{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,9,27]],"date-time":"2026-09-27T14:58:01Z","timestamp":1790521081108,"version":"4.1.0"},"posted":{"date-parts":[[2025,9,30]]},"group-title":"Biophysics","reference-count":80,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,9,30]],"date-time":"2025-09-30T00:00:00Z","timestamp":1759190400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"name":"NIH","award":["R01AG089561"],"award-info":[{"award-number":["R01AG089561"]}]},{"name":"NIH","award":["R01EY029169"],"award-info":[{"award-number":["R01EY029169"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,20]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Eph receptors, the largest family of receptor tyrosine kinases (RTKs), orchestrate diverse developmental processes, including axon guidance, tissue patterning, and cell positioning, while also contributing to adult functions such as synaptogenesis and tissue homeostasis. Proteolytic cleavage of Eph receptors by neurodegeneration-associated proteases generates a membrane-detached intracellular region (ICR) fragment whose structural organization and signaling remain poorly understood. Here, we characterize a model for such a fragment from EphA2 and discover that the juxtamembrane region, the kinase domain, and the C-terminally linked sterile \u03b1 motif (SAM) domain comprise a configurational ensemble of domain contacts and orientations which inhibit the intermolecular interactions and tyrosine cross-phosphorylation. Recombinant EphA2 ICR expressed in\n                  <jats:italic>E. coli<\/jats:italic>\n                  retains catalytic activity and undergoes autophosphorylation at multiple tyrosine residues which have previously been identified in eukaryotic systems. Phosphorylation weakens intramolecular interactions, as measured by microscale thermophoresis, indicating a phosphorylation-dependent remodeling of the ICR. While AlphaFold2-Multimer (AF2M) and Alphafold3 (AF3) predictions as well as available crystal structures provide only limited insight into these configurational states, coarse-grained molecular dynamics simulations using the Martini 3 force field reveal highly dynamic and predominantly transient interactions between the kinase and SAM domains that cluster at two discrete regions on the kinase surface. NMR spectroscopy of isotopically labeled EphA2 SAM suggests non-canonical intramolecular contacts that preserve the canonical SAM interaction interface for binding of partner proteins. Addition of the SHIP2 SAM domain promotes formation of the canonical EphA2\u2013SHIP2 SAM complex while also revealing previously unrecognized interactions of the kinase domain on the SHIP2 SAM domain. Collectively, these findings establish the EphA2 ICR as a dynamic, phosphorylation-responsive signaling module in which transient intramolecular interactions regulate accessibility of several interfaces for binding. Thus, the proteolytically released EphA2 intracellular fragment is likely to have distinct characteristics in cancer and neurodegenerative disease, compared to the membrane bound form in the full receptor.\n                <\/jats:p>","DOI":"10.1101\/2025.09.29.679228","type":"posted-content","created":{"date-parts":[[2025,10,1]],"date-time":"2025-10-01T05:05:24Z","timestamp":1759295124000},"source":"Crossref","is-referenced-by-count":2,"title":["Tyrosine phosphorylation and the inhibitory C-terminal SAM domain regulate the EphA2 intracellular fragment in solution: A combined experimental and molecular modeling study"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7982-5085","authenticated-orcid":false,"given":"Pravesh","family":"Shrestha","sequence":"first","affiliation":[{"name":"Department of Physiology & Biophysics, Case Western Reserve University, Cleveland, OH, USA,"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2190-9518","authenticated-orcid":false,"given":"Amita Rani","family":"Sahoo","sequence":"additional","affiliation":[{"name":"Department of Physiology & Biophysics, Case Western Reserve University, Cleveland, OH, USA,"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Iannucci","sequence":"additional","affiliation":[{"name":"Department of Physiology & Biophysics, Case Western Reserve University, Cleveland, OH, USA,"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6884-6369","authenticated-orcid":false,"given":"Belinda","family":"Willard","sequence":"additional","affiliation":[{"name":"Proteomics Center, Cleveland Clinic Foundation, Cleveland, OH, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2958-0403","authenticated-orcid":false,"given":"Matthias","family":"Buck","sequence":"additional","affiliation":[{"name":"Department of Physiology & Biophysics, Case Western Reserve University, Cleveland, OH, USA,"},{"name":"Department of Neurosciences, Case Western Reserve University, Cleveland, OH 44106, USA"},{"name":"Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA"},{"name":"Case Cancer Comprehensive Center, Case Western Reserve University, Cleveland, OH 44106, USA"},{"name":"Center for Artificial Intelligence in Drug Discovery, Case Western Reserve University, Cleveland, OH 44106, 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