was calculated with the programs OVRLAP [M. G. Rossman and P. Argos, J. Biol. Chem. 250, 7525 (1975)] and ELBOW (D. H. Fremont and T. O. Yeates, personal communication). All molecular surface areas buried by interaction were calculated with the program MS [M. L. Connolly, J. Appl. Crystallogr. 16, 439 (1983)] with a 1.7 Å probe sphere and standard atomic radii [S. Sheriff, Immunomethods 3, 191 (1993)].
37. H1 domains and was calculated with the programs OVRLAP [M. G. Rossman and P. Argos, J. Biol. Chem. 250, 7525 (1975)] and ELBOW (D. H. Fremont and T. O. Yeates, personal communication). All molecular surface areas buried by interaction were calculated with the program MS [M. L. Connolly, J. Appl. Crystallogr. 16, 439 (1983)] with a 1.7 Å probe sphere and standard atomic radii [S. Sheriff, Immunomethods 3, 191 (1993)].
38. I. A. Wilson and R. L. Stanfield, Curr. Opin. Struct. Biol. 3, 113 (1993); ibid. 4, 857 (1994).
39. I. A. Wilson and R. L. Stanfield, Curr. Opin. Struct. Biol. 3, 113 (1993); ibid. 4, 857 (1994).
40. J. N. Herron, X. M. He, M. L. Mason, E. W. Voss, A. B. Edmundson, Proteins Struct. Funct. Genet. 5, 271 (1989).
41. E. A. Kabat, T. T. Wu, H. M. Perry, K. Gottesman, C. Foeller, Sequences of Proteins of Immunological Interest (Department of Health and Human Services, National Institutes of Health, Bethesda, MD, ed. 5, 1991).
42. The ferrocene derivative 9 was crystallized from acetone at 4°C in space group P1 with cell dimensions a = 5.99 Å, b = 12.41 Å, c = 17.29 Å, α = 90.81°, β = 93.6°, γ = 93.34°. The structure was solved with Direct Methods and SHELXS (27) and refined with SHELXL (27).
43. F. Takusagawa and T. F. Koetzle, Acta Crystallogr. Sect. B 35, 2888 (1979); G. J. Palenik, Inorg. Chem. 8, 2744 (1969).
44. F. Takusagawa and T. F. Koetzle, Acta Crystallogr. Sect. B 35, 2888 (1979); G. J. Palenik, Inorg. Chem. 8, 2744 (1969).
45. All ab initio and density functional theory (DFT) calculations were carried out with the Gaussian 94 program [M. J. Frisch et al., Gaussian 94 Revision D.2 (Gaussian, Inc., Pittsburgh, PA, 1995).
46. W. J. Hehre, L. Radom, P. v. R. Schleyer, J. A. Pople, Ab Initio Molecular Orbital Theory (Wiley-Interscience, New York, 1986), pp. 76-79.
47. A. D. Becke, J. Chem. Phys. 98, 5648 (1993); C. Lee, W. Yang, R. G. Parr, Phys. Rev. B 37, 785 (1988).
48. A. D. Becke, J. Chem. Phys. 98, 5648 (1993); C. Lee, W. Yang, R. G. Parr, Phys. Rev. B 37, 785 (1988).
49. Hybrid Hartree-Fock (HF)-DFT methods give excellent results for Diels-Alder reactions. See, for example, V. Barone and R. Arnaud, J. Chem. Phys. 106, 8727 (1997); B. R. Beno, K. N. Houk, D. A. Singleton, J. Am. Chem. Soc. 118, 9984 (1996); E. Goldstein, B. Beno, K. N. Houk, ibid., p. 6036, and references therein.
50. Hybrid Hartree-Fock (HF)-DFT methods give excellent results for Diels-Alder reactions. See, for example, V. Barone and R. Arnaud, J. Chem. Phys. 106, 8727 (1997); B. R. Beno, K. N. Houk, D. A. Singleton, J. Am. Chem. Soc. 118, 9984 (1996); E. Goldstein, B. Beno, K. N. Houk, ibid., p. 6036, and references therein.
