New computational protein design methods forde novosmall molecule binding sites

Author summary Despite the variety of contexts where small molecule recognition by proteins is paramount, computational methods for designing this functionality are limited in scope. Current methods are limited by the availability of structurally characterized protein-ligand complexes and the inability of design methods to incorporate appropriate residues at the protein-ligand interface that predictably yield a functional binding site. In this work we introduce a new set of methods that combines information from the Protein Data Bank (PDB) with the Rosetta Macromolecular Modeling Suite to improve our ability to design new binding sites for arbitrary small molecules. We demonstrate that highly represented protein contacts in the PDB with fragments that compose the target ligand can be identified and recombined to form hundreds of thousands of new binding sites for ligands where less than a dozen characterized complexes exist. Commonly observed protein residue-ligand contacts can also be applied in simulations to optimize the sequences of protein-ligand interfaces, resulting in improved sequence recovery and design metrics predictive of success. We hope that the methods introduced here will open protein design to new applications in small molecule sense and response systems. Protein binding to small molecules is fundamental to many biological processes, yet it remains challenging to predictively design this functionalityde novo. Current state-of-the-art computational design methods typically rely on existing small molecule binding sites or protein scaffolds with existing shape complementarity for a target ligand. Here we introduce new methods that utilize pools of discrete contacts between protein side chains and defined small molecule ligand substructures (ligand fragments) observed in the Protein Data Bank. We use the Rosetta Molecular Modeling Suite to recombine protein side chains in these contact pools to generate hundreds of thousands of energetically favorable binding sites for a target ligand. These composite binding sites are built into existing scaffold proteins matching the intended binding site geometry with high accuracy. In addition, we apply pools of side chain rotamers interacting with the target ligand to augment Rosetta's conventional design machinery and improve key metrics known to be predictive of design success. We demonstrate that our method reliably builds diverse binding sites into different scaffold proteins for a variety of target molecules. Our generalizablede novoligand binding site design method provides a foundation for versatile design of protein to interface previously unattainable molecules for applications in medical diagnostics and synthetic biology.