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

Nat Commun. 2021 Jul 15;12(1):4339. doi: 10.1038/s41467-021-24639-y.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Animals
  • Binding Sites
  • Computational Biology / methods
  • HEK293 Cells
  • Humans
  • Mice
  • Microscopy, Fluorescence
  • NIH 3T3 Cells
  • Phosphatidylinositols / chemistry
  • Phosphatidylinositols / metabolism*
  • Phosphatidylserines / chemistry
  • Phosphatidylserines / metabolism
  • Pleckstrin Homology Domains*
  • Proteins / chemistry*
  • Proteins / genetics
  • Proteins / metabolism*
  • Rho Guanine Nucleotide Exchange Factors / chemistry
  • Rho Guanine Nucleotide Exchange Factors / genetics
  • Rho Guanine Nucleotide Exchange Factors / metabolism
  • Sensitivity and Specificity
  • rhoA GTP-Binding Protein / metabolism

Substances

  • ARHGEF3 protein, human
  • Phosphatidylinositols
  • Phosphatidylserines
  • Proteins
  • Rho Guanine Nucleotide Exchange Factors
  • rhoA GTP-Binding Protein