| Institution: | 1 Department of Biochemistry and Howard Hughes Medical Institute (HHMI), University of Washington, Seattle, WA 98195, USA;2 Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden;3 Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Northeast Structural Genomics Consortium, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA;4 Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027, USA;5 Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel;6 Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA |
| Abstract: | While there has been considerable progress in designing protein–protein interactions, the design of proteins that bind polar surfaces is an unmet challenge. We describe the computational design of a protein that binds the acidic active site of hen egg lysozyme and inhibits the enzyme. The design process starts with two polar amino acids that fit deep into the enzyme active site, identifies a protein scaffold that supports these residues and is complementary in shape to the lysozyme active-site region, and finally optimizes the surrounding contact surface for high-affinity binding. Following affinity maturation, a protein designed using this method bound lysozyme with low nanomolar affinity, and a combination of NMR studies, crystallography, and knockout mutagenesis confirmed the designed binding surface and orientation. Saturation mutagenesis with selection and deep sequencing demonstrated that specific designed interactions extending well beyond the centrally grafted polar residues are critical for high-affinity binding. |
