The antibody response has evolved under constant pressure to recognize common pathogens and also remain adaptable to novel threats. Given the limited size of the germline antibody repertoire, adaptability requires that some antibodies must be polyspecific for multiple distinct antigens. Despite the profound importance of polyspecificity in the antibody response, the structural features that allow it are not well understood.
Antibodies raised against glycoconjugates of Chlamydiaceae LPS oligosaccharides of the inner-core sugar Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) have been shown to cross-react with several inner-core oligosaccharides through conserved recognition of single Kdo residues in a germline-encoded pocket, with additional sugars accommodated by flexible side-chains. Two of these antibodies, S25-2 and S25-39, were observed to bind several Kdo oligosaccharides with an identical binding site conformation, but adopted unique conformations of the heavy chain complementarity determining region loop 3 (CDR H3) in the absence of ligand.
Conformational flexibility of germline antibodies is believed to facilitate polyspecificity by generating multiple unique binding sites in a single antibody. This thesis research further explores the conformational flexibility of the antibodies S25-2 and S25-39 to gain insight into mechanisms of antigen recognition and how this feature may allow polyspecificity. This was achieved first by solving structures of S25-39 from crystals grown in unique conditions to observe alternate CDR H3 conformations, and second by designing synthetic Kdo-based antigens so as both to inhibit interaction with the previously observed liganded conformation of S25-2 and S25-39 and to be accommodated by their observed unliganded conformations.
These structures reveal an unprecedented level of structural diversity of CDR H3, notably including the exact ‘liganded’ conformation in the absence of ligand. This is the first direct structural evidence that CDR H3 can exist in a conformational equilibrium with antigen binding through a selection mechanism, as opposed to induced fit where antigen causes the observed conformational change. Definitive evidence for binding the synthetic antigens was not obtained, however the resulting structures revealed several additional unique conformations of CDR H3 suggesting that ligands can alter conformational equilibria during crystallization. A unique conformation was also observed with CDR H3 coordinating multiple iodide ions, revealing another potential source of polyspecificity with unique binding paratopes generated by ion coordination.
Finally, the unparalleled level of conformational diversity observed for these antibodies highlights the challenges of antibody structure classification and prediction, and stresses the need for additional in-depth studies of conformational diversity and binding mechanisms to advance these fields for therapeutic application.
This is the first targeted structural study of flexibility in antibodies and provides insight into their conformational dynamics and antigen-binding mechanisms. These are of fundamental importance in understanding antibody structure and function, a critical consideration in practical applications such as modelling and design of therapeutic or diagnostic antibodies. / Graduate / 2019-11-27
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/7305 |
| Date | 20 May 2016 |
| Creators | Blackler, Ryan J. |
| Contributors | Evans, S. V. (Stephen V.) |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
| Rights | Available to the World Wide Web |
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