51. Hybrid Hartree-Fock (HF)-DFT methods give excellent results for Diels-Alder reactions. See, for example, V. Barone and R. Arnaud, J. Chem. Phys. 106, 8727 (1997); B. R. Beno, K. N. Houk, D. A. Singleton, J. Am. Chem. Soc. 118, 9984 (1996); E. Goldstein, B. Beno, K. N. Houk, ibid., p. 6036, and references therein.
52. Each of the four transition states was docked with Fab 13G5 by using the program AUTODOCK [D. S. Goodsell and A. J. Olson, Proteins Struct. Funct. Genet. 8, 195 (1990); D. S. Goodsell, G. M. Morris, A. J. Olson, J. Mol. Recognit. 9, 1 (1996)]. This program uses simulated annealing to locate low-energy binding modes; the antibody structure is held fixed in the x-ray geometry. Similar results from the docking experiments were obtained with coordinates of both the unliganded antibody (28) and the inhibitor-bound antibody. Charges on the transition state atoms were obtained with AM1 semi-empirical calculations. The positions of the transition states were varied starting from 100 different, randomly chosen starting orientations in a 22.5 Å cube encompassing the antibody variable region. Each simulation involved 150 cycles of simulated annealing. Five flexible dihedral angles of the diene substituent in each transition state were allowed to vary. The energy of interaction was calculated with a potential function composed of van der Waals and Coulombic terms. As a check on this procedure, the ferrocene inhibitor 9 was docked into the antibody; AUTODOCK reproduces the binding position found experimentally to a root mean square (rms) of 0.6 Å.
53. Each of the four transition states was docked with Fab 13G5 by using the program AUTODOCK [D. S. Goodsell and A. J. Olson, Proteins Struct. Funct. Genet. 8, 195 (1990); D. S. Goodsell, G. M. Morris, A. J. Olson, J. Mol. Recognit. 9, 1 (1996)]. This program uses simulated annealing to locate low-energy binding modes; the antibody structure is held fixed in the x-ray geometry. Similar results from the docking experiments were obtained with coordinates of both the unliganded antibody (28) and the inhibitor-bound antibody. Charges on the transition state atoms were obtained with AM1 semi-empirical calculations. The positions of the transition states were varied starting from 100 different, randomly chosen starting orientations in a 22.5 Å cube encompassing the antibody variable region. Each simulation involved 150 cycles of simulated annealing. Five flexible dihedral angles of the diene substituent in each transition state were allowed to vary. The energy of interaction was calculated with a potential function composed of van der Waals and Coulombic terms. As a check on this procedure, the ferrocene inhibitor 9 was docked into the antibody; AUTODOCK reproduces the binding position found experimentally to a root mean square (rms) of 0.6 Å.
54. C. Cannizzaro and K. N. Houk, personal communication.
55. E. A. Stura, G. G. Fieser, I. A. Wilson, Immunomethods 3, 164 (1993).
56. The x-ray crystallographic programs and methods used were the following. (i) DENZO and SCALE-PACK: Z. Otwinowski, in Data Collection and Processing, L. Sawyer, N. Isaacs, S. Bailey, Eds. (SERC Daresbury Laboratory, Warrington, UK, 1993), p. 56; MERLOT: P. M. D. Fitzgerald, J. Appl. Crystallogr. 21, 273 (1988); AMORE: J. Navaza, Acta Crystallogr. A50, 157 (1994); O: T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991); X-PLOR: A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, X-PLOR, Version 3.1: A System for X-ray and NMR (Yale Univ. Press, New Haven, CT, 1992); SHELXL,SHELXS,SHELX-PRO: G. M. Sheldrick, SHELXL-96, Program for crystal structure refinement, University of Göttingen (1996); sigma-A: R. J. Read, Acta Crystallogr. A42, 140 (1986).
57. The x-ray crystallographic programs and methods used were the following. (i) DENZO and SCALE-PACK: Z. Otwinowski, in Data Collection and Processing, L. Sawyer, N. Isaacs, S. Bailey, Eds. (SERC Daresbury Laboratory, Warrington, UK, 1993), p. 56; (ii) MERLOT: P. M. D. Fitzgerald, J. Appl. Crystallogr. 21, 273 (1988); AMORE: J. Navaza, Acta Crystallogr. A50, 157 (1994); O: T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991); X-PLOR: A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, X-PLOR, Version 3.1: A System for X-ray and NMR (Yale Univ. Press, New Haven, CT, 1992); SHELXL,SHELXS,SHELX-PRO: G. M. Sheldrick, SHELXL-96, Program for crystal structure refinement, University of Göttingen (1996); sigma-A: R. J. Read, Acta Crystallogr. A42, 140 (1986).
