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

Abstract

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 (Adktm; 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 Adktm. Two OPEN form‐specific clones (OP‐2 and OP‐4) inhibited Adktm kinase activity. Epitope mapping with a yeast‐surface display/flow cytometry indicated that OP‐2 binds to the substrate‐entry pathway of Adktm, 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 Adktm, retaining the whole OPEN‐form structure of Adktm. Titration of the OP‐4–Adktm complex with Ap5A, a transition‐state analog of Adktm, showed that the conformational shift to the CLOSED form was suppressed although Adktm 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.

Engineering a monobody specific to monomeric Cu/Zn‐superoxide dismutase associated with amyotrophic lateral sclerosis

“…As described in a recent review and the references therein, a large number of conformation‐specific monobodies targeting different states within the conformational ensemble of the target protein have been developed (Hantschel et al, 2020 ). In addition to these precedents, we recently demonstrated the development of monobodies highly specific to the respective conformation states using the example of Escherichia coli adenylate kinase with a large conformational change between OPEN and CLOSED forms (Nakamura et al, 2023 ).…”

“…In SOD1(FG), Glu is substituted for Phe50 and Gly51, shown in red (i.e., F50E/G51E). specific to the respective conformation states using the example of Escherichia coli adenylate kinase with a large conformational change between OPEN and CLOSED forms (Nakamura et al, 2023).…”