Antigen‐Detected NMR for Minimal Epitope Engineering and Structure‐Guided Selection of a Na V 1.7‐Selective Nanobody

Year: 2026

Type: article

Abstract: ABSTRACT Selective molecular recognition of membrane proteins is challenging because they contain few solvent‐exposed extracellular epitopes, which often depend on their native environment for structure, making them difficult to isolate faithfully for binder discovery. Here, we show that antigen‐detected NMR is well suited both to characterizing the folding of engineered minimal epitopes from the human voltage‐gated sodium channel Na V 1.7 and to selecting binders that recognize their solvent‐exposed surfaces. Isotope labelling of the antigen enables NMR resonance assignment to assess retained local secondary structure, while 15 N titration and zz‐exchange mapping provide binding and interface information. Combined with AlphaFold2 complex prediction, this creates a practical method for screening and ranking candidate binders. The approach was further validated by a high‐resolution x‐ray structure of an antigen–nanobody complex. Applying this workflow identified R4C8, a subtype‐ and species‐selective nanobody whose binding to the extracellular surface of human Na V 1.7 is supported by zz‐exchange mapping, modelling, and cellular recognition, and which has minimal effects on channel gating. R4C8 detected Na V 1.7 in engineered cell lines and in primary osteoarthritis‐derived chondrocytes, providing a useful tool for selective target detection. These results show how antigen‐detected NMR can support peptide engineering and structure‐guided protein binder selection against minimal epitopes.

Source: Advanced Science

DOI: 10.1002/advs.77611

Institutions: The University of Queensland, Institute for Molecular Bioscience, Queensland University of Technology, Walter and Eliza Hall Institute of Medical Research, Monash University, Australian National University, The University of Melbourne

Language: English

Topic: Advanced NMR Techniques and Applications

Subfield: Spectroscopy

Field: Chemistry

Domain: Physical Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $6,730

Funders: Chan Zuckerberg Initiative, Australian Cancer Research Foundation, Australian Synchrotron, University of Queensland, Medical Research Council, Australian Research Council, National Health and Medical Research Council

Awards: From structural mechanisms of endosomal trafficking to therapies for neurodegenerative diseases, Novel analgesics for treating chronic pain, G1722660635, Antibody-based therapies against infectious diseases, Defining a new family of sodium channel accessory proteins, Understanding how toxins interact with lipid membranes and ion channels, A pharmacological approach to define the contribution of Nav1.7 to pain pathways, Developing the toolbox of compounds that target acid-sensing proteins, A new source of bivalent molecules from nature

OpenAlex ID: W7211899801