58. The x-ray crystallographic programs and methods used were the following. (i) DENZO and SCALE-PACK: Z. Otwinowski, in Data Collection and Processing, L. Sawyer, N. Isaacs, S. Bailey, Eds. (SERC Daresbury Laboratory, Warrington, UK, 1993), p. 56; MERLOT: P. M. D. Fitzgerald, J. Appl. Crystallogr. 21, 273 (1988); (iii) AMORE: J. Navaza, Acta Crystallogr. A50, 157 (1994); O: T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991); X-PLOR: A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, X-PLOR, Version 3.1: A System for X-ray and NMR (Yale Univ. Press, New Haven, CT, 1992); SHELXL,SHELXS,SHELX-PRO: G. M. Sheldrick, SHELXL-96, Program for crystal structure refinement, University of Göttingen (1996); sigma-A: R. J. Read, Acta Crystallogr. A42, 140 (1986).
59. The x-ray crystallographic programs and methods used were the following. (i) DENZO and SCALE-PACK: Z. Otwinowski, in Data Collection and Processing, L. Sawyer, N. Isaacs, S. Bailey, Eds. (SERC Daresbury Laboratory, Warrington, UK, 1993), p. 56; MERLOT: P. M. D. Fitzgerald, J. Appl. Crystallogr. 21, 273 (1988); AMORE: J. Navaza, Acta Crystallogr. A50, 157 (1994); (iv) O: T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991); X-PLOR: A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, X-PLOR, Version 3.1: A System for X-ray and NMR (Yale Univ. Press, New Haven, CT, 1992); SHELXL,SHELXS,SHELX-PRO: G. M. Sheldrick, SHELXL-96, Program for crystal structure refinement, University of Göttingen (1996); sigma-A: R. J. Read, Acta Crystallogr. A42, 140 (1986).
60. The x-ray crystallographic programs and methods used were the following. (i) DENZO and SCALE-PACK: Z. Otwinowski, in Data Collection and Processing, L. Sawyer, N. Isaacs, S. Bailey, Eds. (SERC Daresbury Laboratory, Warrington, UK, 1993), p. 56; MERLOT: P. M. D. Fitzgerald, J. Appl. Crystallogr. 21, 273 (1988); AMORE: J. Navaza, Acta Crystallogr. A50, 157 (1994); O: T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991); (v) X-PLOR: A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, X-PLOR, Version 3.1: A System for X-ray and NMR (Yale Univ. Press, New Haven, CT, 1992); SHELXL,SHELXS,SHELX-PRO: G. M. Sheldrick, SHELXL-96, Program for crystal structure refinement, University of Göttingen (1996); sigma-A: R. J. Read, Acta Crystallogr. A42, 140 (1986).
61. The x-ray crystallographic programs and methods used were the following. (i) DENZO and SCALE-PACK: Z. Otwinowski, in Data Collection and Processing, L. Sawyer, N. Isaacs, S. Bailey, Eds. (SERC Daresbury Laboratory, Warrington, UK, 1993), p. 56; MERLOT: P. M. D. Fitzgerald, J. Appl. Crystallogr. 21, 273 (1988); AMORE: J. Navaza, Acta Crystallogr. A50, 157 (1994); O: T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991); X-PLOR: A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, X-PLOR, Version 3.1: A System for X-ray and NMR (Yale Univ. Press, New Haven, CT, 1992); SHELXL,SHELXS,SHELX-PRO: G. M. Sheldrick, SHELXL-96, Program for crystal structure refinement, University of Göttingen (1996); sigma-A: R. J. Read, Acta Crystallogr. A42, 140 (1986).
