Design and Characterization of Proteins Rationally Engineered to Domain Swap by Mutually Exclusive Folding

Abstract

Domain swapping is a mechanism for proteins to form dimers and higher order oligomers through the exchange of a section of their 3D structures. The backbone peptides of domain swapped oligomers are intertwined, but their 3D structures remain identical to their monomeric state, except for where they cross-over, termed a hinged region. We have developed a technique to engineer domain swapping interfaces with mutually exclusive folding (MEF). MEF achieves this by inserting a ‘lever’ protein into the surface loops of a host ‘target’ protein to form a target-lever fusion. This target-leveris conformationally strained with the lever and the target in a thermodynamic tug-of-war. When the lever is folded, the long distance between its N- and C-termini stretches apart the target and splits it in half. Conversely, when the target is folded, the short length of the loop where the lever was inserted compresses the lever and unfolds it. Domain swapping provides an escape from this tug-of-war as it allows the split target to refold and bypass the conformational strain. Because the lever is external to the target, adjusting the stability of the lever, through well-established thermodynamic principles, allows the propensity for domain swapping to be modulated without affecting the binding interface. This enables the design of “triggerable” levers which can reversibly induce domain swapping in response to a signal. Further, we can use domain swapping to turn the function of a target domain on and off. Two target-lever constructs are created with functional mutants in the target domain, one N-terminal to the lever and the other one C-terminal to the lever. Individually, both of these mutants are inactive, however if they are mixed and allowed to domain swap, then up to half of the target domains can swap out the functional mutations into the native active form. This bimolecular system in combination with induced domain swapping enables the design of modular bioswitches and biosensors.