CDR-H3 loop ensemble in solution – conformational selection upon antibody binding

ABSTRACT

We analyzed pairs of protein-binding, peptide-binding and hapten-binding antibodies crystallized as complex and in the absence of the antigen with and without conformational differences upon binding in the complementarity-determining region (CDR)-H3 loop. Here, we introduce a molecular dynamics-based approach to capture a diverse conformational ensemble of the CDR-H3 loop in solution. The results clearly indicate that the inherently flexible CDR-H3 loop indeed needs to be characterized as a conformational ensemble. The conformational changes of the CDR-H3 loop in all antibodies investigated follow the paradigm of conformation selection, because we observe the experimentally determined binding competent conformation without the presence of the antigen within the ensemble of pre-existing conformational states in solution before binding. We also demonstrate for several examples that the conformation observed in the antibody crystal structure without antigen present is actually selected to bind the carboxyterminal tail region of the antigen-binding fragment (Fab). Thus, special care must be taken when characterizing antibody CDR-H3 loops by Fab X-ray structures, and the possibility that pre-existing conformations are present should always be considered.

KEYWORDS:

• CDR-H3 loop
• conformational ensemble
• crystal structure
• dominant solution structure
• conformational selection
• molecular dynamics
• Markov-state models

Abbreviations

Table

Acknowledgments

We are grateful to Radu A. Talmazan for fruitful discussion and input on the visualization of the data.

Author contributions

The manuscript was discussed and written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Supplementary Material

Supplemental data for this article can be accessed on the publisher’s website.

Additional information

Funding

This work was supported by the Austrian Science Fund (FWF) via the grant [P30565] “Characterization of Promiscuity and Specificity in Proteases.”