Small G-proteins of the Ras superfamily control the temporal and spatial coordination of intracellular signaling networks by acting as molecular on/off switches. Guanine nucleotide exchange factors (GEFs) regulate the activation of these G-proteins through catalytic replacement of GDP by GTP. During nucleotide exchange, three distinct substrate·enzyme complexes occur: a ternary complex with GDP at the start of the reaction (G-protein·GEF·GDP), an intermediary nucleotide-free binary complex (G-protein·GEF), and a ternary GTP complex after productive G-protein activation (G-protein·GEF·GTP). Here, we show structural snapshots of the full nucleotide exchange reaction sequence together with the G-protein substrates and products using Rabin8/GRAB (GEF) and Rab8 (G-protein) as a model system. Together with a thorough enzymatic characterization, our data provide a detailed view into the mechanism of Rabin8/GRAB-mediated nucleotide exchange.
Background: The GEFs Rabin8 and GRAB are activators of the vesicular trafficking regulator Rab8.
Results: The catalytic mechanism of Rabin8/GRAB in Rab8 has been elucidated in biophysical and structural detail.
Conclusion: Rabin8 and GRAB are catalytically moderately efficient enzymes and act by disturbing Mg2+ binding and Rab8-guanine base interactions.
Significance: Obtaining snapshots of the nucleotide exchange reaction is crucial to understanding the mechanism of Rab GEFs.
