Crystal Structure of the Catalytic Domains of Adenylyl Cyclase in a Complex with G sα ·GTPγS
10.1126/science.278.5345.1907
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

The crystal structure of a soluble, catalytically active form of adenylyl cyclase in a complex with its stimulatory heterotrimeric G protein α subunit (G s α ) and forskolin was determined to a resolution of 2.3 angstroms. When P-site inhibitors were soaked into native crystals of the complex, the active site of adenylyl cyclase was located and structural elements important for substrate recognition and catalysis were identified. On the basis of these and other structures, a molecular mechanism is proposed for the activation of adenylyl cyclase by G s α .

Bibliography

Tesmer, J. J. G., Sunahara, R. K., Gilman, A. G., & Sprang, S. R. (1997). Crystal Structure of the Catalytic Domains of Adenylyl Cyclase in a Complex with G sα ·GTPγS. Science, 278(5345), 1907–1916.

Authors 4
  1. John J. G. Tesmer (first)
  2. Roger K. Sunahara (additional)
  3. Alfred G. Gilman (additional)
  4. Stephen R. Sprang (additional)
References 56 Referenced 674
  1. 10.1146/annurev.pa.36.040196.002333
  2. Iyengar R., FASEB J. 7, 768 (1993). (10.1096/fasebj.7.9.8330684) / FASEB J. by Iyengar R. (1993)
  3. Xia Z., Storm D. R., Curr. Opin. Neurobiol. 7, 391 (1997). (10.1016/S0959-4388(97)80068-2) / Curr. Opin. Neurobiol. by Xia Z. (1997)
  4. Krupinski J., Coussen F., Bakalyar H. A., Tang W.-J., Feinstein P. G., Science 244, 1558 (1989). (10.1126/science.2472670) / Science by Krupinski J. (1989)
  5. Chinkers M., Garbers D. L., Annu. Rev. Biochem. 60, 553 (1991). (10.1146/annurev.bi.60.070191.003005) / Annu. Rev. Biochem. by Chinkers M. (1991)
  6. Dessauer C. W., Gilman A. G., J. Biol. Chem. 271, 16967 (1996). (10.1074/jbc.271.28.16967) / J. Biol. Chem. by Dessauer C. W. (1996)
  7. 10.1126/science.7792604
  8. Whisnant R. E., Gilman A. G., Dessauer C. W., Proc. Natl. Acad. Sci. U.S.A. 93, 6621 (1996). (10.1073/pnas.93.13.6621) / Proc. Natl. Acad. Sci. U.S.A. by Whisnant R. E. (1996)
  9. Yan S.-Z., Hahn D., Huang Z.-H., Tang W.-J., J. Biol. Chem. 271, 10941 (1996). (10.1074/jbc.271.18.10941) / J. Biol. Chem. by Yan S.-Z. (1996)
  10. Tang W.-J., Stanzel M., Gilman A. G., Biochemistry 34, 14563 (1995). (10.1021/bi00044a035) / Biochemistry by Tang W.-J. (1995)
  11. Sunahara R. K., Dessauer C. W., Whisnant R. E., Kleuss C., Gilman A. G., J. Biol. Chem. 272, 22265 (1997). (10.1074/jbc.272.35.22265) / J. Biol. Chem. by Sunahara R. K. (1997)
  12. C. W. Dessauer T. T. Scully A. G. Gilman ibid. p. 22272.
  13. Desaubry L., Shoshani I., Johnson R. A., ibid. 271, 2380 (1996). / ibid. by Desaubry L. (1996)
  14. Zhang G., Liu Y., Ruoho A. E., Hurley J. H., Nature 386, 247 (1997). (10.1038/386247a0) / Nature by Zhang G. (1997)
  15. Sunahara R. K., Tesmer J. J. G., Gilman A. G., Sprang S. R., Science 278, 1943 (1997). (10.1126/science.278.5345.1943) / Science by Sunahara R. K. (1997)
  16. The VC 1 ·IIC 2 ·G s α ·GTPγS complex was purified by gel filtration as described (11) in the presence of forskolin. The running buffer contained 20 mM sodium Hepes (pH 8.0) 2 mM MgCl 2 1 mM EDTA 2 mM dithiothreitol (DTT) 100 mM NaCl 25 μM forskolin and 10 μM GTPγS. After the appropriate fractions were pooled DTT MPFsk and GTPγS were added to final concentrations of 5 mM 200 μM and 500 μM respectively. The complex was concentrated to ∼8 mg ml –1 with an Amicon Centricon with a 50-kD cutoff and the protein was divided into samples and frozen in liquid nitrogen. Crystals of the complex were obtained by vapor diffusion at room temperature in which 4- to 6-μl drops containing concentrated protein mixed 1:1 with well solution were suspended over a 1-ml well containing 7.2 to 7.5% polyethylene glycol (PEG) 8K 500 mM NaCl and 100 mM MES (pH 5.4 to 5.6). Crystals appeared as stacks of thin