摘要
Autophagy is a fundamental cellular process,conserved across species from yeast to mammals,that plays a crucial role in maintaining cellular homeostasis.The functionally conserved MON1-CCZ1(MC1)complex serves as a guanine nucleotide exchange factor(GEF)for the RAB GTPase RAB7A and is indispensable for directing RAB7A recruitment to autophagosome or lysosomal membranes.Despite its critical role,the precise molecular mechanism underlying the assembly of the human MON1A-CCZ1(HsMC1)complex and its specific GEF activity towards RAB7A has remained unclear.In this study,we report the high-resolution cryo-electron microscopy(cryo-EM)structure of the HsMC1 GEF domain in a complex with the nucleotide-free RAB7A^(N125I)at 2.85 A resolution.Our structural data demonstrate that engagement with the HsMC1 complex induces marked conformational shifts in the phosphate-binding loop(P-loop)and SwitchⅠ/Ⅱregions of RAB7A.A striking feature of this complex is the direct interaction between the P-loop of RAB7A and CCZ1,a structural detail not previously observed.Furthermore,biochemical assays targeting residues within InterfaceⅠorⅡof the HsMC1-RAB7A complex highlight their critical role in mediating the interaction and suggest a unique mechanism for nucleotide exchange facilitated by the HsMC1 complex.These findings provide novel molecular insights into the functional mechanisms of the HsMC1-RAB7A complex,offering a robust structural framework to inform future investigations into disease-related targets and therapeutic development.
基金
supported by the grants from the National Natural Science Foundation of China(32201025 to D.T.,32071214 to S.Q.,32470738 to S.Q.,and 32471311 to D.T.)。
