Structure-based analysis of catalysis and substrate definition in the HIT protein family

Science

Volumn 278, Issue 5336, 1997, Pages 286-290

  Lima, Christopher D ^a   Klein, Michael G ^b   Hendrickson, Wayne A ^a  

^a Columbia University ^*  (United States)

^b Clinical Nutrition   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

HISTIDINE; HYDROLASE; PROTEIN; PROTEIN KINASE C; TRANSFERASE;

ANALYTIC METHOD; ARTICLE; CATALYSIS; CRYSTALLOGRAPHY; ENZYME MECHANISM; ENZYME STRUCTURE; ENZYME SUBSTRATE; HUMAN; PRIORITY JOURNAL; PROTEIN ANALYSIS; PROTEIN FAMILY;

ACID ANHYDRIDE HYDROLASES; ADENOSINE; ADENOSINE DIPHOSPHATE; ADENOSINE MONOPHOSPHATE; ADENOSINE TRIPHOSPHATE; BINDING SITES; CATALYSIS; CRYSTALLOGRAPHY, X-RAY; DIMERIZATION; DINUCLEOSIDE PHOSPHATES; HYDROGEN BONDING; NEOPLASM PROTEINS; NERVE TISSUE PROTEINS; PROTEIN STRUCTURE, SECONDARY; PROTEINS; STRUCTURE-ACTIVITY RELATIONSHIP; SUBSTRATE SPECIFICITY; TUNGSTEN COMPOUNDS;

EID: 0030831990     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.278.5336.286     Document Type: Article

References (37)

