Rab3GAP的生化和结构特征揭示了Rab18核苷酸交换活性的见解-动脉网

Biochemical and structural characterization of Rab3GAP reveals insights into Rab18 nucleotide exchange activity

Nature 等信源发布 2025-01-08 18:52

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可切换为仅中文

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Abstract

摘要

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.

异二聚体Rab3GAP复合物是Rab18 GTPase的鸟嘌呤核苷酸交换因子（GEF），可调节脂滴代谢，ER到高尔基体的运输，分泌和自噬。为什么Rab3GAP的两个亚基都是Rab18 GEF活性所必需的，以及Rab3GAP如何参与并激活其同源底物的分子基础尚不清楚。

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.

在这里，我们显示人Rab3GAP在构象上是柔性的，并且可能被其Rab3GAP2亚基的C末端结构域自动抑制。我们通过cryo-EM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结合Rab3GAP1。

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..

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Warburg Micro综合征相关的错义突变不会影响Rab3GAP的整体结构，而是可能干扰底物结合。。

Introduction

简介

Different membrane trafficking pathways mediate the movement of proteins and other macromolecules between different subcellular compartments, which is critical to the maintenance of organelle identity and the execution of complex processes from protein secretion to protein glycosylation

不同的膜运输途径介导蛋白质和其他大分子在不同亚细胞区室之间的运动，这对于维持细胞器身份和执行从蛋白质分泌到蛋白质糖基化的复杂过程至关重要

. These pathways employ a common mechanism that involves selecting and packaging cargo into a membrane-bound transport vesicle at the donor organelle, moving the cargo-laden vesicle along the cytoskeletal filaments, and docking and fusion of the incoming transport vesicle with the membrane of the acceptor organelle where the content is delivered.

这些途径采用了一种常见的机制，包括选择货物并将其包装到供体细胞器的膜结合运输囊泡中，沿着细胞骨架丝移动装载货物的囊泡，以及将进入的运输囊泡与传递内容物的受体细胞器的膜对接和融合。

. The different steps of membrane trafficking rely on the coordinated activity of specialized factors. These factors include adapter proteins that are involved in cargo selection, coat proteins that mediate vesicle budding, motor proteins that move transport vesicles, tethers that link the incoming vesicle to the acceptor membrane, and soluble N-ethylmaleimide-sensitive factor activating receptors (SNAREs) that bring two membranes close, reducing the energy barrier for fusion.

.膜运输的不同步骤取决于专业因素的协调活动。这些因素包括参与货物选择的衔接蛋白，介导囊泡出芽的外壳蛋白，移动运输囊泡的运动蛋白，将进入的囊泡连接到受体膜的系链，以及可溶性N-乙基马来酰亚胺敏感因子激活受体（SNARE），使两个膜靠近，减少融合的能量屏障。

. Fundamental to these membrane trafficking steps are members of the Ras superfamily of GTPases, including the Arf, Arl, and Rab subfamilies. The coordinated activation and inactivation of these GTPases control the targeted recruitment of their effectors which is critical in providing spatiotemporal control of membrane transport.

这些膜运输步骤的基础是GTPases Ras超家族的成员，包括Arf，Arl和Rab亚家族。这些GTP酶的协调激活和失活控制了其效应子的靶向募集，这对于提供膜运输的时空控制至关重要。

Amongst the different families of small GTPases, the Rab family GTPases are considered master regulators of membrane trafficking

在不同的小GTPases家族中，Rab家族GTPases被认为是膜运输的主要调节因子

. With 11 members in yeast and over 60 members in human cells, different Rabs are uniquely distributed across distinct membrane compartments. Like other members of the Ras superfamily, Rabs switch between a GDP-bound inactive state and a GTP-bound active state, as well as between a cytoplasmic form and a membrane-bound form through their prenylated C-termini and the action of chaperone-like GDP dissociation inhibitors (GDI).

