Solution structure of the cytohesin-1 (B2-1) Sec7 domain and its interaction with the GTPase ADP ribosylation factor 1

Cytohesin-1 (B2-1) is a guanine nucleotide exchange factor for human ADP ribosylation factor (Arf) GTPases, which are important for vesicular protein trafficking and coatamer assembly in the cell. Cytohesin-1 also has been reported to promote cellular adhesion via binding to the beta 2 integrin cytoplasmic domain. The solution structure of the Sec7 domain of cytohesin-1, which is responsible for both the protein's guanine nucleotide exchange factor function and beta 2 integrin binding, was determined by NMR spectroscopy. The structure consists of 10 alpha-helices that form a unique tertiary fold, The binding between the Sec7 domain and a soluble, truncated version of human Arf-1 was investigated by examining H-1-N-15 and H-1-C-13 chemical shift changes between the native protein and the Sec7/Arf-1 complex. We show that the binding to Arf-l occurs through a large surface on the C-terminal subdomain that is composed of both hydrophobic and polar residues. Structure-based mutational analysis of the cytohesin-1 Sec7 domain has been used to identify residues important for binding to Arf and for mediating nucleotide exchange. Investigations into the interaction between the Sec7 domain and the beta 2 integrin cytoplasmic domain suggest that the two proteins do not interact in the solution phase.