1. B. Seraphin, J. DNA Seq. 3, 177 (1992); K. Robinson and A. Aitken, Biochem. J. 304, 662 (1994).
2. B. Seraphin, J. DNA Seq. 3, 177 (1992); K. Robinson and A. Aitken, Biochem. J. 304, 662 (1994).
3. C. D. Lima et al., Structure 5, 763 (1997).
4. C. D. Lima, M. G. Klein, I. B. Weinstein, W. A. Hendrickson, Proc. Natl. Acad. Sci. U.S.A. 93, 5355 (1996).
5. M. Ohta et al., Cell 84, 587 (1996). References to subsequent studies of the FHIT locus as related to human cancer can be found in (2).
6. C. Brenner et al., Nature Struct. Biol. 4, 231 (1997).
7. A. K. Robinson, C. E. Pena, L. D. Barnes, Biochim. Biophys. Acta 1161, 139 (1993); L. D. Barnes et al., Biochemistry 36, 11529 (1996).
8. A. K. Robinson, C. E. Pena, L. D. Barnes, Biochim. Biophys. Acta 1161, 139 (1993); L. D. Barnes et al., Biochemistry 36, 11529 (1996).
9. A systematic approach was used to identify adenosine as the nucleoside with highest affinity for the binding site by soaking pairs of various nucleoside molecules into the crystal in equal concentrations (∼0.1 to 1.0 mM), collecting data, and observing density in the binding site. Adenosine nucleosides were always found to bind to PKCI with higher affinity than any other nucleoside.
10. 5A hydrolysis for FHIT.
11. Y. F. Wei and H. R. Matthews, Methods Enzymol. 200, 388 (1991); S. J. Pilkis et al., J. Biol. Chem. 258, 6135 (1983); C. A. Hasemann, E. S. Istvan, K. Uyeda, J. Deisenhofer, Structure 4, 1017 (1996); Z. B. Rose, Methods Enzymol. 87, 42 (1982); S. H. Thrall, A. F. Mehl, L. J. Carroll, D. Dunaway-Marino, Biochemistry 32, 1803 (1983); S. Morera, M. Chiadmi, G. LeBras, I. Lascu, J. Janin, ibid. 34, 11062 (1995); J. E. Wedekind, P. A. Frey, I. Rayment, ibid. 35, 11560 (1996).
12. Y. F. Wei and H. R. Matthews, Methods Enzymol. 200, 388 (1991); S. J. Pilkis et al., J. Biol. Chem. 258, 6135 (1983); C. A. Hasemann, E. S. Istvan, K. Uyeda, J. Deisenhofer, Structure 4, 1017 (1996); Z. B. Rose, Methods Enzymol. 87, 42 (1982); S. H. Thrall, A. F. Mehl, L. J. Carroll, D. Dunaway-Marino, Biochemistry 32, 1803 (1983); S. Morera, M. Chiadmi, G. LeBras, I. Lascu, J. Janin, ibid. 34, 11062 (1995); J. E. Wedekind, P. A. Frey, I. Rayment, ibid. 35, 11560 (1996).
13. Y. F. Wei and H. R. Matthews, Methods Enzymol. 200, 388 (1991); S. J. Pilkis et al., J. Biol. Chem. 258, 6135 (1983); C. A. Hasemann, E. S. Istvan, K. Uyeda, J. Deisenhofer, Structure 4, 1017 (1996); Z. B. Rose, Methods Enzymol. 87, 42 (1982); S. H. Thrall, A. F. Mehl, L. J. Carroll, D. Dunaway-Marino, Biochemistry 32, 1803 (1983); S. Morera, M. Chiadmi, G. LeBras, I. Lascu, J. Janin, ibid. 34, 11062 (1995); J. E. Wedekind, P. A. Frey, I. Rayment, ibid. 35, 11560 (1996).
14. Y. F. Wei and H. R. Matthews, Methods Enzymol. 200, 388 (1991); S. J. Pilkis et al., J. Biol. Chem. 258, 6135 (1983); C. A. Hasemann, E. S. Istvan, K. Uyeda, J. Deisenhofer, Structure 4, 1017 (1996); Z. B. Rose, Methods Enzymol. 87, 42 (1982); S. H. Thrall, A. F. Mehl, L. J. Carroll, D. Dunaway-Marino, Biochemistry 32, 1803 (1983); S. Morera, M. Chiadmi, G. LeBras, I. Lascu, J. Janin, ibid. 34, 11062 (1995); J. E. Wedekind, P. A. Frey, I. Rayment, ibid. 35, 11560 (1996).
15. Y. F. Wei and H. R. Matthews, Methods Enzymol. 200, 388 (1991); S. J. Pilkis et al., J. Biol. Chem. 258, 6135 (1983); C. A. Hasemann, E. S. Istvan, K. Uyeda, J. Deisenhofer, Structure 4, 1017 (1996); Z. B. Rose, Methods Enzymol. 87, 42 (1982); S. H. Thrall, A. F. Mehl, L. J. Carroll, D. Dunaway-Marino, Biochemistry 32, 1803 (1983); S. Morera, M. Chiadmi, G. LeBras, I. Lascu, J. Janin, ibid. 34, 11062 (1995); J. E. Wedekind, P. A. Frey, I. Rayment, ibid. 35, 11560 (1996).
16. Y. F. Wei and H. R. Matthews, Methods Enzymol. 200, 388 (1991); S. J. Pilkis et al., J. Biol. Chem. 258, 6135 (1983); C. A. Hasemann, E. S. Istvan, K. Uyeda, J. Deisenhofer, Structure 4, 1017 (1996); Z. B. Rose, Methods Enzymol. 87, 42 (1982); S. H. Thrall, A. F. Mehl, L. J. Carroll, D. Dunaway-Marino, Biochemistry 32, 1803 (1983); S. Morera, M. Chiadmi, G. LeBras, I. Lascu, J. Janin, ibid. 34, 11062 (1995); J. E. Wedekind, P. A. Frey, I. Rayment, ibid. 35, 11560 (1996).
