The N-terminal alpha-helix of pancreatic phospholipase A(2) determines productive-mode orientation of the enzyme at the membrane surface

    Authors

    S. Qin; A. H. Pande; K. N. Nemec;S. A. Tatulian

    Comments

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    Abbreviated Journal Title

    J. Mol. Biol.

    Keywords

    phospholipase A(2); membrane binding; orientation; interfacial; activation; polarized infrared spectroscopy; RELAXATION MAGNETIC-RESONANCE; SITE-DIRECTED MUTAGENESIS; SECONDARY; STRUCTURE; C2 DOMAIN; INTERFACIAL ACTIVATION; AGKISTRODON PISCIVORUS; CONFORMATIONAL-CHANGES; INFRARED-SPECTROSCOPY; CATALYTIC MECHANISM; CIRCULAR-DICHROISM; Biochemistry & Molecular Biology

    Abstract

    Phospholipase A(2) (PLA(2)) hydrolyzes glycerophospholipids to free fatty acid and lyso-phospholipid, which serve as precursors for the biosynthesis of eicosanoids and other lipid-derived mediators of inflammation and allergy. PLA2 activity strongly increases upon binding to the surface of aggregated phospho ipid. The N-terminal similar to ten residue alpha-helix of certain PLA(2) isoforms play important roles in the interfacial activation of the enzyme by providing residues for membrane binding of PLA(2) and by contributing to the formation of the substrate-binding pocket. However, the relative contributions of the N-terminal alpha-helix and the rest of the protein in membrane binding of PLA(2) and its productive-mode orientation at the membrane surface are not well understood. Here we use a variety of biophysical approaches to determine the role of the N-terminal helix in membrane binding strength, orientation, and activity of human pancreatic PLA(2). While the full-length PLA(2) binds to membranes with a defined orientation, an engineered PLA(2) fragment DeltaN10 that lacks the N-terminal ten residues binds to membranes with weaker affinity and at random orientation, and exhibits similar to 100-fold lower enzymatic activity compared to the full-length PLA(2), indicating the key role of the N terminus in PLA(2) function. The results of polarized infrared spectroscopic experiments permit determination of the orientation of membrane-bound PLA(2) and identification of its interfacial binding surface. Moreover, the full-length PLA(2) demonstrates increased conformational flexibility in solution and is stabilized upon membrane binding, while the DeltaN10 fragment is more rigid than the full-length PLA(2) both in free and membrane-bound states. Our results suggest that the N-terminal alpha-helix supports the activation of PLA(2) by (a) enhancing the membrane binding strength, (b) facilitating a productive-mode orientation of PLA(2) at the membrane surface, and (c) conferring conformational integrity and plasticity to the enzyme. (C) 2004 Elsevier Ltd. All rights reserved.

    Journal Title

    Journal of Molecular Biology

    Volume

    344

    Issue/Number

    1

    Publication Date

    1-1-2004

    Document Type

    Article

    Language

    English

    First Page

    71

    Last Page

    89

    WOS Identifier

    WOS:000224926700006

    ISSN

    0022-2836

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