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We have previously identified the interaction between mammalian V-ATPase a2-subunit isoform and cytohesin-2 (CTH2) and studied molecular details of binding between these proteins. In particular, we found that six peptides derived from the N-terminal cytosolic domain of a2 subunit (a2N) are involved in interaction with CTH2 (Merkulova, Bakulina, Thaker, Grüber, & Marshansky, 2010). However, the actual 3D binding interface was not determined in that study due to the lack of high-resolution structural information about a-subunits of V-ATPase. Here, using a combination of homology modeling and NMR analysis, we generated the structural model of complete a2N and uncovered the CTH2-binding interface. First, using the crystal-structure of the bacterial M. rubber I-subunit of A-ATPase as a template (Srinivasan, Vyas, Baker, & Quiocho, 2011), we built a homology model of mammalian a2N fragment. Next, we combined it with the determined NMR structures of peptides a2N and a2N of the C-terminal section of a2N. The complete molecular model of a2N revealed that six CTH2 interacting peptides are clustered in the distal and proximal lobe sub-domains of a2N. Our data indicate that the proximal lobe sub-domain is the major interacting site with the Sec7 domain of first CTH2 protein, while the distal lobe sub-domain of a2N interacts with the PH-domain of second CTH2. Indeed, using Sec7/Arf-GEF activity assay we experimentally confirmed our model. The interface formed by peptides a2N and a2N is involved in specific interaction with Sec7 domain and regulation of GEF activity. These data are critical for understanding of the cross-talk between V-ATPase and CTH2 as well as for the rational drug design to regulate their function.

current topics in membranes

Structural model of a2-subunit N-terminus and its binding interface for Arf-GEF CTH2: Implication for regulation of V-ATPase, CTH2 function and rational drug design