Two-Metal-Ion Catalysis in Adenylyl Cyclase (original) (raw)

Abstract

Adenylyl cyclase (AC) converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate, a ubiquitous second messenger that regulates many cellular functions. Recent structural studies have revealed much about the structure and function of mammalian AC but have not fully defined its active site or catalytic mechanism. Four crystal structures were determined of the catalytic domains of AC in complex with two different ATP analogs and various divalent metal ions. These structures provide a model for the enzyme-substrate complex and conclusively demonstrate that two metal ions bind in the active site. The similarity of the active site of AC to those of DNA polymerases suggests that the enzymes catalyze phosphoryl transfer by the same two-metal-ion mechanism and likely have evolved from a common ancestor.

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REFERENCES AND NOTES

Sunahara R. K., Dessauer C. W., Gilman A. G., Annu. Rev. Pharmacol. Toxicol. 36, 461 (1996);

; D. M. F. Cooper, Ed., Advances in Second Messenger and Phosphoprotein Research, vol. 32 (Lippincott-Raven, Philadelphia, PA, 1998).

Sunahara R. K., Dessauer C. W., Whisnant R. E., Kleuss C., Gilman A. G., J. Biol. Chem. 272, 22265 (1997).

Whisnant R. E., Gilman A. G., Dessauer C. W., Proc. Natl. Acad. Sci. U.S.A. 93, 6621 (1996);

Yan S.-Z., Hahn D., Huang Z.-H., Tang W.-J., J. Biol. Chem. 271, 10941 (1996).

Dessauer C. W., Tesmer J. J., Sprang S. R., Gilman A. G., J. Biol. Chem. 273, 25831 (1998).

Tesmer J. J. G. T., Sunahara R. K., Gilman A. G., Sprang S. R., Science 278, 1907 (1997).

Garbers D. L., Johnson R. A., J. Biol. Chem. 250, 8449 (1975);

Zimmermann G., Zhou D., Taussig R., 273, 19650 (1998).

Artymiuk P. J., Poirrette A. R., Rice D. W., Willett P., Nature 388, 33 (1997).

Doublié S., Tabor S., Long A. M., Richardson C. C., Ellenberger T., 391, 251 (1998).

Huang H., Chopra R., Verdine G. L., Harrison S. C., Science 282, 1669 (1998).

Li Y., Korolev S., Waksman G., EMBO J. 17, 7514 (1998).

Kaushik N., et al., Biochemistry 35, 11536 (1996);

; A. Y. Woody, S. S. Eaton, P. A. Osumi-Davis, R. W. Woody, ibid., p. 144.

I. Shoshani, V. Boudou, C. Pierra, G. Gosselin, R. A. Johnson, unpublished data.

R. K. Sunahava and J. J. G. Tesmer, data not shown.

Eckstein F., Romaniuk P., Heideman W., Storm D., J. Biol. Chem. 256, 9118 (1981).

The recombinant C1a domain from canine type V AC, the C2a domain from rat type II AC, and bovine Gsα were expressed in E. coli and purified as previously described (30). Gsα was activated with Mg2+ · GTPγS and then subjected to limited proteolysis by trypsin. Crystals of the ternary complex between C1a, C2a, and Gsα were grown as previously described (5). All crystals were harvested in cryoprotectant (5) to which was added various combinations of inhibitors and metal ions, described as follows: for AC · βLddATP · Mg, 5 mM MgCl2 and 380 μM β-L-2′,3′-dd-5′-ATP; for AC · βLddATP · Mn, 250 μM MgCl2, 500 μM MnCl2, and 380 μM β-L-2′,3′-dd-5′-ATP; for AC · βLddATP · Zn, 250 μM MgCl2, 500 μM ZnCl2, and 380 μM β-L-2′,3′-dd-5′-ATP; for AC · ATPαS-Rp · Mn, 500 μM MgCl2, 500 μM MnCl2, and 1 mM ATPαS-Rp. Crystals of AC · βLddATP · Mg, AC · βLddATP · Mn, and AC · βLddATP · Zn were soaked for 1 to 2 hours; the conformation of AC in these complexes was closed. AC · ATPαS-Rp · Mn was soaked for 6 hours to achieve a closed conformation; 2-hour soaks resulted in structures of the open conformation of AC. AC complexes with inhibitors such as ATP · Zn2+, ATPαS-Sp · Zn2+, and 2′-iodo-ATP · Mg2+ also adopt the open conformation, and each metal site exhibits the same preference for either Mn2+ or Zn2+ as observed in the closed conformation (13). Strong electron density is observed for the β and γ phosphates of these inhibitors in the presence of Mg2+ alone or of Mn2+ and Mg2+, and for the entire triphosphate in the presence of Zn2+ and Mg2+. Much weaker density corresponding to a purine ring is observed in the purine binding pocket of these complexes. However, the purine and triphosphate densities are not continuous, and a single model of ATP cannot be built to accommodate both purine and triphosphate binding sites.