酵母中有11个成员，人类细胞中有60多个成员，不同的Rabs独特地分布在不同的膜区室中。像Ras超家族的其他成员一样，Rabs通过其异戊二烯化的C末端和伴侣样GDP解离抑制剂（GDI）的作用，在GDP结合的非活性状态和GTP结合的活性状态之间，以及在细胞质形式和膜结合形式之间切换。

Upon activation and localization to the proper membrane compartments, Rab GTPases coordinate different transport steps by recruiting diverse effectors.

激活并定位到适当的膜区室后，Rab GTPases通过募集不同的效应子来协调不同的转运步骤。

. The activation of Rab GTPases is controlled by guanine nucleotide exchange factors (GEF) that catalyze the exchange of bound GDP with GTP and GTPase activating proteins (GAP) that accelerate the intrinsically slow GTP hydrolysis rate of the Rab

。Rab GTPases的激活受鸟嘌呤核苷酸交换因子（GEF）控制，该因子催化结合的GDP与GTP和GTPase激活蛋白（GAP）的交换，从而加速Rab固有的缓慢GTP水解速率

. Some GEFs also play a role in assisting recruitment of their substrate Rabs to specific membrane compartments

。一些GEF还可以帮助将其底物Rabs募集到特定的膜区室中

. While almost all known Rab GAPs belong to the TBC (Tre-2/Bub/Cdc16) domain family, Rab GEFs are divergent in primary sequence and structure

尽管几乎所有已知的Rab缺口都属于TBC（Tre-2/Bub/Cdc16）域家族，但Rab GEF在一级序列和结构上存在差异

. Interestingly, despite the diversity in composition and structure of Rab GEFs, all GEFs characterized structurally thus far were observed to utilize a similar strategy to promote nucleotide exchange of their substrate Rabs

有趣的是，尽管Rab GEF的组成和结构多种多样，但到目前为止，观察到所有在结构上表征的GEF都利用类似的策略来促进其底物Rab的核苷酸交换

. This involves binding the nucleotide-bound Rab at its switch I/switch II and interswitch regions and inducing conformational changes that open the nucleotide-binding pocket and hinder the binding of a magnesium ion, leading to the release of the bound nucleotide

。这涉及在其开关I/开关II和开关间区域结合核苷酸结合的Rab，并诱导构象变化，从而打开核苷酸结合口袋并阻碍镁离子的结合，从而导致结合核苷酸的释放

. After this, the GEF forms a high-affinity complex with the nucleotide-free Rab. The higher cellular concentration of GTP compared to GDP results in the conversion of the Rab to the GTP-bound form and displacement of the GEF

在此之后，GEF与无核苷酸的Rab形成高亲和力复合物。与GDP相比，GTP的细胞浓度更高，导致Rab转化为GTP结合形式，并取代了GEF

Rab3GAP is a heterodimeric complex composed of the 130 kDa Rab3GAP1 and the 150 kDa Rab3GAP2 subunits. Originally isolated from rat brain tissues and identified to be the GAP for brain-specific Rab3, Rab3GAP was later shown to exhibit GEF activity on Rab18, a Rab that is ubiquitously expressed in all human tissues and is one of the six primordial Rabs found in the last eukaryotic common ancestor.

Rab3GAP是由130 kDa Rab3GAP1和150 kDa Rab3GAP2亚基组成的异二聚体复合物。Rab3GAP最初是从大鼠脑组织中分离出来的，被鉴定为大脑特异性Rab3的GAP，后来被证明对Rab18具有GEF活性，Rab18是一种在所有人体组织中普遍表达的Rab，是在最后一个真核生物共同祖先中发现的六个原始Rab之一。

. Subsequent studies revealed that mutations in genes encoding Rab3GAP1, Rab3GAP2, and Rab18 cause Warburg Micro Syndrome (WMS), an autosomal recessive genetic disorder characterized by postnatal growth retardation, microcephaly, congenital cataracts, optical atrophy, spastic paraplegia, hypogonadism, and delayed motor and intellectual development.

