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J. Biol. Chem., Vol. 282, Issue 22, 16644-16653, June 1, 2007

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Binding of Phosphoinositide-specific Phospholipase C- (PLC-) to Phospholipid Membranes

POTENTIAL ROLE OF AN UNSTRUCTURED CLUSTER OF BASIC RESIDUES^*

Michail Nomikos¹, Anna Mulgrew-Nesbitt¹, Payal Pallavi^¶, Gyongyi Mihalyne^¶, Irina Zaitseva^¶, Karl Swann^||, F. Anthony Lai, Diana Murray, and Stuart McLaughlin^¶2

From the Cell Signaling Laboratory, Wales Heart Research Institute and the ^||Department of Obstetrics and Gynaecology, School of Medicine, Cardiff University, Cardiff CF14 4XN, United Kingdom, the Department of Microbiology and Immunology, Weill Medical College of Cornell University, New York, New York 10021, and the ^¶Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York 11794

Phospholipase C- (PLC-) is a sperm-specific enzyme that initiates the Ca²⁺ oscillations in mammalian eggs that activate embryo development. It shares considerable sequence homology with PLC-1, but lacks the PH domain that anchors PLC-1 to phosphatidylinositol 4,5-bisphosphate, PIP₂. Thus it is unclear how PLC- interacts with membranes. The linker region between the X and Y catalytic domains of PLC-, however, contains a cluster of basic residues not present in PLC-1. Application of electrostatic theory to a homology model of PLC- suggests this basic cluster could interact with acidic lipids. We measured the binding of catalytically competent mouse PLC- to phospholipid vesicles: for 2:1 phosphatidylcholine/phosphatidylserine (PC/PS) vesicles, the molar partition coefficient, K, is too weak to be of physiological significance. Incorporating 1% PIP₂ into the 2:1 PC/PS vesicles increases K about 10-fold, to 5 x 10³ M^-1, a biologically relevant value. Expressed fragments corresponding to the PLC- X-Y linker region also bind with higher affinity to polyvalent than monovalent phosphoinositides on nitrocellulose filters. A peptide corresponding to the basic cluster (charge =+7) within the linker region, PLC--(374-385), binds to PC/PS vesicles with higher affinity than PLC-, but its binding is less sensitive to incorporating PIP₂. The acidic residues flanking this basic cluster in PLC- may account for both these phenomena. FRET experiments suggest the basic cluster could not only anchor the protein to the membrane, but also enhance the local concentration of PIP₂ adjacent to the catalytic domain.

Received for publication, February 5, 2007 , and in revised form, April 6, 2007.

^* This work was supported in part by Grant GM24971 from the National Institutes of Health (to S. M.) and Grants MCB0212362 and MCB030028 for supercomputing resources (to D. M.) and an NSF Graduate Research Fellowship (to A. M.-N.) from the National Science Foundation, a grant from the ELWa Knowledge Exploitation Fund and Cardiff Partnership Fund (to A. L.), a grant from the Wellcome Trust (to K. S.), and a Cardiff School of Medicine PhD studentship (to M. N.). 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 supplemental Figs. S1-S3 and Eq. S1.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Stony Brook University, Stony Brook, NY 11794-8661. Tel.: 631-444-3615; Fax: 631-444-3432; E-mail: stuart.mclaughlin{at}stonybrook.edu.

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