Differential Roles of Ionic, Aliphatic, and Aromatic Residues in Membrane−Protein Interactions:  A Surface Plasmon Resonance Study on Phospholipases A₂

Abstract

The roles of cationic, aliphatic, and aromatic residues in the membrane association and dissociation of five phospholipases A(2) (PLA(2)), including Asp-49 PLA(2) from the venom of Agkistrodon piscivorus piscivorus, acidic PLA(2) from the venom of Naja naja atra, human group IIa and V PLA(2)s, and the C2 domain of cytosolic PLA(2), were determined by surface plasmon resonance analysis. Cationic interfacial binding residues of A. p. piscivorus PLA(2) (Lys-10) and human group IIa PLA(2) (Arg-7, Lys-10, and Lys-16), which mediate electrostatic interactions with anionic membranes, primarily accelerate the membrane association. In contrast, an aliphatic side chain of the C2 domain of cytosolic PLA(2) (Val-97), which penetrates into the hydrophobic core of the membrane and forms hydrophobic interactions, mainly slows the dissociation of membrane-bound protein. Aromatic residues of human group V PLA(2) (Trp-31) and N. n. atra PLA(2) (Trp-61, Phe-64, and Tyr-110) contribute to both membrane association and dissociation steps, and the relative contribution to these processes depends on the chemical nature and the orientation of the side chains as well as their location on the interfacial binding surface. On the basis of these results, a general model is proposed for the interfacial binding of peripheral proteins, in which electrostatic interactions by ionic and aromatic residues initially bring the protein to the membrane surface and the subsequent membrane penetration and hydrophobic interactions by aliphatic and aromatic residues stabilize the membrane-protein complexes, thereby elongating the membrane residence time of protein.

pH-dependent Binding of the Epsin ENTH Domain and the AP180 ANTH Domain to PI(4,5)P2-containing Bilayers

“…Concurrently a decrease of k a and an increase of k d were observed, most likely due to the reduced electrostatic interactions and a lesser degree of membrane penetration by the ENTH domain. 19,20 A similar enhancement of the membrane binding affinity upon acidification was seen for the AP180 ANTH domain. We monitored the ANTH domain association with PtdIns(4,5)P 2 -containing vesicles at pH 6.0, 7.4, and 8.0.…”

“…The association and dissociation phases of sensorgrams were globally fit to a 1:1 Langmuir binding model: protein + (protein binding site on vesicle) ↔ (complex) using BIAevalutation software (Biacore) as described previously. 19 The dissociation constant (K d ) was calculated from the equation…”

The Molecular Basis of Differential Subcellular Localization of C2 Domains of Protein Kinase C-α and Group IVa Cytosolic Phospholipase A2

“…SPR Measurements-The preparation of vesicle-coated Pioneer L1 sensor chip (Biacore) was described in detail elsewhere (29,30). In control experiments, the sensor chip was coated with vesicles (e.g.…”

“…We have shown that the SPR analysis allows direct determination of membrane association (k a ) and dissociation (k d ) rate constants for peripheral proteins (29,31). As summarized in Table I, PKC-␣-C2 has higher affinity (in terms of K d ) for POPC/POPS (7:3) than for POPC/POPG (7:3), and the PS selectivity of PKC-␣-C2 expressed in term of the ratio of 1/K d for POPC/POPS (7:3) to 1/K d for POPC/POPG (7:3) is about 11.…”

A Dibasic Amino Acid Pair Conserved in the Activation Loop Directs Plasma Membrane Localization and Is Necessary for Activity of Plant Type I/II Phosphatidylinositol Phosphate Kinase

“…A role of basic amino acid residues in plasma membrane targeting has also been found in the C2 domains of phospholipase A2 and protein kinase Ca, for which it was proposed that electric interactions by cationic residues initially bring proteins to anionic membrane surfaces (Stahelin and Cho, 2001;Stahelin et al, 2003). On the basis of these findings, it is postulated that the molecular basis of plasma membrane localization via basic amino acid residues is widely conserved among membrane-targeting proteins in eukaryotes.…”