[2P-2] Poster 2 (2P-38ー2P-88)

[2P-88(YSA-5)] De Novo Design of Allosteric Sites into Rotary Molecular Motor V₁-ATPase by Restoring Lost Function

^○Takahiro Kosugi^1,2,3,4, Tatsuya Iida^1,2, Mikio Tanabe⁵, Ryota Iino^1,2, Nobuyasu Koga^1,2,3 (1.IMS, 2.SOKENDAI, 3.ExCELLS, NINS, 4.PRESTO, JST, 5.IMSS, KEK)

Allostery produces concerted functions of protein complexes by orchestrating the cooperative work between the constituent subunits. We describe an approach to creating artificial allosteric sites into protein complexes. The key idea is to restore lost functions of pseudo-active sites, which are inferred to have lost their catalytic abilities in evolutionary history, using computational protein design. Molecular motor V₁-ATPase has two different types of interface in the hetero hexametric ring. One is catalytic interface where ATP is hydrolyzed. The other, non-catalytic interface, contains a pseudo-active site which evolutionally lost the ATP- hydrolysis or even binding ability. We created an ATP binding site in the pseudo-active site and performed single-molecule experiments and X-ray crystallography analyses. The results showed that ATP binding to the created ATP binding site promotes ADP-release at the original catalytic site away from the created site. This designed allosteric mechanism made it possible to control and accelerate the rotation. Since pseudo-active sites are widespread in nature, engineering the pseudo-active sites should be one of the approaches for the de novo design of allosteric sites into protein complexes.