17. Y. F. Wei and H. R. Matthews, Methods Enzymol. 200, 388 (1991); S. J. Pilkis et al., J. Biol. Chem. 258, 6135 (1983); C. A. Hasemann, E. S. Istvan, K. Uyeda, J. Deisenhofer, Structure 4, 1017 (1996); Z. B. Rose, Methods Enzymol. 87, 42 (1982); S. H. Thrall, A. F. Mehl, L. J. Carroll, D. Dunaway-Marino, Biochemistry 32, 1803 (1983); S. Morera, M. Chiadmi, G. LeBras, I. Lascu, J. Janin, ibid. 34, 11062 (1995); J. E. Wedekind, P. A. Frey, I. Rayment, ibid. 35, 11560 (1996).
18. 3H). A similar approach was used to trap covalent phosphotyrosine intermediates in topoisomerase reactions [Y.-C. Tse-Dinh, K. Kirkegaard, J. Wang, J. Biol. Chem. 255, 5560 (1980)]. Radioactive labeling was coincident with the position of FHIT in SDS-PAGE.
19. 12) and was solved using a partial model (3) in molecular replacement with AMORE [J. Navaza, Acta Crystallogr. A50, 157 (1994)] and modeled with the program O [T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991)]. Isomorphous, related space groups were solved by a similar approach in AMORE. All models were refined with X-Plor using the cross-validation test [A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, Nature 355, 472 (1992)]. Each FHIT model roughly includes residues 2 to 108 and 125 to 147. Each PKCI model roughly includes residues 14 to 126. Occupancies for the a and β tung-state molecules refined to 0.50 and 0.54, respectively, for PKCI and to 0.47 for FHIT.
20. 12) and was solved using a partial model (3) in molecular replacement with AMORE [J. Navaza, Acta Crystallogr. A50, 157 (1994)] and modeled with the program O [T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991)]. Isomorphous, related space groups were solved by a similar approach in AMORE. All models were refined with X-Plor using the cross-validation test [A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, Nature 355, 472 (1992)]. Each FHIT model roughly includes residues 2 to 108 and 125 to 147. Each PKCI model roughly includes residues 14 to 126. Occupancies for the a and β tung-state molecules refined to 0.50 and 0.54, respectively, for PKCI and to 0.47 for FHIT.
21. 12) and was solved using a partial model (3) in molecular replacement with AMORE [J. Navaza, Acta Crystallogr. A50, 157 (1994)] and modeled with the program O [T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991)]. Isomorphous, related space groups were solved by a similar approach in AMORE. All models were refined with X-Plor using the cross-validation test [A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, Nature 355, 472 (1992)]. Each FHIT model roughly includes residues 2 to 108 and 125 to 147. Each PKCI model roughly includes residues 14 to 126. Occupancies for the a and β tung-state molecules refined to 0.50 and 0.54, respectively, for PKCI and to 0.47 for FHIT.
22. 12) and was solved using a partial model (3) in molecular replacement with AMORE [J. Navaza, Acta Crystallogr. A50, 157 (1994)] and modeled with the program O [T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991)]. Isomorphous, related space groups were solved by a similar approach in AMORE. All models were refined with X-Plor using the cross-validation test [A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, Nature 355, 472 (1992)]. Each FHIT model roughly includes residues 2 to 108 and 125 to 147. Each PKCI model roughly includes residues 14 to 126. Occupancies for the a and β tung-state molecules refined to 0.50 and 0.54, respectively, for PKCI and to 0.47 for FHIT.
23. 12) and was solved using a partial model (3) in molecular replacement with AMORE [J. Navaza, Acta Crystallogr. A50, 157 (1994)] and modeled with the program O [T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991)]. Isomorphous, related space groups were solved by a similar approach in AMORE. All models were refined with X-Plor using the cross-validation test [A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, Nature 355, 472 (1992)]. Each FHIT model roughly includes residues 2 to 108 and 125 to 147. Each PKCI model roughly includes residues 14 to 126. Occupancies for the a and β tung-state molecules refined to 0.50 and 0.54, respectively, for PKCI and to 0.47 for FHIT.
