Design of allosteric sites into rotary motor V1-ATPase by restoring lost function of pseudo-active sites

Allostery produces concerted functions of protein complexes by orchestrating the cooperative work between the constituent subunits. Here we describe an approach to create artificial allosteric sites in protein complexes. Certain protein complexes contain subunits with pseudo-active sites, which are believed to have lost functions during evolution. Our hypothesis is that allosteric sites in such protein complexes can be created by restoring the lost functions of pseudo-active sites. We used computational design to restore the lost ATP-binding ability of the pseudo-active site in the B subunit of a rotary molecular motor, V-1-ATPase. Single-molecule experiments with X-ray crystallography analyses revealed that binding of ATP to the designed allosteric site boosts this V-1's activity compared with the wild-type, and the rotation rate can be tuned by modulating ATP's binding affinity. Pseudo-active sites are widespread in nature, and our approach shows promise as a means of programming allosteric control over concerted functions of protein complexes.

Automated summary

Allostery allows for the coordinated function of protein complexes by facilitating cooperative interaction between subunits. This study presents an approach to artificially create allosteric sites in protein complexes. By restoring the lost functions of pseudo-active sites, it is possible to generate allosteric sites within certain protein complexes. Computational design was used to restore ATP-binding ability in the pseudo-active site of the B subunit of V-1-ATPase. Single-molecule experiments with X-ray crystallography analysis demonstrated that binding of ATP to the designed allosteric site enhances the activity of V-1 and allows for modulation of rotation rate through ATP's binding affinity. This approach holds promise for programmable allosteric control of protein complex functions.