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J. Biol. Chem., Vol. 282, Issue 34, 24825-24832, August 24, 2007

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Catalysis-associated Conformational Changes Revealed by Human CD38 Complexed with a Non-hydrolyzable Substrate Analog^*

Qun Liu, Irina A. Kriksunov, Christelle Moreau, Richard Graeff^¶, Barry V. L. Potter, Hon Cheung Lee^¶||1, and Quan Hao²

From the MacCHESS, Cornell High Energy Synchrotron Source, Cornell University, Ithaca, New York 14853, Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom, ^¶Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455, and ^||Department of Physiology, University of Hong Kong, Hong Kong, China

Cyclic ADP-ribose (cADPR) is a calcium mobilization messenger important for mediating a wide range of physiological functions. The endogenous levels of cADPR in mammalian tissues are primarily controlled by CD38, a multifunctional enzyme capable of both synthesizing and hydrolyzing cADPR. In this study, a novel non-hydrolyzable analog of cADPR, N1-cIDPR (N1-cyclic inosine diphosphate ribose), was utilized to elucidate the structural determinants involved in the hydrolysis of cADPR. N1-cIDPR inhibits CD38-catalyzed cADPR hydrolysis with an IC₅₀ of 0.26 mM. N1-cIDPR forms a complex with CD38 or its inactive mutant in which the catalytic residue Glu-226 is mutated. Both complexes have been determined by x-ray crystallography at 1.7 and 1.76 Å resolution, respectively. The results show that N1-cIDPR forms two hydrogen bonds (2.61 and 2.64Å₎ with Glu-226, confirming our previously proposed model for cADPR catalysis. Structural analyses reveal that both the enzyme and substrate cADPR undergo catalysis-associated conformational changes. From the enzyme side, residues Glu-146, Asp-147, and Trp-125 work collaboratively to facilitate the formation of the Michaelis complex. From the substrate side, cADPR is found to change its conformation to fit into the active site until it reaches the catalytic residue. The binary CD38-cADPR model described here represents the most detailed description of the CD38-catalyzed hydrolysis of cADPR at atomic resolution. Our structural model should provide insights into the design of effective cADPR analogs.

Received for publication, February 26, 2007 , and in revised form, June 11, 2007.

^* This work was supported by National Institutes of Health Grants RR01646 (to MacCHESS) and GM061568 (to H. C. L. and Q. H.) and by the Wellcome Trust Value in People program (to B. V. L. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (code 2PGJ, 2PGL) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence may be addressed. Tel.: 852-2819-9163; Fax: 852-2819-9230; E-mail: leehc{at}hku.hk. ² To whom correspondence may be addressed. Tel.: 607-254-8983; Fax: 607-255-9001; E-mail: qh22{at}cornell.edu.

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