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.