24. 12) and was solved using a partial model (3) in molecular replacement with AMORE [J. Navaza, Acta Crystallogr. A50, 157 (1994)] and modeled with the program O [T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991)]. Isomorphous, related space groups were solved by a similar approach in AMORE. All models were refined with X-Plor using the cross-validation test [A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, Nature 355, 472 (1992)]. Each FHIT model roughly includes residues 2 to 108 and 125 to 147. Each PKCI model roughly includes residues 14 to 126. Occupancies for the a and β tung-state molecules refined to 0.50 and 0.54, respectively, for PKCI and to 0.47 for FHIT.
25. 12) and was solved using a partial model (3) in molecular replacement with AMORE [J. Navaza, Acta Crystallogr. A50, 157 (1994)] and modeled with the program O [T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991)]. Isomorphous, related space groups were solved by a similar approach in AMORE. All models were refined with X-Plor using the cross-validation test [A. T. Brünger, J. Kuriyan, M. Karplus, Science 235, 458 (1987); A. T. Brünger, Nature 355, 472 (1992)]. Each FHIT model roughly includes residues 2 to 108 and 125 to 147. Each PKCI model roughly includes residues 14 to 126. Occupancies for the a and β tung-state molecules refined to 0.50 and 0.54, respectively, for PKCI and to 0.47 for FHIT.
26. Vanadate and molybdate pentacovalent metal sites were identified in structures of chloroperoxidase, rat acid phosphatase, bovine low - molecular weight phosphotyrosyl phosphatase, ribonuclease A, and a vanadate-ADP transition-state complex of S1 myosin [B. Borah et al., Biochemistry 24, 2058 (1985); Y. Lindqvist, G. Schneider, P. Vihko, Eur. J. Biochem. 221, 139 (1994); A. Messerschmidt and R. Wever, Proc. Natl. Acad. Sci. U.S.A. 93, 392 (1996); C. A. Smith and I. Rayment, Biochemistry 35, 5404 (1996); A. Wlodawer, M. Miller, L. Sjolin, Proc. Natl. Acad. Sci. U.S.A. 80, 3628 (1983); M. Zhang, M. Zhou, R. L. Van Etten, C. V. Stauffacher, Biochemistry 36, 15 (1997)]. The active sites of PKCI and FHIT share several structural similarities and characteristics with protein phosphatases, particularly rat acid phosphatase. A search of the small-molecule database revealed a pentacovalent tungstate structure with similar characteristics to those observed in our enzyme complex [I. Feinstein-Jaffe, J. C. Dewan, R. R. Schrock, Organometallics 4, 1189 (1985)]. The bond lengths and angles observed in our crystal structures are in agreement with those observed in several other tungsten-containing molecules found in the database. We know of no reported protein structure that describes a similar pentacovalent tungstate complex.
27. Vanadate and molybdate pentacovalent metal sites were identified in structures of chloroperoxidase, rat acid phosphatase, bovine low - molecular weight phosphotyrosyl phosphatase, ribonuclease A, and a vanadate-ADP transition-state complex of S1 myosin [B. Borah et al., Biochemistry 24, 2058 (1985); Y. Lindqvist, G. Schneider, P. Vihko, Eur. J. Biochem. 221, 139 (1994); A. Messerschmidt and R. Wever, Proc. Natl. Acad. Sci. U.S.A. 93, 392 (1996); C. A. Smith and I. Rayment, Biochemistry 35, 5404 (1996); A. Wlodawer, M. Miller, L. Sjolin, Proc. Natl. Acad. Sci. U.S.A. 80, 3628 (1983); M. Zhang, M. Zhou, R. L. Van Etten, C. V. Stauffacher, Biochemistry 36, 15 (1997)]. The active sites of PKCI and FHIT share several structural similarities and characteristics with protein phosphatases, particularly rat acid phosphatase. A search of the small-molecule database revealed a pentacovalent tungstate structure with similar characteristics to those observed in our enzyme complex [I. Feinstein-Jaffe, J. C. Dewan, R. R. Schrock, Organometallics 4, 1189 (1985)]. The bond lengths and angles observed in our crystal structures are in agreement with those observed in several other tungsten-containing molecules found in the database. We know of no reported protein structure that describes a similar pentacovalent tungstate complex.
