Conformation state-specific monobodies regulate the functions of flexible proteins through conformation trapping

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 side of Adk(tm), whereas OP-4 binding occurs at another site. Small angle X-ray scattering coupled with size-exclusion chromatography (SEC-SAXS) 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.