An Integrated Approach for Determining a Protein-Protein Binding Interface in Solution and an Evaluation of HDX Kinetics for Adjudicating Candidate Docking Models

• Published in: Analytical chemistry (Washington) 2019-11, Vol.91 (24), p.15709-15717
• Main Authors: Zhang, Mengru Mira, Beno, Brett R., Huang, Richard Y.-C., Adhikari, Jagat, Deyanova, Ekaterina G., Li, Jing, Chen, Guodong, Gross, Michael L.
• Format: Article
• Language: English
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/acs.analchem.9b03879

We describe an integrated approach of using hydrogen deuterium exchange mass spectrometry (HDX-MS), chemical crosslinking mass spectrometry (XL-MS), and molecular docking to characterize the binding interface and to predict the three-dimensional quaternary structure of a protein-protein complex in solution. Interleukin 7 (IL-7) and its α-receptor, IL-7Rα, serving as essential mediators in the immune system, are the model system. HDX kinetics report widespread protection on IL-7Rα but show no differential evidence of binding-induced protection or remote conformational change. Crosslinking with reagents that differ in spacer lengths and targeting residues increases the spatial resolution. Using five cross-links as distance restraints for protein-protein docking, we generated a high-confidence model of the IL-7/IL-7Rα complex. Both the predicted binding interface and regions with direct contacts agree well with those in the solid-state structure, as confirmed by previous X-ray crystallography. An additional binding region was revealed to be the C-terminus of helix B of IL-7, highlighting the value of solution-based characterization. To generalize the integrated approach, protein-protein docking was executed with a different number of cross-links. Combining cluster analysis and HDX kinetics adjudication, we found that two intermolecular crosslink-derived restraints are sufficient to generate a high-confidence model with root mean square distance (r.m.s.d.) value of all alpha carbons below 2.0 Å relative to the crystal structure. The remarkable results of binding-interface determination and quaternary structure prediction highlight the effectiveness and capability of the integrated approach, which will allow more efficient and comprehensive analysis of inter-protein interactions with broad applications in the multiple stages of design, implementation, and evaluation for protein therapeutics.