Fig. 1: Overview of the computational design of broad-spectrum mini-protein binders against TcdB variants.

Our computational design approach consists of two steps. In step 1, we performed the de novo design of mini-protein binders targeting TcdB1. Initially, we utilized RIFDock to generate the initial positions of the mini-protein binders relative to the RBI-1 of TcdB1. Interface design was conducted using Rosetta, along with the utilization of the MotifGraft algorithm for sampling additional backbone geometries and interface compositions. In total, 69,752 mini-protein binders against the RBI-1 of TcdB1 were designed and progressed to step 2. In step 2, we selected binders with the capability to bind the RBI-1 of TcdB2 from the previously designed pool of mini-protein binders targeting TcdB1. For each design model, TcdB2 was superimposed onto the RBI-1 of TcdB1, and the resulting binder-TcdB2 coordinates were considered as the binder-TcdB2 complex design model. These models were further subjected to structure relaxation using Rosetta. Subsequently, the designed models underwent in silico evaluation, which led to the selection of 3,434 final mini-protein designs for targeting both TcdB1 and TcdB2 (Methods).