The Pleckstrin Homology (PH) Domain of the Arf Exchange Factor Brag2 Is an Allosteric Binding Site

Brag2, a Sec7 domain (sec7d)-containing guanine nucleotide exchange factor, regulates cell adhesion and tumor cell invasion. Brag2 catalyzes nucleotide exchange, converting Arf·GDP to Arf·GTP. Brag2 contains a pleckstrin homology (PH) domain, and its nucleotide exchange activity is stimulated by phosphatidylinositol 4,5-bisphosphate (PIP2). Here we determined kinetic parameters for Brag2 and examined the basis for regulation by phosphoinositides. Using myristoylated Arf1·GDP as a substrate, the kcat was 1.8 ± 0.1/s as determined by single turnover kinetics, and the Km was 0.20 ± 0.07 μm as determined by substrate saturation kinetics. PIP2 decreased the Km and increased the kcat of the reaction. The effect of PIP2 required the PH domain of Brag2 and the N terminus of Arf and was largely independent of Arf myristoylation. Structural analysis indicated that the linker between the sec7d and the PH domain in Brag2 may directly contact Arf. In support, we found that a Brag2 fragment containing the sec7d and the linker was more active than sec7d alone. We conclude that Brag2 is allosterically regulated by PIP2 binding to the PH domain and that activity depends on the interdomain linker. Thus, the PH domain and the interdomain linker of Brag2 may be targets for selectively regulating the activity of Brag2. Background: Brag2 is a PH domain-containing Arf guanine nucleotide exchange factor (GEF) that regulates cell adhesion. Results: PIP2 association with the PH domain stimulated Brag2 activity. Regulation was dependent on the N terminus of Arf and independent of the N-terminal myristate. Conclusion: PIP2 binding to the PH domain allosterically modifies Brag2 activity. Significance: A novel regulatory mechanism for GEFs was identified.