28. Vanadate and molybdate pentacovalent metal sites were identified in structures of chloroperoxidase, rat acid phosphatase, bovine low - molecular weight phosphotyrosyl phosphatase, ribonuclease A, and a vanadate-ADP transition-state complex of S1 myosin [B. Borah et al., Biochemistry 24, 2058 (1985); Y. Lindqvist, G. Schneider, P. Vihko, Eur. J. Biochem. 221, 139 (1994); A. Messerschmidt and R. Wever, Proc. Natl. Acad. Sci. U.S.A. 93, 392 (1996); C. A. Smith and I. Rayment, Biochemistry 35, 5404 (1996); A. Wlodawer, M. Miller, L. Sjolin, Proc. Natl. Acad. Sci. U.S.A. 80, 3628 (1983); M. Zhang, M. Zhou, R. L. Van Etten, C. V. Stauffacher, Biochemistry 36, 15 (1997)]. The active sites of PKCI and FHIT share several structural similarities and characteristics with protein phosphatases, particularly rat acid phosphatase. A search of the small-molecule database revealed a pentacovalent tungstate structure with similar characteristics to those observed in our enzyme complex [I. Feinstein-Jaffe, J. C. Dewan, R. R. Schrock, Organometallics 4, 1189 (1985)]. The bond lengths and angles observed in our crystal structures are in agreement with those observed in several other tungsten-containing molecules found in the database. We know of no reported protein structure that describes a similar pentacovalent tungstate complex.
29. Vanadate and molybdate pentacovalent metal sites were identified in structures of chloroperoxidase, rat acid phosphatase, bovine low - molecular weight phosphotyrosyl phosphatase, ribonuclease A, and a vanadate-ADP transition-state complex of S1 myosin [B. Borah et al., Biochemistry 24, 2058 (1985); Y. Lindqvist, G. Schneider, P. Vihko, Eur. J. Biochem. 221, 139 (1994); A. Messerschmidt and R. Wever, Proc. Natl. Acad. Sci. U.S.A. 93, 392 (1996); C. A. Smith and I. Rayment, Biochemistry 35, 5404 (1996); A. Wlodawer, M. Miller, L. Sjolin, Proc. Natl. Acad. Sci. U.S.A. 80, 3628 (1983); M. Zhang, M. Zhou, R. L. Van Etten, C. V. Stauffacher, Biochemistry 36, 15 (1997)]. The active sites of PKCI and FHIT share several structural similarities and characteristics with protein phosphatases, particularly rat acid phosphatase. A search of the small-molecule database revealed a pentacovalent tungstate structure with similar characteristics to those observed in our enzyme complex [I. Feinstein-Jaffe, J. C. Dewan, R. R. Schrock, Organometallics 4, 1189 (1985)]. The bond lengths and angles observed in our crystal structures are in agreement with those observed in several other tungsten-containing molecules found in the database. We know of no reported protein structure that describes a similar pentacovalent tungstate complex.
30. Vanadate and molybdate pentacovalent metal sites were identified in structures of chloroperoxidase, rat acid phosphatase, bovine low - molecular weight phosphotyrosyl phosphatase, ribonuclease A, and a vanadate-ADP transition-state complex of S1 myosin [B. Borah et al., Biochemistry 24, 2058 (1985); Y. Lindqvist, G. Schneider, P. Vihko, Eur. J. Biochem. 221, 139 (1994); A. Messerschmidt and R. Wever, Proc. Natl. Acad. Sci. U.S.A. 93, 392 (1996); C. A. Smith and I. Rayment, Biochemistry 35, 5404 (1996); A. Wlodawer, M. Miller, L. Sjolin, Proc. Natl. Acad. Sci. U.S.A. 80, 3628 (1983); M. Zhang, M. Zhou, R. L. Van Etten, C. V. Stauffacher, Biochemistry 36, 15 (1997)]. The active sites of PKCI and FHIT share several structural similarities and characteristics with protein phosphatases, particularly rat acid phosphatase. A search of the small-molecule database revealed a pentacovalent tungstate structure with similar characteristics to those observed in our enzyme complex [I. Feinstein-Jaffe, J. C. Dewan, R. R. Schrock, Organometallics 4, 1189 (1985)]. The bond lengths and angles observed in our crystal structures are in agreement with those observed in several other tungsten-containing molecules found in the database. We know of no reported protein structure that describes a similar pentacovalent tungstate complex.
