Fairlie Gage M J, Nguyen Kha M, Nam Sung-Eun, Shaw Alexandria L, Parson Matthew A H, Shariati Hannah R, Wang Xinyin, Jenkins Meredith L, Gong Michael, Burke John E, Yip Calvin K
Life Sciences Institute, Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, BC, V6T 1Z3, Canada.
Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 2Y2, Canada.
Nat Commun. 2025 Jan 8;16(1):479. doi: 10.1038/s41467-025-55828-8.
The heterodimeric Rab3GAP complex is a guanine nucleotide exchange factor (GEF) for the Rab18 GTPase that regulates lipid droplet metabolism, ER-to-Golgi trafficking, secretion, and autophagy. Why both subunits of Rab3GAP are required for Rab18 GEF activity and the molecular basis of how Rab3GAP engages and activates its cognate substrate are unknown. Here we show that human Rab3GAP is conformationally flexible and potentially autoinhibited by the C-terminal domain of its Rab3GAP2 subunit. Our high-resolution structure of the catalytic core of Rab3GAP, determined by cryo-EM, shows that the Rab3GAP2 N-terminal domain binds Rab3GAP1 via an extensive interface. AlphaFold3 modelling analysis together with targeted mutagenesis and in vitro activity assay reveal that Rab3GAP likely engages its substrate Rab18 through an interface away from the switch and interswitch regions. Lastly, we find that three Warburg Micro Syndrome-associated missense mutations do not affect the overall architecture of Rab3GAP but instead likely interfere with substrate binding.
异源二聚体Rab3GAP复合物是Rab18 GTP酶的鸟嘌呤核苷酸交换因子（GEF），可调节脂滴代谢、内质网到高尔基体的运输、分泌和自噬。Rab18 GEF活性为何需要Rab3GAP的两个亚基以及Rab3GAP如何结合并激活其同源底物的分子基础尚不清楚。在这里，我们表明人类Rab3GAP在构象上具有灵活性，并且可能被其Rab3GAP2亚基的C末端结构域自抑制。我们通过冷冻电镜确定的Rab3GAP催化核心的高分辨率结构表明，Rab3GAP2 N末端结构域通过广泛的界面与Rab3GAP1结合。AlphaFold3建模分析以及靶向诱变和体外活性测定表明，Rab3GAP可能通过远离开关和开关间区域的界面与其底物Rab18结合。最后，我们发现三个与沃伯格微综合征相关的错义突变不会影响Rab3GAP的整体结构，但可能会干扰底物结合。