随后的研究表明，编码Rab3GAP1，Rab3GAP2和Rab18的基因突变会导致Warburg Micro Syndrome（WMS），这是一种常染色体隐性遗传疾病，其特征是出生后发育迟缓，小头畸形，先天性白内障，视神经萎缩，痉挛性截瘫，性腺功能减退以及运动和智力发育迟缓。

. These observations suggest that activating Rab18 might be the primary function for Rab3GAP in most cell types. Rab3GAP facilitates the recruitment of Rab18 to lipid droplets (LDs), the endoplasmic reticulum (ER), and the Golgi apparatus

这些观察结果表明，在大多数细胞类型中，激活Rab18可能是Rab3GAP的主要功能。Rab3GAP促进Rab18向脂滴（LDs）、内质网（ER）和高尔基体的募集

. Rab18 has been implicated in LD metabolism, establishing and/or maintaining ER morphology, ER-to-Golgi trafficking, autophagy, secretion, peroxisome regulation, and viral assembly

Rab18与LD代谢，ER形态的建立和/或维持，ER到高尔基体的运输，自噬，分泌，过氧化物酶体调节和病毒组装有关

. Rab18 has also been shown to promote the formation of ER-LD contact sites by recruiting the NAG-RINT1-ZW10 (NRZ) tethering complex and ER-associated SNAREs, Syntaxin18, Use1, and BNIP1, to facilitate lipid transfer between these organelles

Rab18还被证明可以通过募集NAG-RINT1-ZW10（NRZ）束缚复合物和ER相关的SNARE，Syntaxin18，Use1和BNIP1来促进ER-LD接触位点的形成，以促进这些细胞器之间的脂质转移

. Apart from the findings that both subunits are required for the Rab18 GEF activity of Rab3GAP and that three WMS-associated point mutations disrupt this activity, little is known about the biochemical and structural properties of Rab3GAP

除了发现Rab3GAP的Rab18 GEF活性需要两个亚基以及三个与WMS相关的点突变破坏了该活性外，对Rab3GAP的生化和结构特性知之甚少

. Furthermore, how this heterodimeric complex engages its cognate substrate and promotes GDP-to-GTP exchange and how WMS-associated point mutations affect this critical function remain obscure due to the lack of sequence and structural similarities to other Rab GEFs. Lastly, what allows Rab3GAP to perform two seemingly opposite functions in Rab3 inactivation and Rab18 activation is still a mystery..

此外，由于缺乏与其他Rab GEF的序列和结构相似性，这种异二聚体复合物如何与其同源底物结合并促进GDP与GTP的交换以及WMS相关的点突变如何影响这一关键功能仍然不清楚。最后，是什么使Rab3GAP在Rab3失活和Rab18激活中执行两个看似相反的功能仍然是一个谜。。

In this work, we characterize the biochemical and structural properties of human Rab3GAP using a combination of negative stain electron microscopy (EM) and cryo-EM, in vitro GEF assays, hydrogen-deuterium exchange mass spectrometry (HDX-MS), and in silico structural modeling. We find that full-length Rab3GAP is conformationally flexible and adopts a range of open-to-closed conformations.

在这项工作中，我们使用负染电子显微镜（EM）和冷冻EM，体外GEF测定，氢-氘交换质谱（HDX-MS）和计算机结构建模的组合来表征人Rab3GAP的生化和结构特性。我们发现全长Rab3GAP在构象上是灵活的，并且采用了一系列从开放到闭合的构象。

We observe that Rab3GAP exhibits enhanced Rab18 GEF activity in the presence of a membrane. We also find that core Rab3GAP composed of Rab3GAP1 and RabGAP2 devoid of its C-terminal domain, shows increased Rab18 GEF activity, raising the possibility that the full-length complex is autoinhibited. We subsequently determine the high-resolution structure of core Rab3GAP by cryo-EM and validate the observed intersubunit interface by hydrogen-deuterium exchange mass spectrometry (HDX-MS).

