{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T05:17:49Z","timestamp":1787807869301,"version":"build-2784847793"},"reference-count":33,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T00:00:00Z","timestamp":1787616000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000038","name":"Food and Drug Administration","doi-asserted-by":"publisher","award":["R01FD007481"],"award-info":[{"award-number":["R01FD007481"]}],"id":[{"id":"10.13039\/100000038","id-type":"DOI","asserted-by":"publisher"}]},{"award":["R01FD007481"],"award-info":[{"award-number":["R01FD007481"]}],"id":[{"id":"https:\/\/ror.org\/034xvzb47","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100014726","name":"National Institute for Innovation in Manufacturing Biopharmaceuticals","doi-asserted-by":"crossref","award":["PC8.1-135"],"award-info":[{"award-number":["PC8.1-135"]}],"id":[{"id":"10.13039\/100014726","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100014726","name":"National Institute for Innovation in Manufacturing Biopharmaceuticals","doi-asserted-by":"crossref","award":["PC9.1-105"],"award-info":[{"award-number":["PC9.1-105"]}],"id":[{"id":"10.13039\/100014726","id-type":"DOI","asserted-by":"crossref"}]},{"award":["PC8.1-135"],"award-info":[{"award-number":["PC8.1-135"]}],"id":[{"id":"https:\/\/ror.org\/04wzf4f82","id-type":"ROR","asserted-by":"publisher"}]},{"award":["PC9.1-105"],"award-info":[{"award-number":["PC9.1-105"]}],"id":[{"id":"https:\/\/ror.org\/04wzf4f82","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["CBET 2449235"],"award-info":[{"award-number":["CBET 2449235"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"award":["CBET 2449235"],"award-info":[{"award-number":["CBET 2449235"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Triangle Universities Center for Advanced Studies Inc."},{"name":"North Carolina Viral Vector Initiative in Research and Learning (NC-VVIRAL) at NC State University"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceutics"],"abstract":"<jats:p>Background\/Objectives: Adeno-associated virus (AAV) capsids undergo pH-dependent conformational gating at the 5-fold symmetry pore, but how these structural dynamics shape serotype-specific behavior and affinity-ligand recognition remains unclear, particularly for the clinically important serotypes AAV8 and AAV9. This study aimed to establish a pH-resolved structural framework linking 5-fold pore dynamics to peptide-ligand recognition and to translate this framework into sequence-based design principles for affinity capture of gene therapy vectors. Methods: AAV8 and AAV9 5-fold capsid assemblies were subjected to 500 ns molecular dynamics simulations under acidic (pH 5), neutral (pH 7), and basic (pH 9) conditions, with analysis of pore volume, inter-residue contact networks, electrostatic potential, and solvent-accessible surface area. In parallel, affinity chromatography using three mixed-mode peptide ligands (RVVAVYRI, TTFRAHHI, and TYHHHHII) was performed on clarified HEK293 lysates containing AAV8 or AAV9, with capsid yield, host-cell-protein clearance, and transduction activity assessed by ELISA, SEC-HPLC, and flow-cytometry-based transduction assays. Results: AAV8 displayed a heterogeneous, bimodal pore conformational landscape at pH 7, whereas AAV9 exhibited a discrete gate-like transition with maximal pore constriction at physiological pH; both serotypes showed pore-proximal contact remodeling with distinct network topologies. Experimentally, TYHHHHII achieved the highest selectivity for genome-containing capsids at pH 7, with transduction activity enrichment factors of 2.82 (AAV8) and 5.61 (AAV9), while TTFRAHHI provided the broadest operational pH range for bulk capsid recovery. Conclusions: These findings establish a structural framework linking pH-dependent pore dynamics to affinity ligand recognition and suggest practical sequence-design rules for ligand engineering: clustered histidines for neutral-pH selectivity, Arg-containing motifs for broad-pH robustness, and aromatic or hydrophobic residues for reinforcement of capsid binding.<\/jats:p>","DOI":"10.3390\/pharmaceutics18091053","type":"journal-article","created":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T10:04:55Z","timestamp":1787652295000},"page":"1053","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Probing the Capsid: pH-Driven Gating at the AAV 5-Fold Pore and Its Role in Peptide Ligand Binding"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4504-9567","authenticated-orcid":false,"given":"Arianna","family":"Minzoni","sequence":"first","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering, NC State University, 911 Partners Way, Raleigh, NC 27606, USA"},{"name":"North Carolina Viral Vector Initiative in Research and Learning (NC-VVIRAL), NC State University, 911 Oval Dr, Raleigh, NC 27695, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4775-3686","authenticated-orcid":false,"given":"Benjamin","family":"Bobay","sequence":"additional","affiliation":[{"name":"Duke University NMR Center, Department of Chemistry, Duke University Medical Center, 308 Research Dr, Durham, NC 27710, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9164-9398","authenticated-orcid":false,"given":"Shriarjun","family":"Shastry","sequence":"additional","affiliation":[{"name":"Department of Chemical and Biomolecular Engineering, NC State University, 911 Partners Way, Raleigh, NC 27606, USA"},{"name":"North Carolina Viral Vector Initiative in Research and Learning 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