Conformation state-specific monobodies regulate the functions of flexible proteins through conformation trapping.
Ibuki NakamuraHiroshi AmesakaMizuho HaraKento YonezawaKeisuke OkamotoHironari KamikuboShun-Ichi TanakaTakashi MatsuoPublished in: Protein science : a publication of the Protein Society (2023)
Synthetic binding proteins have emerged as modulators of protein functions through protein-protein interactions (PPIs). Because PPIs are influenced by the structural dynamics of targeted proteins, investigating whether the synthetic-binders-based strategy is applicable for proteins with large conformational changes is important. This study demonstrates the applicability of monobodies (fibronectin type-III domain-based synthetic binding proteins) in regulating the functions of proteins that undergo tens-of-angstroms-scale conformational changes, using an example of the A55C/C77S/V169C triple mutant (Adk tm ; a phosphoryl transfer-catalyzing enzyme with a conformational change between OPEN/CLOSED forms). Phage display successfully developed monobodies that recognize the OPEN form (substrate-unbound form), but not the CLOSED form of Adk tm . Two OPEN form-specific clones (OP-2 and OP-4) inhibited Adk tm kinase activity. Epitope mapping with a yeast-surface display/flow cytometry indicated that OP-2 binds to the substrate-entry pathway of Adk tm , whereas OP-4 binding occurs at another site. SEC-SAXS analysis indicated that OP-4 binds to the hinge side opposite to the substrate-binding site of Adk tm , retaining the whole OPEN-form structure of Adk tm . Titration of the OP-4-Adk tm complex with Ap 5 A, a transition-state analog of Adk tm , showed that the conformational shift to the CLOSED form was suppressed although Adk tm retained the OPEN-form (i.e., substrate-binding ready form). These results show that OP-4 captures and stabilizes the OPEN-form state, thereby affecting the hinge motion. These experimental results indicate that monobody-based modulators can regulate the functions of proteins that show tens-of-angstroms-scale conformational changes, by trapping specific conformational states generated during large conformational change process that is essential for function exertion. This article is protected by copyright. All rights reserved.