title: Computational design of a protein-based enzyme inhibitor.
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.
authors.affiliation: Department of Biochemistry and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA. procko@u.washington.edu
authors.forename: Erik
authors.initials: E
authors.lastname: Procko
authors.forename: Rickard
authors.initials: R
authors.lastname: Hedman
authors.forename: Keith
authors.initials: K
authors.lastname: Hamilton
authors.forename: Jayaraman
authors.initials: J
authors.lastname: Seetharaman
authors.forename: Sarel J
authors.initials: SJ
authors.lastname: Fleishman
authors.forename: Min
authors.initials: M
authors.lastname: Su
authors.forename: James
authors.initials: J
authors.lastname: Aramini
authors.forename: Gregory
authors.initials: G
authors.lastname: Kornhaber
authors.forename: John F
authors.initials: JF
authors.lastname: Hunt
authors.forename: Liang
authors.initials: L
authors.lastname: Tong
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
authors.forename: David
authors.initials: D
authors.lastname: Baker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D019295:Computational Biology; D004791:Enzyme Inhibitors / Q000737:chemistry*; D008958:Models, Molecular; D062105:Molecular Docking Simulation / Q000379:methods; D009113:Muramidase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D060066:Protein Interaction Maps
chemical_list: D004791:Enzyme Inhibitors; C421864:hen egg lysozyme; D009113:Muramidase
keywords: BSA; FACS; HA; HEL; MW; PAK1; PBS; PDB; Protein Data Bank; Rosetta molecular modeling program; SEC; VNAR; bovine serum albumin; fluorescence-activated cell sorting; hemagglutinin; hen egg lysozyme; hot spot; molecular weight; p21-activated kinase 1; phosphate-buffered saline; protein engineering and design; protein–protein interactions; size-exclusion chromatography; variable new antigen receptor