Mapping of discontinuous conformational epitopes by amide hydrogen/deuterium exchange mass spectrometry and computational docking

Abstract

Understanding antigen-antibody interactions at the sub-molecular level is of particular interest for scientific, regulatory, and intellectual property reasons, especially with increasing demand for monoclonal antibody therapeutic agents. Although various techniques are available for the determination of an epitope, there is no widely applicable, high-resolution, and reliable method available. Here, a combination approach using amide hydrogen/deuterium exchange coupled with proteolysis and mass spectrometry (HDX-MS) and computational docking was applied to investigate antigen-antibody interactions. HDX-MS is a widely applicable, medium-resolution, medium-throughput technology that can be applied to epitope identification. First, the epitopes of cytochrome c-E8, IL-13-CNTO607, and IL-17A-CAT-2200 interactions identified using the HDX-MS method were compared with those identified by X-ray co-crystal structures. The identified epitopes are in good agreement with those identified using high-resolution X-ray crystallography. Second, the HDX-MS data were used as constraints for computational docking. More specifically, the non-epitope residues of an antigen identified using HDX-MS were designated as binding ineligible during computational docking. This approach, termed HDX-DOCK, gave more tightly clustered docking poses than stand-alone docking for all antigen-antibody interactions examined and improved docking results significantly for the cytochrome c-E8 interaction.

Small-molecule ligand docking into comparative models with Rosetta

“…Numerous types of experimental data have been incorporated into such protocols, including electron density from X-ray crystallography 62 and electron microscopy, NMR distance and orientation data 60,63 , EPR distance data 59,61 , cross-linking restraints 64 , small-angle X-ray scattering data 65 and deuterium-exchange mass spectrometry data 66 . Although these types of data are more often applied to de novo protein structure elucidation, they can also be of some use in loop building 67 , reorientation of domains during comparative modeling or identification of residues involved in ligand binding.…”

Structural analysis of the interleukin-8/glycosaminoglycan interactions by amide hydrogen/deuterium exchange mass spectrometry

“…A sig- 42 nificantly reduced H/D exchange, especially of the C-terminal a-helical region comprising amino acids 43 70-77 and to the loop comprising amino acids 27-29 was observed in the presence of chondroitin sulfate. 44 HDX MS data were used to model the IL-8/chondroitin sulfate complex. The binding interface of IL-8 and 45 chondroitin sulfate determined this way correlated excellently with the corresponding NMR based ato- 46 mistic model previously published.…”

Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing

“…Co-crystallization provides unambiguous epitope identification but can require considerable effort and generation of many antigen variants to identify one that is compatible with crystallization (2). Mass spectrometry-based methods utilizing hydrogen/ deuterium exchange identify epitopes to a ϳ5-amino acid resolution only under rigorous control experiments that limit throughput (16,17). Competing display-based methods use many sorts (18), identify only partial epitopes (19,20), or are limited by restricting mutations to alanine (21).…”