Computer-guided Binding Mode Identification and Affinity Improvement of an LRR Protein Binder without Structure Determination

Precise binding mode identification and subsequent affinity improvement without structure determination remain a challenge in the development of therapeutic proteins. However, relevant experimental techniques are generally quite costly, and purely computational methods have been unreliable. Here, we show that integrated computational and experimental epitope localization followed by full-atom energy minimization can yield an accurate complex model structure which ultimately enables effective affinity improvement and redesign of binding specificity. As proof-of-concept, we used a leucine-rich repeat (LRR) protein binder, called a repebody (Rb), that specifically recognizes human IgG 1 (hIgG 1 ). We performed computationally-guided identification of the Rb:hIgG 1 binding mode and leveraged the resulting model to reengineer the Rb so as to significantly increase its binding affinity for hIgG 1 as well as redesign its specificity toward multiple IgGs from other species. Experimental structure determination verified that our Rb:hIgG 1 model closely matched the co-crystal structure. Using a benchmark of other LRR protein complexes, we further demonstrated that the present approach may be broadly applicable to proteins undergoing relatively small conformational changes upon target binding. Author Summary It is quite challenging for computational methods to determine how proteins interact and to design mutations to alter their binding affinity and specificity. Despite recent advances in computational methods, however, in silico evaluation of binding energies has proven to be extremely difficult. We show that, in the case of protein-protein interactions where only small structural changes occur upon target binding, an integrated computational and experimental approach can identify a binding mode and drive reengineering efforts to improve binding affinity or specificity. Using as a model system a leucine-rich repeat (LRR) protein binder that recognizes human IgG 1 , our approach yielded a model of the protein complex that was very similar to the subsequently experimentally determined co-crystal structure, and enabled design of variants with significantly improved IgG 1 binding affinity and with the ability to recognize IgG 1 from other species.