62. The x-ray crystallographic programs and methods used were the following. (i) DENZO and SCALE-PACK: Z. Otwinowski, in Data Collection and Processing, L. Sawyer, N. Isaacs, S. Bailey, Eds. (SERC Daresbury Laboratory, Warrington, UK, 1993), p. 56; MERLOT: P. M. D. Fitzgerald, J. Appl. Crystallogr. 21, 273 (1988); AMORE: J. Navaza, Acta Crystallogr. A50, 157 (1994); O: T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991); X-PLOR: A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, X-PLOR, Version 3.1: A System for X-ray and NMR (Yale Univ. Press, New Haven, CT, 1992); (vi) SHELXL,SHELXS,SHELX-PRO: G. M. Sheldrick, SHELXL-96, Program for crystal structure refinement, University of Göttingen (1996); sigma-A: R. J. Read, Acta Crystallogr. A42, 140 (1986).
63. The x-ray crystallographic programs and methods used were the following. (i) DENZO and SCALE-PACK: Z. Otwinowski, in Data Collection and Processing, L. Sawyer, N. Isaacs, S. Bailey, Eds. (SERC Daresbury Laboratory, Warrington, UK, 1993), p. 56; MERLOT: P. M. D. Fitzgerald, J. Appl. Crystallogr. 21, 273 (1988); AMORE: J. Navaza, Acta Crystallogr. A50, 157 (1994); O: T. A. Jones, J.-Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991); X-PLOR: A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, X-PLOR, Version 3.1: A System for X-ray and NMR (Yale Univ. Press, New Haven, CT, 1992); SHELXL,SHELXS,SHELX-PRO: G. M. Sheldrick, SHELXL-96, Program for crystal structure refinement, University of Göttingen (1996); (vii) sigma-A: R. J. Read, Acta Crystallogr. A42, 140 (1986).
64. A. Heine, E. A. Stura, K. D. Janda, I. A. Wilson, unpublished data.
65. R. L. Stanfield, T. M. Fieser, R. A. Lerner, I. A. Wilson, Science 248, 712 (1990).
66. J. H. Arevalo, E. A. Stura, M. J. Taussig, I. A. Wilson, J. Mol. Biol. 231, 103 (1993).
67. The graphics programs used were the following. (i) AVS: C. Upson et al., IEEE Comput. Graph. Appl. 9, 30 (1989); XP: SHELXTL 5.03, Siemens Industrial Automation, Inc., Madison, WI, 1990-1995; MOLMOL: R. Koradj, M. Billeter, K. Wüthrich, J. Mol. Graph. 14, 51 (1996).
68. The graphics programs used were the following. (i) AVS: C. Upson et al., IEEE Comput. Graph. Appl. 9, 30 (1989); (ii) XP: SHELXTL 5.03, Siemens Industrial Automation, Inc., Madison, WI, 1990-1995; MOLMOL: R. Koradj, M. Billeter, K. Wüthrich, J. Mol. Graph. 14, 51 (1996).
69. The graphics programs used were the following. (i) AVS: C. Upson et al., IEEE Comput. Graph. Appl. 9, 30 (1989); XP: SHELXTL 5.03, Siemens Industrial Automation, Inc., Madison, WI, 1990-1995; MOLMOL: R. Koradj, M. Billeter, K. Wüthrich, J. Mol. Graph. 14, 51 (1996).
70. G. D. Purvis and C. Culberson, J. Mol. Graph. 4 (1986).
71. We thank M. Pique for production of figures, the Stanford Synchrotron Radiation Laboratory for data collection time, and the staff of beamline 7-1 for their assistance; G. Fieser and R. Stefanko for technical assistance, C.-H. Lo for Fab sequence determination, R. Stanfield and K. Gruber for discussions and comments, K. Renner for computational assistance, D. McRee for map generation in XFIT, R. Lerner for support and encouragement, and R. Chadha for data collection of 9. Supported by NIH grants PO1 CA27489 (I.A.W. and K.D.J.) and GM-43858 (K.D.J.), NSF grant CHE9120588 (K.N.H.), and a fellowship by TEKES, Technology Development Centre, The Finnish Centre for International Mobility and Exchange Programmes, Helsinki, Finland (J.Y-K). This is publication 11111-MB from The Scripps Research Institute. The coordinates have been deposited in the Brookhaven databank with access code 1a3l and are available immediately from, wilson@scripps.edu or aheine@scripps.edu.