plates within 2 days and grew to their maximum size over a period of 2 weeks. Single crystals of suitable size for data collection typically 200 μm by 100 μm by 10 μm were obtained by breaking up the stacks and then soaking them in a harvesting solution containing 100 mM MES (pH 5.4) 9% PEG 8K 500 mM NaCl 5 mM MgCl 2 20 mM sodium Hepes pH 8.0 1 mM EDTA 2 mM DTT 200 μM MPFsk and 100 μM GTPγS plus 30% PEG 400 as the cryoprotectant. For the P-site–inhibited complex crystals were soaked in a harvesting solution that also included either 3.5 mM 2′ d3′-AMP plus 3.5 mM pyrophosphate or 100 μM 2′ d3′-ATP plus 2 mM pyrophosphate for 2 hours. Other compounds that we attempted to soak into native crystals included 1.4 mM Ap(CH 2 )pp 7 mM ATP and 7 mM 2′-iodo-ATP. The diffraction amplitudes collected from these crystals were not significantly different from those of the native complex although the ATP-soaked crystals suffered a considerable increase in mosaicity and loss of high-resolution diffraction. The 2′-iodo-ATP data set was actually superior to that of the native data and thus is treated as “native.”
  17. Graziano M. P., Casey P. J., Gilman A. G., J. Biol. Chem. 262, 11375 (1987). (10.1016/S0021-9258(18)60970-6) / J. Biol. Chem. by Graziano M. P. (1987)
  18. The IIC 2 we used was initially derived by limited proteolysis of a longer COOH-terminal fragment of rat ACII (spanning residues 847 to 1090) by using the protease clostripain (Sigma). The resulting 23-kD fragment which retained full adenylyl cyclase activity when reconstituted with the C 1 domain from ACI was purified by Mono Q anion exchange chromatography. The NH 2 -terminal proteolysis site and length of this fragment were determined by NH 2 -terminal sequencing and mass spectroscopy. Subsequently a new expression vector (PQE60-ArgC-IIC 2 ) was created to produce the same C 2 fragment (IIC 2 ) without a hexahistidine tag. Recombinant IIC 2 was purified from the bacterial lysate by Q-Sepharose anion exchange followed by filtration through a hydroxyapatite column and then Mono Q and phenyl Sepharose fast protein liquid chromatography steps.
  19. Coleman D. E., et al., Science 265, 1405 (1994). (10.1126/science.8073283) / Science by Coleman D. E. (1994)
  20. 10.1038/355472a0
  21. 10.1126/science.7761832
  22. Chen Y., et al., Proc. Natl. Acad. Sci. U.S.A. 94, 2711 (1997). (10.1073/pnas.94.6.2711) / Proc. Natl. Acad. Sci. U.S.A. by Chen Y. (1997)
  23. 10.1126/science.3775366
  24. 10.1016/0022-2836(71)90324-X
  25. 10.1038/379311a0
  26. Lichtarge O., Bourne H., Cohen F., Proc. Natl. Acad. Sci. U.S.A. 93, 7507 (1996). (10.1073/pnas.93.15.7507) / Proc. Natl. Acad. Sci. U.S.A. by Lichtarge O. (1996)
  27. Yan S.-Z., Huang Z.-H., Rao V.-D., Hurley J. H., Tang W.-J., J. Biol. Chem. 272, 18849 (1997). (10.1074/jbc.272.30.18849) / J. Biol. Chem. by Yan S.-Z. (1997)
  28. Berlot C. H., Bourne H. R., Cell 68, 911 (1992). (10.1016/0092-8674(92)90034-A) / Cell by Berlot C. H. (1992)
  29. Itoh H., Gilman A. G., J. Biol. Chem. 266, 16226 (1991). (10.1016/S0021-9258(18)98539-X) / J. Biol. Chem. by Itoh H. (1991)
  30. Grishina G., Berlot C. H., ibid. 272, 20619 (1997). / ibid. by Grishina G. (1997)
  31. R. Taussig W.-J. Tang J. R. Hepler A. G. Gilman ibid. 269 6093 (1994). (10.1016/S0021-9258(17)37574-9)
  32. C. W. Dessauer and A. G. Gilman ibid. 272 27787 (1997). (10.1074/jbc.272.44.27787)
  33. S.-Z. Yan Z.-H. Huang R. S. Shaw W.-J. Tang ibid. p. 12342.
  34. F. Eckstein P. Romaniuk W. Heideman D. Storm ibid. 256 9118 (1981). (10.1016/S0021-9258(19)52516-9)
  35. J. Gerlt J. Coderre M. Wolin ibid. 255 331 (1980). (10.1016/S0021-9258(19)86171-9)
  36. K. Koch F. Eckstein L. Stryer ibid. 265 9659 (1990). (10.1016/S0021-9258(19)38720-4)
  37. P. Senter F. Eckstein A. Mulsch E. Bohme ibid. 258 6741 (1983). (10.1016/S0021-9258(18)32282-8)
