title: Optimization of protease-inhibitor interactions by randomizing adventitious contacts.
abstract: Polypeptide protease inhibitors are often found to inhibit targets with which they did not coevolve, as in the case of high-affinity inhibition of bacterial subtilisin by the leech inhibitor eglin c. Two kinds of contacts exist in such complexes: (i) reactive site loop-active site contacts and (ii) interactions outside of these that form the broader enzyme-inhibitor interface. We hypothesized that the second class of "adventitious" contacts could be optimized to generate significant increases in affinity for a target enzyme or discrimination of an inhibitor for closely related target proteases. We began with a modified eglin c, Arg-42-Arg-45-eglin, in which the reactive site loop had been optimized for subtilisin-related processing proteases of the Kex2/furin family. We randomized 10 potential adventitious contact residues and screened for inhibition of soluble human furin. Substitutions at one of these sites, Y49, were also screened against yeast Kex2 and human PC7. These screens identified not only variants that exhibited increased affinity (up to 20-fold), but also species that exhibited enhanced selectivity, that is, increased discrimination between the target enzymes (up to 41-fold for furin versus PC7 and 20-fold for PC7 versus furin). One variant, Asp-49-Arg-42-Arg-45-eglin, exhibited a Ki of 310 pM for furin and blocked furin-dependent processing of von Willebrand factor in COS-1 cells when added to the culture medium of the cells. The exploitation of adventitious contact sites may provide a versatile technique for developing potent, selective inhibitors for newly discovered proteases and could in principle be applied to optimize numerous protein-protein interactions.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Komiyama
authors.forename: Bryan
authors.initials: B
authors.lastname: VanderLugt
authors.forename: Martin
authors.initials: M
authors.lastname: Fugère
authors.forename: Robert
authors.initials: R
authors.lastname: Day
authors.forename: Randal J
authors.initials: RJ
authors.lastname: Kaufman
authors.forename: Robert S
authors.initials: RS
authors.lastname: Fuller
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003062:Codon / Q000235:genetics; D003470:Culture Media; D015195:Drug Design; D045683:Furin; D015723:Gene Library; D006801:Humans; D007865:Leeches / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D043484:Proprotein Convertases; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D025941:Protein Interaction Mapping; D011499:Protein Processing, Post-Translational; D011506:Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000037:antagonists & inhibitors; D015843:Serpins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013381:Subtilisins / Q000037:antagonists & inhibitors; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D003062:Codon; D003470:Culture Media; D011480:Protease Inhibitors; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D015843:Serpins; C028656:eglin proteinase inhibitors; D014841:von Willebrand Factor; C099101:PCSK7 protein, human; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae; D045683:Furin