Probing the Capsid: pH-Driven Gating at the AAV 5-Fold Pore and Its Role in Peptide Ligand Binding

Document Type

Article

Publication Date

9-1-2026

Abstract

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.

Publication Title

Pharmaceutics

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