我们观察到Rab3GAP在膜存在下表现出增强的Rab18 GEF活性。我们还发现，由Rab3GAP1和RabGAP2组成的核心Rab3GAP没有其C端结构域，显示出Rab18 GEF活性增加，从而增加了全长复合物被自动抑制的可能性。随后，我们通过cryo-EM确定了核心Rab3GAP的高分辨率结构，并通过氢-氘交换质谱（HDX-MS）验证了观察到的亚基间界面。

Furthermore, by combining AlphaFold3 modeling, targeted mutagenesis in conjunction with in vitro GEF assays, confocal microscopy, and WMS disease mutation mapping, we find that Rab3GAP likely binds its substrate Rab18 through a platform opposite of the critical switch and interswitch regions. Collectively, our work provides biochemical and structural insights on human Rab3GAP and how this complex may engage its substrate Rab18, and presents a molecular framework to predict how WMS disease mutants interfere with Rab18 binding and activation..

此外，通过将AlphaFold3建模，靶向诱变与体外GEF分析，共聚焦显微镜和WMS疾病突变作图相结合，我们发现Rab3GAP可能通过与关键开关和开关间区域相对的平台结合其底物Rab18。总的来说，我们的工作提供了关于人类Rab3GAP的生化和结构见解，以及该复合物如何与其底物Rab18结合，并提供了一个分子框架来预测WMS疾病突变体如何干扰Rab18的结合和激活。。

Results

结果

Human Rab3GAP adopts a flexible bilobal overall architecture

人类Rab3GAP采用灵活的双叶整体结构

To facilitate biochemical and structural analyses, we established a baculovirus-insect cell-based method to reconstitute the dimeric complex of human Rab3GAP1-Rab3GAP2. In brief, we constructed a plasmid encoding a Rab3GAP1-Rab3GAP2 two-gene cassette using the biGBac system and then generated baculoviruses for infecting and co-expressing the two Rab3GAP subunits in Sf9 cells.

为了促进生化和结构分析，我们建立了基于杆状病毒-昆虫细胞的方法来重建人Rab3GAP1-Rab3GAP2的二聚体复合物。简而言之，我们使用biGBac系统构建了编码Rab3GAP1-Rab3GAP2双基因盒的质粒，然后产生了杆状病毒，用于感染和共表达Sf9细胞中的两个Rab3GAP亚基。

. Reconstituted human Rab3GAP was subsequently purified using anti-FLAG affinity chromatography and size exclusion chromatography. Rab3GAP elutes at a volume corresponding to the predicted mass for a ~300 kDa complex, which is consistent with the previously proposed 1:1 subunit stoichiometry for this complex (Fig. .

随后使用抗FLAG亲和色谱法和尺寸排阻色谱法纯化重构的人Rab3GAP。Rab3GAP以与〜300kDa复合物的预测质量相对应的体积洗脱，这与先前提出的该复合物的1:1亚基化学计量一致（图）。

1a级

)

. We next examined the ability of the reconstituted complex to promote nucleotide exchange by carrying out an in vitro GEF assay on recombinant human Rabs loaded with the fluorescent GDP analog 3-(N-methyl-anthraniloyl)-2-deoxy-GDP (Mant-GDP) (Fig.

接下来，我们通过对载有荧光GDP类似物3-（N-甲基-邻氨基苯甲酰基）-2-脱氧-GDP（Mant-GDP）的重组人Rabs进行体外GEF测定，检查了重组复合物促进核苷酸交换的能力（图）。

1b级

). The fluorescent signal of Mant-GDP bound to a Rab is substantially stronger than free Mant-GDP, allowing nucleotide dissociation to be monitored as a decrease in fluorescence

)。与Rab结合的Mant-GDP的荧光信号明显强于游离的Mant-GDP，从而可以监测核苷酸解离作为荧光的减少

. We found that recombinant Rab3GAP promotes GDP release of its cognate substrate Rab18 but not Rab11a (Supplementary Fig.

我们发现重组Rab3GAP促进其同源底物Rab18的GDP释放，但不促进Rab11a的GDP释放（Supplementary Fig.）。

). We also validated the previous finding that both subunits of Rab3GAP are required for this Rab18 GEF activity as Rab3GAP1 subunit alone cannot promote nucleotide exchange (Fig.

