Author ORCID Identifier
https://orcid.org/0000-0001-8175-1196
Date of Award
Winter 11-13-2025
Document Type
Thesis (Ph.D.)
Department or Program
Biochemistry and Cell Biology
First Advisor
Dean R. Madden
Abstract
Nanobodies are immune fragments derived from heavy-chain only antibodies and are attractive tools for biomedical research due to their ease of production, small size, stability, and facile functionalization. Nanobodies have also garnered attention for their ability to modulate enzymatic activities through competitive and allosteric mechanisms. In the context of microbial virulence factors and toxins, these attributes give nanobodies therapeutic and diagnostic potential while simultaneously providing new opportunities to explore fundamental principles pertaining to protein function. For these reasons, a nanobody library was constructed for Cif, an epoxide hydrolase with virulence properties produced by Pseudomonas aeruginosa that has implications for people with Cystic Fibrosis.
Preliminary data indicated that a subset of nanobodies showed inhibitory potential against Cif and were selected for further characterization using a combination of structural and biochemical techniques. The co-crystal structures of several Cif:nanobody complexes revealed CDR3-centric and CDR2-centric mechanisms for direct steric competitive inhibition. In both cases, a conserved pentad motif in the nanobodies position a hydrophobic, often aromatic, side chain into the active-site entrance of Cif. Nanobodies in both classes also utilize highly similar stereochemistries for antigen recognition despite low sequence identity.
Allosteric nanobody modulators of Cif were also identified and described for the first time. The first is nanobody VHH108, which exerts substrate-dependent enhancement of Cif. Co-crystal structures of the Cif:VHH108 complex reveal subtle structural changes at the binding interface that are accompanied with global changes to protein dynamics, as inferred by an analysis of normalized ADPs. In contrast, the second allosteric nanobody, VHH116, functions as an allosteric inhibitor of Cif, and ongoing work to uncover the structural details governing the interaction is underway.
Collectively, these allosteric nanobodies add to a growing network of long-range communications previously identified in Cif. More generally, the detailed understanding of these Cif:nanobody interactions serves as a primer for the development of research tools that will aid in clarifying the clinical impact Cif has in the scope of Cystic Fibrosis lung infections. The inhibitory nanobodies also have potential for immediate therapeutic applications.
Recommended Citation
Simard, Adam R., "Exploration of inhibition and modulation of an epoxide hydrolase by nanobodies." (2025). Dartmouth College Ph.D Dissertations. 441.
https://digitalcommons.dartmouth.edu/dissertations/441