Kim E. E., Wyckoff H. W., J. Mol. Biol. 218, 449 (1991).

Derbyshire V., Pinsonneault J. K., Joyce C. M., Methods Enzymol. 262, 363 (1995).

Piccirilli J. A., Vyle J. S., Caruthers M. H., Cech T. R., Nature 361, 85 (1993).

Pelletier H., Sawaya M. R., Wolfle W., Wilson S. H., Kraut J., Biochemistry 35, 12762 (1996);

Slaby I., Lind B., Holmgren A., Biochem. Biophys. Res. Commun. 122, 1410 (1984);

King G. C., Martin C. T., Pham T. T., Coleman J. E., Biochemistry 25, 36 (1986);

Ferrari E., et al., J. Virol. 73, 1649 (1999).

Yan S.-Z., Huang Z.-H., Shaw R. S., Tang W.-J., J. Biol. Chem. 272, 12342 (1997).

Liu Y., Ruoho A. E., Rao V. D., Hurley J. H., Proc. Natl. Acad. Sci. U.S.A. 94, 13414 (1997).

Sheldrick W. S., Rieke E., Acta Crystallogr. B34, 2324 (1978);

Varughese K. I., Lu C. T., Kartha G., J. Am. Chem. Soc. 104, 3398 (1982);

Padmaja N., Ramakumar S. R., Viswamitra M. A., Bull. Chem. Soc. Jpn. 64, 1359 (1991).

Dessauer C. W., Gilman A. G., J. Biol. Chem. 272, 27787 (1997).

Tang W.-J., Stanzel M., Gilman A. G., Biochemistry 34, 14563 (1995).

Tesmer J. J., Sprang S. R., Curr. Opin. Struct. Biol. 8, 713 (1998).

Beese L. S., Steitz T. A., EMBO J. 10, 25 (1991);

Steitz T. A., Curr. Opin. Struct. Biol. 3, 31 (1993);

Pelletier H., Sawaya M. R., Kumar A., Wilson S. H., Kraut J., Science 264, 1891 (1994).

Doublié S., Sawaya M. R., Ellenberger T., Structure 7, R31 (1999).

Doublié S., Ellenberger T., Curr. Opin. Struct. Biol. 8, 704 (1998).

Sarafianos S. G., Pandey V. N., Kaushik N., Modak M. J., J. Biol. Chem. 270, 19729 (1995);

Pandey V. N., Kaushik N., Modak M. J., 269, 13259 (1994).

Sunahara R. K., et al., J. Biol. Chem. 273, 16332 (1998).

Single-letter abbreviations for the amino acid residues are as follows: D, Asp; I, Ile; K, Lys; N, Asn; R, Arg.

A. G. W. Leslie, MOSFLM 5.50 for image plate data (1997).

Bailey S., Acta Crystallogr. D50, 760 (1994).

Brünger A. T., et al., D54, 905 (1998).

T. A. Jones, J. Y. Zou, S. W. Cowan, M. Kjeldgaard, ibid. A47, 110 (1991).

We thank F. Eckstein at the Max-Planck Institute (Göttingen) for pure ATPαS-Rp; J. Collins for technical assistance; C. Brautigam for helpful discussion and for reading the manuscript; D. Coleman and the MacCHESS staff for their assistance with data collection at the Cornell High Energy Synchrotron Source (CHESS); and L. Esser for his assistance in preparing figures. R.K.S. was supported by a postdoctoral fellowship from the Medical Research Council of Canada. This work was supported by grants from the Agence Nationale de Recherche sur le SIDA (ANRS, France) to G.G.; by NIH grants DK38828 (R.A.J.), GM34497 (A.G.G.), and DK46371 (S.R.S.); by an Innovative Technology Grant from the Stony Brook Center for Biotechnology to R.A.J.; by Welch Foundation grants I-1271 (A.G.G.) and I-1229 (S.R.S.); and by the Raymond and Ellen Willie Distinguished Chair of Molecular Neuropharmacology (A.G.G.). Coordinates for the four models have been deposited in the Protein Data Bank with the codes 1CJT, 1CJU, 1CJV, and 1CJK.