)。我们还验证了先前的发现，即Rab3GAP的两个亚基都是这种Rab18 GEF活性所必需的，因为单独的Rab3GAP1亚基不能促进核苷酸交换（图）。

1c级

)

. By measuring Mant-GDP release at different concentrations of Rab3GAP, we determined that Rab3GAP has a catalytic efficiency of 2.5 × 10

通过测量不同浓度Rab3GAP下Mant GDP的释放，我们确定Rab3GAP的催化效率为2.5×10

−1

, a value that is comparable to those reported for other Rab GEFs (Fig.

，该值与其他Rab GEF报告的值相当（图）。

1维

)

Fig. 1: In vitro GEF assays and negative stain EM analysis reveal that reconstituted human Rab3GAP is a Rab18 GEF exhibiting conformational flexibility.

图1：体外GEF测定和阴性染色EM分析表明，重组人Rab3GAP是具有构象柔性的Rab18 GEF。

一

Analytical gel filtration elution profile of Rab3GAP on an ENrich™ SEC 650 column and representative SDS-PAGE gel of the reconstituted complex stained with Coomassie Blue. Analytical gel filtration and SDS-PAGE gels were performed in biological triplicate (

Rab3GAP在ENrich™SEC 650色谱柱上的分析凝胶过滤洗脱曲线和用考马斯蓝染色的重构复合物的代表性SDS-PAGE凝胶。分析凝胶过滤和SDS-PAGE凝胶一式三份进行(

= 3).

b类

Schematic of in vitro GEF assays to assess nucleotide exchange activity towards Rabs using the fluorescent GDP analog, Mant-GDP.

使用荧光GDP类似物Mant-GDP评估针对Rabs的核苷酸交换活性的体外GEF测定的示意图。

c级

GEF assays on Rab18 with Rab3GAP1 and Rab3GAP1/2. Nucleotide exchange was detected by measuring fluorescent decrease in reactions containing 0 nM GEF (Mock) or 300 nM GEF with 4 µM Mant-GDP loaded Rab18 and 100 µM GTPγS. Data are presented as mean with error bars showing SEM for assays performed in technical triplicate (.

用Rab3GAP1和Rab3GAP1/2对Rab18进行GEF分析。通过测量含有0 nM GEF（模拟物）或300 nM GEF与4µM Mant-GDP负载的Rab18和100µM GTPγS的反应中的荧光减少来检测核苷酸交换。数据以平均值表示，误差线显示SEM用于技术一式三份进行的测定（。

= 3).

Determination of catalytic efficiency (

催化效率的测定(

cat

猫

) using reactions with Rab3GAP1 + 2 (0–300 nM), 4 µM Mant-GDP loaded Rab18 and 100 µM GTPγS. Reactions were conducted in technical triplicate (

)使用与Rab3GAP1+ 2（0–300 nM）的反应，4µM Mant-GDP负载的Rab18和100µM GTPγS。反应一式三份进行(

= 3). Data are presented as mean with error bars showing SEM and

数据以平均值表示，误差线显示SEM和

cat

猫

was calculated as described previously

如前所述进行计算

2D class averages showing the general architecture of Rab3GAP and schematic representation of core Rab3GAP (pink) and the flexible arm (orange) adopting closed, V-shaped, and extended conformations. Source data are provided as a Source Data file.

2D类平均值显示了Rab3GAP的一般结构，以及采用闭合，V形和扩展构象的核心Rab3GAP（粉红色）和柔性臂（橙色）的示意图。源数据作为源数据文件提供。

Full size image

全尺寸图像

Having successfully reconstituted Rab3GAP, we next examined the overall architecture of this complex by negative stain single-particle EM. Class averages generated from our two-dimensional (2D) analysis revealed that Rab3GAP is composed of an extended but rigid arm region attached to the head of a tadpole-shaped body (Fig. .

成功重建Rab3GAP后，我们接下来通过负染色单粒子EM检查了该复合物的整体结构。从我们的二维（2D）分析产生的类平均值显示，Rab3GAP由一个延伸但刚性的臂区域组成，该臂区域连接到蝌蚪形身体的头部（图。

1e级

). The gallery of class averages revealed that the arm can rotate around the junction point at the head of up to 180° with respect to the body and this results in a range of architectures from fully clasped V-shaped to fully opened I-shaped.

