{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,26]],"date-time":"2026-09-26T16:01:20Z","timestamp":1790438480987,"version":"4.1.0"},"posted":{"date-parts":[[2026,9,22]]},"group-title":"Biochemistry","reference-count":61,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T00:00:00Z","timestamp":1790035200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["R35 GM122485"],"award-info":[{"award-number":["R35 GM122485"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,22]]},"abstract":"<jats:title>SUMMARY<\/jats:title>\n                <jats:p>\n                  Pleckstrin homology (PH) domains are typically assumed to be phosphoinositide-binding modules, although most lack strong lipid specificity and their broader ligand repertoire remains poorly defined. We find that many yeast PH domains bind Nsr1p, the ortholog of nucleolin \u2013 also identified as a PH domain ligand \u2013 suggesting widespread recognition of negatively charged protein regions. The phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)\n                  <jats:italic>P<\/jats:italic>\n                  <jats:sub>2<\/jats:sub>\n                  )-binding PLC\u03b4\n                  <jats:sub>1<\/jats:sub>\n                  PH domain also binds a highly phosphorylated region of IRBIT through the same site that recognizes PtdIns(4,5)\n                  <jats:italic>P<\/jats:italic>\n                  <jats:sub>2<\/jats:sub>\n                  . Using high-throughput integrated phosphopeptide (Hi-P) screening, we surveyed 38,624 mammalian sequences containing three documented phosphoserines.\n                <\/jats:p>\n                <jats:p>\n                  PLC\u03b4\n                  <jats:sub>1<\/jats:sub>\n                  -PH bound numerous phosphorylated and unphosphorylated acidic peptides, and phosphorylation generally strengthened pre-existing interactions rather than conferring strict specificity \u2013 without requiring a fixed phosphoserine spacing. Instead, favored phosphopeptides combined a key phosphoserine with upstream acidic residues. Structural modeling suggests that this phosphoserine occupies the canonical inositol phosphate binding pocket, while adjacent acidic residues make delocalized electrostatic contacts. Thus, PH domains can recognize various patterns of protein negative charge, expanding their potential regulatory roles beyond membrane targeting.\n                <\/jats:p>","DOI":"10.64898\/2026.09.21.753330","type":"posted-content","created":{"date-parts":[[2026,9,22]],"date-time":"2026-09-22T22:50:21Z","timestamp":1790117421000},"source":"Crossref","is-referenced-by-count":0,"title":["Recognition of negative charge arrays by pleckstrin homology (PH) domains"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0430-5632","authenticated-orcid":false,"given":"Christopher K.","family":"Ng","sequence":"first","affiliation":[{"name":"Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA"},{"name":"Yale Cancer Biology Institute, Yale University West Campus, West Haven, CT 06516, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3046-6034","authenticated-orcid":false,"given":"James W.","family":"Murphy","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA"},{"name":"Yale Cancer Biology Institute, Yale University West Campus, West Haven, CT 06516, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeannine M.","family":"Mendrola","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Sarullo","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA"},{"name":"Yale Cancer Biology Institute, Yale University West Campus, West Haven, CT 06516, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1677-8293","authenticated-orcid":false,"given":"Steven E.","family":"Stayrook","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA"},{"name":"Yale Cancer Biology Institute, Yale University West Campus, West Haven, CT 06516, USA"},{"name":"Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7671-5403","authenticated-orcid":false,"given":"Kathryn M.","family":"Ferguson","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA"},{"name":"Yale Cancer Biology Institute, Yale University West Campus, West Haven, CT 06516, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3379-5319","authenticated-orcid":false,"given":"Mark A.","family":"Lemmon","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA"},{"name":"Yale Cancer Biology Institute, Yale University West Campus, West Haven, CT 06516, USA"},{"name":"Department of Biochemistry and Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 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