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J. Biol. Chem., Vol. 283, Issue 15, 9820-9827, April 11, 2008

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Calcium Binding Rigidifies the C2 Domain and the Intradomain Interaction of GIVA Phospholipase A₂ as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry^*

Yuan-Hao Hsu, John E. Burke, Daren L. Stephens, Raymond A. Deems, Sheng Li^¶, Kyle M. Asmus^¶, Virgil L. Woods, Jr.^¶1, and Edward A. Dennis²

From the Departments of Chemistry and Biochemistry, Pharmacology, and ^¶Medicine and the Biomedical Sciences Graduate Program, University of California, San Diego, La Jolla, California 92093-0601

The GIVA phospholipase A₂ (PLA₂) contains two domains: a calcium-binding domain (C2) and a catalytic domain. These domains are linked via a flexible tether. GIVA PLA₂ activity is Ca²⁺-dependent in that calcium binding promotes protein docking to the phospholipid membrane. In addition, the catalytic domain has a lid that covers the active site, presumably regulating GIVA PLA₂ activity. We now present studies that explore the dynamics and conformational changes of this enzyme in solution utilizing peptide amide hydrogen/deuterium (H/D) exchange coupled with liquid chromatographymass spectrometry (DXMS) to probe the solvent accessibility and backbone flexibility of the C2 domain, the catalytic domain, and the intact GIVA PLA₂. We also analyzed the changes in H/D exchange of the intact GIVA PLA₂ upon Ca²⁺ binding. The DXMS results showed a fast H/D-exchanging lid and a slow exchanging central core. The C2 domain showed two distinct regions: a fast exchanging region facing away from the catalytic domain and a slow exchanging region present in the "cleft" region between the C2 and catalytic domains. The slow exchanging region of the C2 domain is in tight proximity to the catalytic domain. The effects of Ca²⁺ binding on GIVA PLA₂ are localized in the C2 domain and suggest that binding of two distinct Ca²⁺ ions causes tightening up of the regions that surround the anion hole at the tip of the C2 domain. This conformational change may be the initial step in GIVA PLA₂ activation.

Received for publication, October 1, 2007 , and in revised form, December 31, 2007.

^* This work was supported in part by National Institutes of Health Grants GM20501(to E. A. D.) and CA099835, CA118595, GM037684, AI0220221, and AI022160 (to V. L. W.) and Discovery Grant UC10591 from the University of California Industry-University Cooperative Research Program (to V. L. W.). 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.

¹ To whom correspondence may be addressed. Tel.: 858-534-2180; Fax: 858-534-2606; E-mail: vwoods{at}ucsd.edu. ² To whom correspondence may be addressed. Tel.: 858-534-3055; Fax: 858-534-7390; E-mail: edennis{at}ucsd.edu.

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