)。平均水平画廊显示，手臂可以围绕头部的连接点旋转，相对于身体高达180°，这导致了一系列结构，从完全扣紧的V形到完全打开的I形。

The C-terminal domain of Rab3GAP2 confers flexibility to Rab3GAP

Rab3GAP2的C末端结构域赋予Rab3GAP灵活性

The conformational heterogeneity of Rab3GAP posed technical challenges to high-resolution cryo-EM analysis. To mitigate this problem, we investigated the basis of the conformational flexibility of the arm region by first examining the AlphaFold predicted structural models of human Rab3GAP1 and Rab3GAP2.

Rab3GAP的构象异质性对高分辨率低温电磁分析提出了技术挑战。为了缓解这个问题，我们通过首先检查人Rab3GAP1和Rab3GAP2的AlphaFold预测结构模型，研究了臂区构象灵活性的基础。

Rab3GAP2 is predicted to be composed of two globular domains that show low predicted alignment error (PAE) joined by an unstructured linker (Fig. .

预计Rab3GAP2由两个球状结构域组成，这些结构域显示出由非结构化接头连接的低预测比对误差（PAE）（图。

2a级

and Supplementary Fig.

和补充图。

2a, b

2a、b

). Based on the predicted Rab3GAP2 structural model, we designed two expression constructs encoding either Rab3GAP2N (N-terminal domain: 1–544) or Rab3GAP2C (C-terminal domain: 545–1393) and then prepared two biGBac vectors encoding Rab3GAP1 and one of the Rab3GAP2 domains. We found that Rab3GAP1 is only capable of binding the N-terminal domain of Rab3GAP2, and we were able to purify the reconstituted Rab3GAP1-Rab3GAP2N complex (Supplementary Fig. .

)。基于预测的Rab3GAP2结构模型，我们设计了两个编码Rab3GAP2N（N端结构域：1-544）或Rab3GAP2C（C端结构域：545-1393）的表达构建体，然后制备了两个编码Rab3GAP1的biGBac载体和一个Rab3GAP2结构域。我们发现Rab3GAP1仅能够结合Rab3GAP2的N末端结构域，并且我们能够纯化重组的Rab3GAP1-Rab3GAP2N复合物（补充图）。

2摄氏度

). This truncated complex eluted as a monodisperse peak with 1:1 subunit stoichiometry in size exclusion chromatography (Fig.

)。这种截短的复合物在尺寸排阻色谱中以1:1亚基化学计量的单分散峰洗脱（图）。

2b级

). We next subjected the Rab3GAP1-Rab3GAP2N complex to an in vitro GEF assay and found that this complex retains the ability to promote Rab18 nucleotide exchange, revealing that the C-terminal domain of Rab3GAP2 is not required for GEF activity (Fig.

)。接下来，我们对Rab3GAP1-Rab3GAP2N复合物进行了体外GEF测定，发现该复合物保留了促进Rab18核苷酸交换的能力，表明Rab3GAP2的C末端结构域不是GEF活性所必需的（图）。

2摄氏度

). In light of this finding, we hereafter refer the Rab3GAP1-Rab3GAP2N complex as “core Rab3GAP” and Rab3GAP1-Rab3GAP2 as “full-length Rab3GAP”. We next analyzed core Rab3GAP by negative stain EM. Our 2D analysis showed that this truncated complex is structurally homogeneous and retains only the tadpole-shaped body without the flexible arm (Fig. .

)。鉴于这一发现，我们在下文中将Rab3GAP1-Rab3GAP2N复合物称为“核心Rab3GAP”，将Rab3GAP1-Rab3GAP2称为“全长Rab3GAP”。接下来，我们通过负染EM分析了核心Rab3GAP。我们的2D分析表明，这种截短的复合物在结构上是均匀的，仅保留了蝌蚪形的身体，而没有柔性臂（图。

二维

). In summary, we were able to identify that the Rab3GAP2 C-terminal domain confers conformational flexibility to full-length Rab3GAP, and this domain is not required for Rab18 GEF activity.

