HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 45, 31227-31236, November 7, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/45/31227    most recent M804492200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Burke, J. E. Articles by Dennis, E. A. Search for Related Content PubMed PubMed Citation Articles by Burke, J. E. Articles by Dennis, E. A. Social Bookmarking                      What's this?

A Phospholipid Substrate Molecule Residing in the Membrane Surface Mediates Opening of the Lid Region in Group IVA Cytosolic Phospholipase A₂^*

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

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

The Group IVA (GIVA) phospholipase A₂ associates with natural membranes in response to an increase in intracellular Ca²⁺ along with increases in certain lipid mediators. This enzyme associates with the membrane surface as well as binding a single phospholipid molecule in the active site for catalysis. Employing deuterium exchange mass spectrometry, we have identified the regions of the protein binding the lipid surface and conformational changes upon a single phospholipid binding in the absence of a lipid surface. Experiments were carried out using natural palmitoyl arachidonyl phosphatidylcholine vesicles with the intact GIVA enzyme as well as the isolated C2 and catalytic domains. Lipid binding produced changes in deuterium exchange in eight different regions of the protein. The regions with decreased exchange included Ca²⁺ binding loop one, which has been proposed to penetrate the membrane surface, and a charged patch of residues, which may be important in interacting with the polar head groups of phospholipids. The regions with an increase in exchange are all located either in the hydrophobic core underneath the lid region or near the lid and hinge regions from 403 to 457. Using the GIVA phospholipase A₂ irreversible inhibitor methyl-arachidonyl fluorophosphonate, we were able to isolate structural changes caused only by pseudo-substrate binding. This produced results that were very similar to natural lipid binding in the presence of a lipid interface with the exception of the C2 domain and region 466-470. This implies that most of the changes seen in the catalytic domain are due to a substrate-mediated, not interface-mediated, lid opening, which exposes the active site to water. Finally experiments carried out with inhibitor plus phospholipid vesicles showed decreases at the C2 domain as well as charged residues on the putative membrane binding surface of the catalytic domain revealing the binding sites of the enzyme to the lipid surface.

Received for publication, June 12, 2008 , and in revised form, August 27, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants GM20501 (to E. A. D.) and CA099835, CA118595, GM037684, AI0220221, and AI022160 (to V. L. W.). This work was also supported by 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.

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

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. EAD: Tel.: 858-534-3055; Fax: 858-534-7390; E-mail: edennis{at}ucsd.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				J. E. Burke and E. A. Dennis Phospholipase A2 structure/function, mechanism, and signaling J. Lipid Res., April 1, 2009; 50(Supplement): S237 - S242. [Abstract] [Full Text] [PDF]