Designing heterotropically activated allosteric conformational switches using supercharging

Significance

We present a general method to imbue proteins with heterotropic allostery by using surface protein supercharging to impart intrinsic disorder to the protein fold, allowing for functional activation by low concentrations of multivalent ions, including Mg(II), Ca(II), and spermine. We have designed two different proteins which exhibit this property and present a protocol which anyone can employ to impart this property to their protein of interest. This method promises to enable future synthetic biology projects which utilize natural fluxes of these ions to directly actuate function at significantly faster rates than that of genetic activation. Such multivalent ionic activation of disordered proteins may be a mechanism utilized by Nature which has yet to be appreciated.

Abstract

Heterotropic allosteric activation of protein function, in which binding of one ligand thermodynamically activates the binding of another, different ligand or substrate, is a fundamental control mechanism in metabolism and as such has been a long-aspired capability in protein design. Here we show that greatly increasing the magnitude of a protein’s net charge using surface supercharging transforms that protein into an allosteric ligand- and counterion-gated conformational molecular switch. To demonstrate this we first modified the designed helical bundle hemoprotein H4, creating a highly charged protein which both unfolds reversibly at low ionic strength and undergoes the ligand-induced folding transition commonly observed in signal transduction by intrinsically disordered proteins in biology. As a result of the high surface-charge density, ligand binding to this protein is allosterically activated up to 1,300-fold by low concentrations of divalent cations and the polyamine spermine. To extend this process further using a natural protein, we similarly modified Escherichia coli cytochrome b ₅₆₂ and the resulting protein behaves in a like manner. These simple model systems not only establish a set of general engineering principles which can be used to convert natural and designed soluble proteins into allosteric molecular switches useful in biodesign, sensing, and synthetic biology, the behavior we have demonstrated––functional activation of supercharged intrinsically disordered proteins by low concentrations of multivalent ions––may be a control mechanism utilized by Nature which has yet to be appreciated.