  38. Zhang G., et al., Protein Sci. 6, 903 (1997). (10.1002/pro.5560060417) / Protein Sci. by Zhang G. (1997)
  39. Sondek J., Lambright D. G., Noel J. P., Hamm H. E., Sigler P. B., Nature 372, 276 (1994). (10.1038/372276a0) / Nature by Sondek J. (1994)
  40. Perona J., Rould M. A., Steitz T. A., Biochemistry 32, 8758 (1993). (10.1021/bi00085a006) / Biochemistry by Perona J. (1993)
  41. Schweins T., et al., Nature Struct. Biol. 2, 36 (1995). (10.1038/nsb0195-36) / Nature Struct. Biol. by Schweins T. (1995)
  42. Artymiuk P. J., Poirrette A. R., Rice D. W., Willett P., Nature 388, 33 (1997). (10.1038/40310) / Nature by Artymiuk P. J. (1997)
  43. Steitz T. A., Smerdon S. J., Jäger J., Joyce C. M., Science 266, 2022 (1994). (10.1126/science.7528445) / Science by Steitz T. A. (1994)
  44. Z. Otwinowski in Data Collection and Processing N. I. L. Sawyer and S. Bailey Eds. (Science and Engineering Research Council Daresbury Laboratory Daresbury UK 1993) pp. 56–62.
  45. 10.1107/S0108767393007597
  46. Bailey S., ibid. D50, 760 (1994). / ibid. by Bailey S. (1994)
  47. T. A. Jones J. Y. Zou S. W. Cowan M. Kjeldgaard ibid. A47 110 (1991). (10.1107/S0108767390010224)
  48. A. T. Brünger X-PLOR Version 3.1. A System for X-ray Crystallography and NMR (Yale Univ. Press New Haven and London 1992).
  49. 10.1107/S0021889892009944
  50. Single-letter abbreviations for the amino acid residues are as follows: A Ala; C Cys; D Asp; E Glu; F Phe; G Gly; H His; I Ile; K Lys; L Leu; M Met; N Asn; P Pro; Q Gln; R Arg; S Ser; T Thr; V Val; W Trp; and Y Tyr.
  51. Esnouf R. M., J. Mol. Graphics 15, 133 (1997). / J. Mol. Graphics by Esnouf R. M. (1997)
  52. Merritt E. A., Murphy E. P., Acta Crystallogr. D50, 869 (1994). / Acta Crystallogr. by Merritt E. A. (1994)
  53. Nakane M., et al., J. Biol. Chem. 265, 16841 (1990). (10.1016/S0021-9258(17)44837-X) / J. Biol. Chem. by Nakane M. (1990)
  54. Feinstein P. G., et al., Proc. Natl. Acad. Sci. U.S.A. 88, 10173 (1991). (10.1073/pnas.88.22.10173) / Proc. Natl. Acad. Sci. U.S.A. by Feinstein P. G. (1991)
  55. Ishikawa Y., et al., J. Biol. Chem. 267, 13553 (1992). (10.1016/S0021-9258(18)42247-8) / J. Biol. Chem. by Ishikawa Y. (1992)
  56. We thank J. Collins for technical assistance; C. Dessauer for helpful discussion; D. Coleman T. Harrell T. Xiao and the MacCHESS staff for assistance with data collection at CHESS; L. Esser for assistance in preparing figures; J. Hurley for providing the coordinates of the IIC 2 homodimer before their release in the Protein Data Bank; R. Johnson for supplying 2′-deoxy-3′-ATP (synthesis supported by NIH grant DK38828); and R. Cooke for supplying 2′-iodoATP. Supported by NIH grant DK46371 and Welch Foundation grant I-1229 to S.R.S.; by NIH grant GM34497 American Cancer Society grant RPG-77-001-21-BE Welch Foundation grant I-1271 and the Raymond and Ellen Willie Distinguished Chair of Molecular Neuropharmacology to A.G.G; and a postdoctoral fellowship from the Medical Research Council of Canada to R.K.S. Atomic coordinates have been submitted to the Protein Data Bank (1AZS).
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:50 a.m.)
Deposited 1 year, 7 months ago (Jan. 12, 2024, 11:46 p.m.)
Indexed 5 days, 5 hours ago (Aug. 27, 2025, 12:15 p.m.)
Issued 27 years, 8 months ago (Dec. 12, 1997)
Published 27 years, 8 months ago (Dec. 12, 1997)
Published Print 27 years, 8 months ago (Dec. 12, 1997)
Funders 0

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@article{Tesmer_1997, title={Crystal Structure of the Catalytic Domains of Adenylyl Cyclase in a Complex with G sα ·GTPγS}, volume={278}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.278.5345.1907}, DOI={10.1126/science.278.5345.1907}, number={5345}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Tesmer, John J. G. and Sunahara, Roger K. and Gilman, Alfred G. and Sprang, Stephen R.}, year={1997}, month=dec, pages={1907–1916} }