Structural study of Parkinson's disease related protein LRRK2 and vaccinia virus protein A6

Abstract

LRRK2 is a multi-domain protein expressed widely in organs. The mutations of LRRK2 are related to Parkinson's disease. The Roc domain in LRRK2 is a Ras GTPase, and its GTP hydrolysis activity has been proved to regulate kinase activity of the kinase domain. With the structure of GDP bound Roc domain (inactive state) solved, we intend to determine the structure of GTP analog bound Roc domain (active state). The change between them should reveal the dynamic conformational changes in the ROC GTPase domain, and provide important clues on the mechanisms of GTP hydrolysis and signaling. We engineered Roc based on surface entropy reduction and used GDP, AlF4- and GppNp as GTP analogs. Mutants of Roc crystallized but only GDP was found in the binding pocket.

A6 is a vaccinia virus protein is expressed during late translation and is packed in the viron core. The absence of A6 dislocates some viron proteins, disrupts the recruitment of virus membrane and stops virus maturation. There is no homologue structure of A6, so we intend to solve the structure and compare it with other known functional domain structures. In this way, we can expand our understanding of the role A6 plays during vaccinia virus replication. A6 and its homologues are recalcitrant to crystallization, but surface entropy reduction and truncation based on limited proteolysis promoted crystallization. The mutant AC crystal diffracted to 4 A and truncated mutant C diffracted to 8 A. Antibodies including Fabs and SdAbs were also used to assist crystallization. But more combination of A6 and antibodies need to be tried.