Lipid Cooperativity as a General Membrane-Recruitment Principle for PH Domains

Abstract

Many cellular processes involve the recruitment of proteins to specific membranes, which are decorated with distinctive lipids that act as docking sites. The phosphoinositides form signaling hubs, and we examine mechanisms underlying recruitment. We applied a physiological, quantitative, liposome microarray-based assay to measure the membrane-binding properties of 91 pleckstrin homology (PH) domains, the most common phosphoinositide-binding target. 10,514 experiments quantified the role of phosphoinositides in membrane recruitment. For most domains examined, the observed binding specificity implied cooperativity with additional signaling lipids. Analyses of PH domains with similar lipid-binding profiles identified a conserved motif, mutations in which-including some found in human cancers-induced discrete changes in binding affinities in vitro and protein mislocalization in vivo. The data set reveals cooperativity as a key mechanism for membrane recruitment and, by enabling the interpretation of disease-associated mutations, suggests avenues for the design of small molecules targeting PH domains.

Allosteric properties of PH domains in Arf regulatory proteins

“…A single PH domain may also bind to 2 ligands simultaneously. In a recent large-scale analysis of the binding of PH domains to liposomes, 10 56 of the 60 PH domains found to bind liposomes did so cooperatively, leading the authors to conclude that cooperative binding of 2 lipids may be common among PH domains.…”

“…[6][7][8][9] However, the model of the PH domain binding a single ligand to mediate protein targeting to specific membranes may not apply to most PH domains. 4,5,10 Membrane-associated proteins commonly bind more than one membrane-constrained ligand simultaneously. 5 Two domains, such as PH, PX, C2 and BAR, that bind membrane components may be constituents of a single protein.…”

“…As described in the introduction, a highthroughput screen indicated that a high proportion cooperatively bind lipids. 10 In earlier work, simultaneous occupancy of the canonical and alternate sites was proposed for the PH domain of Slm1, a target of TORC2 in the PI -3-kinase pathway and of sphingomyelin signaling. 104,105 Akt, a serine/threonine kinase that mediates signaling in the PtdIns 3-kinase pathway, also contains a PH domain that may bind phospholipids cooperatively.…”

“…104,105 Akt, a serine/threonine kinase that mediates signaling in the PtdIns 3-kinase pathway, also contains a PH domain that may bind phospholipids cooperatively. 10,[106][107][108] Akt binds PI(3,4)P 2 and PtdIns(3,4,5)P 3 through a structured canonical site. It also has a patch of positive surface charge contributed by amino acids in the b1/b2 and b5/b6 loops, a position similar to the alternate site seen, for example, in ASAP1, Slm1 and TIAM1.…”

Sensitivity of peripheral membrane proteins to the membrane context: A case study of phosphatidylserine and the TIM proteins

“…The Enzyme-Linked Immunosorbent Assays (ELISA), Surface Plasmon Resonance (SPR), Langmuir monolayer systems, lipid microarrays, liposome microarrays, fluorescence spectroscopy, and Isothermal Titration Calorimetry (ITC) have been integral in measuring the binding affinity of peripheral membrane-binding proteins for lipids [29–32]. Fluorescence spectroscopy and ITC have the advantage of measuring the binding of liposomes, soluble unilamellar lipid vesicles that require no membrane immobilization and serve as models for natural membrane structures of various sizes such as vesicles, organelle membranes, or cell membranes [33].…”