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

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Background: Integrin ␣1 I domain undergoes conformational changes upon collagen binding. Results: Deuterium exchange was used to measure the effects of cations, collagen, or an antibody on the ␣1I solution structure. Conclusion: Full-length collagen and metal ions induce changes that differ in key aspects from previously proposed models for ␣1I activation. Significance: These studies support a new model for integrin I domain activation.

Section: Methodsmentioning

confidence: 99%

Dynamic Structural Changes Are Observed upon Collagen and Metal Ion Binding to the Integrin α1 I Domain

Weinreb

Gao

et al. 2012

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“…The change in exchange upon binding to 500 M lipid vesicles was tested in the presence of 0, 200, and 1000 M Ca 2ϩ . Significant decreases upon exposure to the lipid sur- (35). A significant decrease in exchange due to binding of lipid vesicles was seen in the region 36 -39, as well as a small change in the region 28 -35 in the presence of 1 mM Ca 2ϩ and PAPC lipid vesicles (Fig.…”

Section: Giva Pla 2 Coverage Map Of Pepsin Fragmentation-mentioning

confidence: 92%

“…The protein digestion procedure was optimized to produce a peptide map that yielded the best coverage of GIVA PLA 2 as described previously (35). The optimized condition gave 157 distinct peptides that gave 92% coverage of the GIVA PLA 2 sequence.…”

Section: Giva Pla 2 Coverage Map Of Pepsin Fragmentation-mentioning

confidence: 99%

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

Burke

Hsu

Deems

et al. 2008

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105

The 85-kDa GIVA 3 phospholipase A 2 (GIVA PLA 2 ) is a member of the superfamily of phospholipase A 2 enzymes (1, 2) that cleave fatty acids from the sn-2 position of membrane phospholipids. This enzyme was initially isolated from human platelets, and it is specific for phospholipids containing arachidonic acid in the sn-2 position (3, 4). The release of arachidonic acid is the critical first step in the biosynthesis of eicosanoids, which are potent mediators of inflammation and pain (5). There are a number of enzymes and routes by which arachidonic acid can be released from phospholipids, but experiments with GIVA PLA 2 knock-out mice have demonstrated its importance in many inflammatory processes (6 -8), thereby confirming the key role of the GIVA enzyme.The enzyme is composed of two domains, a Ca 2ϩ binding C2 domain and a ␣/␤-hydrolase domain that contains the catalytic site (9). Crystal structures of both the intact enzyme (PDB 1CJY) (10) and the C2 domain alone (11) (PDB 1RLW) have been solved. For the enzyme to be active, it must be sequestered to a phospholipid interface. Binding Ca 2ϩ to the C2 domain accomplishes this as does the binding of two different lipid mediators, e.g. ceramide 1 phosphate (12, 13) and phosphatidylinositol-(4,5) bis-phosphate (14, 15).The Ca 2ϩ binding C2 domain is a conserved domain that is present on many different lipid-binding proteins (16). Ca 2ϩ binding to this domain sequesters the protein to the lipid surface. Extensive studies have been carried out on the C2 domain using a variety of techniques to determine how Ca 2ϩ binding accomplishes lipid surface binding. These studies have included x-ray reflectivity, site-directed mutagenesis, NMR, EPR, and computational methods (17). These studies have shown (18 -23) that lipid binding entails the penetration of Ca 2ϩ binding loops one and three, composed of amino acids 35-39 and 96 -98, into the interface. However, these studies only deal with C2 binding to the membrane, not the intact cPLA 2 enzyme. How the presence of the ␣/␤-hydrolase domain affects surface binding has not been determined in detail due to the difficulties of working with such a large protein.Unlike the C2 domain, the binding of the catalytic domain of the enzyme to the lipid surface has not been extensively studied. Numerous studies using site-directed mutagenesis of the intact protein have localized amino acids that are important for lipid * 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. 858-534-7390; E-mail: edennis@ucsd.edu.3 The abbreviations used...

“…This approach has traditionally been used to study protein-protein and protein-ligand interactions, but we applied it to look at the interaction of water-soluble proteins with membranes and demonstrated this approach with PLA 2 (136 -138). We have now studied four of the major types of PLA 2 , including the secreted (139), cytosolic (140,141), and Ca 2ϩ -independent forms (142), as well as lipoprotein-associated/platelet-activating factor (PAF) acetyl hydrolase (143) and the interactions of the latter with lipoproteins such as HDL (144). These DXMS methods also led to a better understanding of the catalytic action of an unusual form with a Cys in the catalytic site (145).…”

Section: The Mechanism Comes Togethermentioning

confidence: 99%

Liberating Chiral Lipid Mediators, Inflammatory Enzymes, and LIPID MAPS from Biological Grease

Dennis

2016

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In 1970, it was well accepted that the central role of lipids was in energy storage and metabolism, and it was assumed that amphipathic lipids simply served a passive structural role as the backbone of biological membranes. As a result, the scientific community was focused on nucleic acids, proteins, and carbohydrates as information-containing molecules. It took considerable effort until scientists accepted that lipids also "encode" specific and unique biological information and play a central role in cell signaling. Along with this realization came the recognition that the enzymes that act on lipid substrates residing in or on membranes and micelles must also have important signaling roles, spurring curiosity into their potentially unique modes of action differing from those acting on water-soluble substrates. This led to the creation of the concept of "surface dilution kinetics" for describing the mechanism of enzymes acting on lipid substrates, as well as the demonstration that lipid enzymes such as phospholipase A 2 (PLA 2 ) contain allosteric activator sites for specific phospholipids as well as for membranes. As our understanding of phospholipases advanced, so did the understanding that many of the lipids released by these enzymes are chiral informationcontaining signaling molecules; for example, PLA 2 regulates the generation of precursors for the biosynthesis of eicosanoids and other bioactive lipid mediators of inflammation and resolution underlying disease progression. The creation of the LIPID MAPS initiative in 2003 and the ensuing development of the lipidomics field have revealed that lipid metabolites are central to human metabolism. Today lipids are recognized as key mediators of health and disease as we enter a new era of biomarkers and personalized medicine. This article is my personal "reflection" on these scientific advances.