)。总之，我们能够确定Rab3GAP2 C末端结构域赋予全长Rab3GAP构象灵活性，而Rab18 GEF活性不需要该结构域。

Fig. 2: In vitro GEF assays and negative stain EM analysis reveal that the reconstituted core Rab3GAP complex retains GEF activity towards Rab18 and adopts only one main conformation.

图2：体外GEF测定和阴性染色EM分析表明，重组的核心Rab3GAP复合物保留了对Rab18的GEF活性，并且仅采用一种主要构象。

一

Schematic of Rab3GAP1 with GAP domain annotated, and Rab3GAP2 domain 1 and Rab3GAP2 domain 2 connected by a flexible linker.

注释了GAP域的Rab3GAP1的示意图，以及通过柔性接头连接的Rab3GAP2域1和Rab3GAP2域2的示意图。

b类

Analytical gel filtration elution profile of core Rab3GAP on an ENrich™ SEC 650 column and representative SDS-PAGE gel of the reconstituted complex stained with Coomassie Blue. Analytical gel filtration and SDS-PAGE gels were performed in biological triplicate (

核心Rab3GAP在ENrich™SEC 650色谱柱上的分析凝胶过滤洗脱曲线，以及用考马斯亮蓝染色的重组复合物的代表性SDS-PAGE凝胶。分析凝胶过滤和SDS-PAGE凝胶一式三份进行(

= 3).

c级

GEF assays on core Rab3GAP. Nucleotide exchange was detected by measuring fluorescent decrease in reactions containing 0 nM GEF (Mock) or 300 nM GEF with 4 µM Mant-GDP loaded Rab18 and 100 µM GTPγS. Data are presented as mean with error bars showing SEM for assays performed in technical triplicate (.

核心Rab3GAP的GEF分析。通过测量含有0 nM GEF（模拟物）或300 nM GEF与4µM Mant-GDP负载的Rab18和100µM GTPγS的反应中的荧光减少来检测核苷酸交换。数据以平均值表示，误差线显示SEM用于技术一式三份进行的测定（。

= 3).

Representative 2D class averages comparing the general architecture of core Rab3GAP to full-length Rab3GAP. Schematic showing only a single conformation for core Rab3GAP compared to the open and closed conformation of full-length Rab3GAP, with Rab3GAP1 in blue and Rab3GAP2 in pink. Source data are provided as a Source Data file..

比较核心Rab3GAP与全长Rab3GAP的一般体系结构的代表性2D类平均值。示意图显示，与全长Rab3GAP的开放和闭合构象相比，核心Rab3GAP仅具有单一构象，其中Rab3GAP1为蓝色，Rab3GAP2为粉红色。源数据作为源数据文件提供。。

Full size image

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Rab3GAP GEF activity is enhanced by membrane-anchored Rab18

膜锚定的Rab18增强了Rab3GAP GEF的活性

Having confirmed that core Rab3GAP represents the minimal machinery necessary to catalyze Rab18 nucleotide exchange, we next compared the GEF activity of core Rab3GAP to full-length Rab3GAP. Intriguingly, we found that the core complex exhibits enhanced Rab18 GEF activity, with its catalytic efficiency (5.5 × 10.

在确认核心Rab3GAP代表催化Rab18核苷酸交换所需的最小机制后，我们接下来将核心Rab3GAP的GEF活性与全长Rab3GAP进行了比较。有趣的是，我们发现核心复合物表现出增强的Rab18-GEF活性，其催化效率（5.5×） 10.

−1

) more than twice as high as the full-length complex (Fig.

)超过全长复合体的两倍（图）。

3a, b

3a，b

). This raises the possibility that the activity of full-length Rab3GAP is autoinhibited by the C-terminal domain of Rab3GAP2. Previous studies showed that many Rab GEFs show enhanced rates when their substrates are presented on a membrane

)。这增加了全长Rab3GAP的活性被Rab3GAP2的C端结构域自动抑制的可能性。先前的研究表明，许多Rab GEF在其底物呈现在膜上时显示出增强的速率

. To determine if Rab3GAP activity is affected by a membrane, we incubated Mant-GDP-loaded, C-terminally His-tagged Rab18 with 100 nm-diameter lipid vesicles containing Ni-NTA conjugated lipids and then carried out an in vitro GEF assay using the resulting membrane-anchored Rab18 as a substrate (Fig. .

