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Jönsson M, Mushtaq AU, Nagy TM, von Witting E, Löfblom J, Nam K, Wolf-Watz M, Hober S
J. Biol. Chem. 300 (11) 107795 [2024-09-19; online 2024-09-19]
Designing proteins with tunable activities from easily accessible external cues remains a biotechnological challenge. Here, we set out to create a small antibody-binding domain equipped with a molecular switch inspired by the allosteric response to calcium seen in naturally derived proteins like calmodulin. We have focused on one of the three domains of Protein G that show inherent affinity to antibodies. By combining a semi-rational protein design with directed evolution, we engineered novel variants containing a calcium-binding loop rendering the inherent antibody affinity calcium-dependent. The evolved variants resulted from a designed selection strategy subjecting them to negative and positive selection pressures focused on conditional antibody binding. Hence, these variants contains molecular "on/off" switches, controlling the target affinity towards antibody fragments simply by the presence or absence of calcium. From NMR spectroscopy we found that the molecular mechanism underlying the evolved switching behavior was a coupled calcium-binding and folding event where the target binding surface was intact and functional only in the presence of bound calcium. Notably, it was observed that the response to the employed selection pressures gave rise to the evolution of a cooperative folding mechanism. This observation illustrates why the cooperative folding reaction is an effective solution seen repeatedly in the natural evolution of fine-tuned macromolecular recognition. Engineering binding moieties to confer conditional target interaction has great potential due to the exquisite interaction control that is tunable to application requirements. Improved understanding of the molecular mechanisms behind regulated interactions is crucial to unlock how to engineer switchable proteins useful in a variety of biotechnological applications.
Swedish NMR Centre (SNC) [Service]
PubMed 39305954
DOI 10.1016/j.jbc.2024.107795
Crossref 10.1016/j.jbc.2024.107795
pmc: PMC11532951
pii: S0021-9258(24)02296-8