Studies on Enzymatic Activity and Protein Stability of Rab6A′ and Rab11A by crystal structure

Abstract

The Ras-superfamily of small GTP-binding proteins (GTPase), plays critical roles in various cellular processes. The activity of this family is controlled by the states of nucleotide binding. When GTP binds to GTPase, it becomes the active form. Once GTP is hydrolyzed to GDP + Pi, the GTPase becomes the inactive form. Rab GTPase is Ras-superfamily and plays an especially important role in vesicle trafficking, which is essential for endocytosis, biosynthesis, secretion, cell differentiation and growth. Because a functional loss of the Rab pathways has been implicated in a variety of disease, the Rab GTPase family has been extensively investigated. Mutant form of Rab GTPase has been commonly utilized for biochemical study of Rab and Rab-related functional studies. Generally, most mutants in catalytic region of Rab GTPase have been constructed by sequence alignment from other GTPases which are already known as active- or inactive form by structural study or biochemical study. Since similar structural of GTPases could show different functional profile, such as binding affinity of ligand, rate of enzyme activity and interacting partners, it is possible that mutation of similar position of amino acid likely showed different activity between Rab GTPases. Therefore, functional activity and molecular mechanism of Rab GTPase mutants must be validated by biochemical analysis and structural analysis. Due to this reason, I undertook to characterize the activity of Rab GTPase mutants. In this study, I crystallized mutant form of Rab6A′and Rab11A and solved crystal structures of them. In the first part, I solved crystal structure of active form of Rab6A and I found that active form of GTPase could trap GDP. This observation indicated that binding of ligand could be various dependent on protein-type and environment around protein. In second part, I found that the stability of inactive Rab11A depended on the occupation of GDP. In the third part, active Rab11A could trap GTP instead of hydrolyzing it, because of absence of water molecule around catalytic pocket. These results provide better understanding for Rab-related study.