31. Vanadate and molybdate pentacovalent metal sites were identified in structures of chloroperoxidase, rat acid phosphatase, bovine low - molecular weight phosphotyrosyl phosphatase, ribonuclease A, and a vanadate-ADP transition-state complex of S1 myosin [B. Borah et al., Biochemistry 24, 2058 (1985); Y. Lindqvist, G. Schneider, P. Vihko, Eur. J. Biochem. 221, 139 (1994); A. Messerschmidt and R. Wever, Proc. Natl. Acad. Sci. U.S.A. 93, 392 (1996); C. A. Smith and I. Rayment, Biochemistry 35, 5404 (1996); A. Wlodawer, M. Miller, L. Sjolin, Proc. Natl. Acad. Sci. U.S.A. 80, 3628 (1983); M. Zhang, M. Zhou, R. L. Van Etten, C. V. Stauffacher, Biochemistry 36, 15 (1997)]. The active sites of PKCI and FHIT share several structural similarities and characteristics with protein phosphatases, particularly rat acid phosphatase. A search of the small-molecule database revealed a pentacovalent tungstate structure with similar characteristics to those observed in our enzyme complex [I. Feinstein-Jaffe, J. C. Dewan, R. R. Schrock, Organometallics 4, 1189 (1985)]. The bond lengths and angles observed in our crystal structures are in agreement with those observed in several other tungsten-containing molecules found in the database. We know of no reported protein structure that describes a similar pentacovalent tungstate complex.
32. Vanadate and molybdate pentacovalent metal sites were identified in structures of chloroperoxidase, rat acid phosphatase, bovine low - molecular weight phosphotyrosyl phosphatase, ribonuclease A, and a vanadate-ADP transition-state complex of S1 myosin [B. Borah et al., Biochemistry 24, 2058 (1985); Y. Lindqvist, G. Schneider, P. Vihko, Eur. J. Biochem. 221, 139 (1994); A. Messerschmidt and R. Wever, Proc. Natl. Acad. Sci. U.S.A. 93, 392 (1996); C. A. Smith and I. Rayment, Biochemistry 35, 5404 (1996); A. Wlodawer, M. Miller, L. Sjolin, Proc. Natl. Acad. Sci. U.S.A. 80, 3628 (1983); M. Zhang, M. Zhou, R. L. Van Etten, C. V. Stauffacher, Biochemistry 36, 15 (1997)]. The active sites of PKCI and FHIT share several structural similarities and characteristics with protein phosphatases, particularly rat acid phosphatase. A search of the small-molecule database revealed a pentacovalent tungstate structure with similar characteristics to those observed in our enzyme complex [I. Feinstein-Jaffe, J. C. Dewan, R. R. Schrock, Organometallics 4, 1189 (1985)]. The bond lengths and angles observed in our crystal structures are in agreement with those observed in several other tungsten-containing molecules found in the database. We know of no reported protein structure that describes a similar pentacovalent tungstate complex.
33. L. Holm and C. Sander, Structure 15, 165 (1997); Trends Biochem. Sci. 22, 116 (1997).
34. L. Holm and C. Sander, Structure 15, 165 (1997); Trends Biochem. Sci. 22, 116 (1997).
35. J. E. Wedekind, P. A. Frey, I. Rayment, Biochemistry 34, 11049 (1995).
36. S. V. Evans, J. Mol. Graphics 11, 134 (1993).
37. We thank the staffs of beamline X4A at the National Synchrotron Light Source (NSLS) and beamline 19-ID at the APS. We particularly thank members of the Hendrickson lab for helpful discussion, and especially C. Bingman for discussions about catalysis. We also thank the Pyle, McDermott, and Parkin labs for helpful discussions and for use of resources during the analysis of the catalytic intermediate. Beamline X4A at the NSLS, a U.S. Department of Energy facility, is supported by the Howard Hughes Medical Institute. Use of the APS was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Energy Research. The SBC is supported by the U.S. Department of Energy, Office of Health and Environmental Research, Office of Energy Research. Both the SBC and APS are supported under contract W-31-109-ENG-38. This work was supported in part by National Cancer Institute training grant T32CA09503 (M.G.K.) and by a Helen Hay Whitney Foundation Fellowship to C.D.L.