Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins

Nilmani Singh,Jie Chen,Taekjip Ha,Benjamin J Leslie,Jesus F Moreno,Michael A Compagnone,Adriana Reyes-Ordoñez

doi:10.1038/s41467-021-24639-y

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https://doi.org/10.1038/s41467-021-24639-y

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Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. Here we employ a single-molecule pulldown assay to study interactions of lipid vesicles with full-length proteins in mammalian whole cell lysates. Of 67 human PH domain-containing proteins initially examined, 36 (54%) are found to have affinity for PIPs with various specificity, the majority of which have not been reported before. Further investigation of ARHGEF3 reveals distinct structural requirements for its binding to PI(4,5)P2 and PI(3,5)P2, and functional relevance of its PI(4,5)P2 binding. We generate a recursive-learning algorithm based on the assay results to analyze the sequences of 242 human PH domains, predicting that 49% of them bind PIPs. Twenty predicted binders and 11 predicted non-binders are assayed, yielding results highly consistent with the prediction. Taken together, our findings reveal unexpected lipid-binding specificity of PH domain-containing proteins.

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Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear
In order to investigate binding of PIPs by PH domaincontaining proteins using the lipid-single-molecule pulldown (SiMPull) assay[17], we created cDNA constructs and transfected them in human embryonic kidney (HEK) 293 cells to express 67 full-length human PH domaincontaining proteins with EGFP fused at their C-termini
Given the high likelihood that the PH domains mediate PIP binding of the full-length proteins, we compared the sequences of the PH domains of the 67 proteins we studied plus AKT1-PH as a positive control, excluding the four proteins that bound lipids non

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Introduction

Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. 1234567890():,; Specific phospholipid–protein interactions are critical to the regulation of many signal transduction processes and cellular functions These interactions typically involve lipidbinding domains recognized by specific lipid species and/or physical properties of the membrane such as charge or curvature[1,2,3,4]. The interaction with PIP is so specific for those two PH domains that they have been commonly used as sensors to detect PI(4,5)P2 and PIP3 in cells[12,13] Despite those early examples of PH–PIP interactions, to date only a modest number of PH domain-containing proteins have been demonstrated to bind PIPs with specificity. Protein partners have been identified for PH domains, which may cooperate with lipid–PH interaction or operate independently to regulate or mediate PH domain functions[1,16]

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