Molecular mechanism for Rabex-5 GEF activation by Rabaptin-5

Abstract

Rabex-5 and Rabaptin-5 function together to activate Rab5 and further promote early endosomal fusion in endocytosis. The Rabex-5 GEF activity is autoinhibited by the Rabex-5 CC domain (Rabex-5CC) and activated by the Rabaptin-5 C2-1 domain (Rabaptin-5C21) with yet unknown mechanism. We report here the crystal structures of Rabex-5 in complex with the dimeric Rabaptin-5C21 (Rabaptin-5C212) and in complex with Rabaptin-5C212 and Rab5, along with biophysical and biochemical analyses. We show that Rabex-5CC assumes an amphipathic α-helix which binds weakly to the substrate-binding site of the GEF domain, leading to weak autoinhibition of the GEF activity. Binding of Rabaptin-5C21 to Rabex-5 displaces Rabex-5CC to yield a largely exposed substrate-binding site, leading to release of the GEF activity. In the ternary complex the substrate-binding site of Rabex-5 is completely exposed to bind and activate Rab5. Our results reveal the molecular mechanism for the regulation of the Rabex-5 GEF activity.Cells need to allow various molecules to pass through the plasma membrane on their surface. Some molecules have to enter the cell, whereas others have to leave. Cells rely on a process called endocytosis to move large molecules into the cell. This involves part of the membrane engulfing the molecule to form a ‘bubble’ around it. This bubble, which is called an endosome, then moves the molecule to final destination inside the cell.A protein called Rab5 controls how a new endosome is produced. However, before this can happen, various other molecules—including two proteins called Rabex-5 and Rabaptin-5—must activate the Rab5 protein. Exactly how these three proteins interact with each other was unknown.Zhang et al. used X-ray crystallography to examine the structures of the complexes formed when Rabex-5 and Rabaptin-5 bind to each other, both when Rab5 is present, and also when it is absent. Biochemical and biophysical experiments confirmed that the Rabex-5/Rabaptin-5 complex is able to activate Rab5.Zhang et al. also found that Rabex-5, on its own, folds so that the site that normally binds to Rab5 instead binds to a different part of Rabex-5, thus preventing endocytosis. However, when Rabaptin-5 forms a complex with Rabex-5, the Rab5 binding site is freed up.The Rabex-5/Rabaptin-5 complex can switch between a V shape and a linear structure. Binding to Rab5 stabilizes the linear form of the complex, which then helps activate Rab5, and subsequently the activated Rab5 can interact with other downstream molecules, triggering endocytosis.