TY - JOUR
T1 - Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins
AU - Singh, Nilmani
AU - Reyes-Ordoñez, Adriana
AU - Compagnone, Michael A.
AU - Moreno, Jesus F.
AU - Leslie, Benjamin J.
AU - Ha, Taekjip
AU - Chen, Jie
N1 - Funding Information:
We thank Dr. Lisa Stubbs and Mr. Joe Troy for help with the initial identification of PH domain sequences, and Ms. Lucy Yao and Ms. Eleanor Marcet for assistance with western blotting. This work was supported by grants from the National Institute of General Medical Sciences (R01 GM089771 to JC and R35 GM122569 to TH).
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/12/1
Y1 - 2021/12/1
N2 - 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.
AB - 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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U2 - 10.1038/s41467-021-24639-y
DO - 10.1038/s41467-021-24639-y
M3 - Article
C2 - 34267198
AN - SCOPUS:85110707204
SN - 2041-1723
VL - 12
JO - Nature communications
JF - Nature communications
IS - 1
M1 - 4339
ER -