Protein interfacial pocket engineering via coupled computational filtering and biological focusing criterion

To engineer bio-macromolecular systems, protein-substrate interactions and their configurations need to be understood, harnessed, and utilized. Due to the inherent large numbers of combinatorial configurations and conformational complexity, methods that rely on heuristics or stochastics, such as practical computational filtering (CF) or biological focusing (BF) criterions, when used alone rarely yield insights into these complexes or successes in (re)designing them. Here we use a coupled CF-BF criterion upon an amenable interfacial pocket (IP) of a protein scaffold complexed with its substrate to undergo residue replacement and R-group refinement (R4) to filter out energetically unfavorable residues and R-group conformations, and focus in on those that are evolutionarily favorable. We show that this coupled filtering and focusing can efficiently provide a putative engineered IP candidate and validate it computationally and empirically. The CF-BF criterion may permit holistic understanding of the nuances of existing protein IPs and their scaffolds and facilitate bioengineering efforts to alter substrate specificity. Such approach may contribute to accelerated elucidation of engineering principles of bio-macromolecular systems.