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

Authors

• Arianna Minzoni, NC State College of Engineering
• Benjamin Bobay, Duke University Medical Center
• Shriarjun Shastry, NC State College of Engineering
• Eduardo Barbieri, Michigan Technological UniversityFollow
• Brandon Brino, NC State College of Engineering
• Crystal Collazo, NC State College of Engineering
• Shizuo Kamita, Bio-Rad Laboratories, Inc.
• Danni Wang, Bio-Rad Laboratories, Inc.
• Ciera Khuu, Bio-Rad Laboratories, Inc.
• Alexander Polgar, Bio-Rad Laboratories, Inc.
• Joseph Siino, Bio-Rad Laboratories, Inc.
• Sushmita Koley, Bio-Rad Laboratories, Inc.
• Peyton Russelburg, Bio-Rad Laboratories, Inc.
• Mark Snyder, Bio-Rad Laboratories, Inc.
• Christopher Belisle, Bio-Rad Laboratories, Inc.
• Michael Daniele, NC State University
• Stefano Menegatti, NC State College of Engineering

Document Type

Article

Publication Date

8-25-2026

Department

Health Research Institute

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.

Publisher's Statement

Copyright: © 2026 by the authors. Licensee MDPI, Basel, Switzerland. Publisher’s version of record: https://doi.org/10.3390/pharmaceutics18091053

Publication Title

Pharmaceutics

Recommended Citation

Minzoni, A., Bobay, B., Shastry, S., Barbieri, E., Brino, B., Collazo, C., Kamita, S., Wang, D., Khuu, C., Polgar, A., Siino, J., Koley, S., Russelburg, P., Snyder, M., Belisle, C., Daniele, M., & Menegatti, S. (2026). Probing the Capsid: pH-Driven Gating at the AAV 5-Fold Pore and Its Role in Peptide Ligand Binding. Pharmaceutics, 18(9). http://doi.org/10.3390/pharmaceutics18091053
Retrieved from: https://digitalcommons.mtu.edu/michigantech-p2/2986

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

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