为了确定Rab3GAP活性是否受到膜的影响，我们将Mant-GDP负载的C末端His标记的Rab18与含有Ni-NTA缀合脂质的100 nm直径脂质囊泡孵育，然后使用所得膜锚定的Rab18作为底物进行体外GEF测定（图。

). We found that the GEF activity of both full-length and core Rab3GAP increased more than ten-fold when Rab18 was presented on a membrane, with a catalytic efficiency of 6.6 × 10

)。我们发现，当Rab18出现在膜上时，全长和核心Rab3GAP的GEF活性都增加了十倍以上，催化效率为6.6×10

−1

for FL Rab3GAP and 4.4 × 10

对于FL Rab3GAP和4.4×10

−1

for core Rab3GAP (Fig.

对于核心Rab3GAP（图）。

). Finally, we carried out systematic GEF assays using Rab18 anchored to vesicles with different lipid compositions mimicking different cellular organelles (Fig.

)。最后，我们使用Rab18进行了系统的GEF分析，Rab18锚定在具有不同脂质成分的囊泡上，模拟不同的细胞器（图）。

3e公司

). Ordinary one-way ANOVA showed that varying the lipid composition of the vesicles does not result in significant differences in nucleotide exchange rates (

)。普通的单因素方差分析表明，改变囊泡的脂质组成不会导致核苷酸交换率的显著差异(

obs

obs公司

), suggesting that the enhancement in GEF activity is not induced by a specific type of lipid (Fig.

)，表明GEF活性的增强不是由特定类型的脂质诱导的（图）。

3f级

Fig. 3: In vitro GEF assays reveal that core Rab3GAP has enhanced activity and membrane-presentation of Rab18 causes increased nucleotide exchange rate.

图3：体外GEF测定显示核心Rab3GAP具有增强的活性，Rab18的膜呈递导致核苷酸交换率增加。

一

In vitro GEF assays with full-length Rab3GAP or core Rab3GAP to detect activity towards Rab18. Nucleotide exchange was detected by measuring fluorescent decrease in reactions containing 0 nM GEF (Mock) or 300 nM GEF with 4 µM Mant-GDP loaded Rab18 and 100 µM GTPγS. Data are presented as mean with error bars showing SEM for assays performed in technical triplicate (.

用全长Rab3GAP或核心Rab3GAP进行体外GEF测定，以检测对Rab18的活性。通过测量含有0 nM GEF（模拟物）或300 nM GEF与4µM Mant-GDP负载的Rab18和100µM GTPγS的反应中的荧光减少来检测核苷酸交换。数据以平均值表示，误差线显示SEM用于技术一式三份进行的测定（。

= 3). Data from Fig.

= 3）。来自图的数据。

2摄氏度

was used to generate this plot.

用于生成此图。

b类

Determination of catalytic efficiency (

催化效率的测定(

cat

猫

) using reactions with full-length Rab3GAP or core Rab3GAP (0–300 nM), 4 µM Mant-GDP loaded Rab18 and 100 µM GTPγS. Reactions were conducted in technical triplicate (

)使用与全长Rab3GAP或核心Rab3GAP（0-300nm）的反应，4µM Mant-GDP负载的Rab18和100µM GTPγS。反应一式三份进行(

= 3). Data are presented as mean with error bars showing SEM and

数据以平均值表示，误差线显示SEM和

cat

猫

was calculated as described previously

如前所述进行计算

. Data from Fig.

.来自Fig.的数据。

1维

was used to generate this plot.

用于生成此图。

c级

Schematic of Mant-GDP based GEF activation assay with C-terminally His-tagged Rab18 anchored to NiNTA-containing lipid vesicles.

基于Mant-GDP的GEF激活测定的示意图，其中C末端带有His标签的Rab18锚定在含有NiNTA的脂质囊泡上。

Evaluation of membrane regulation for full-length or core Rab3GAP activity towards Rab18. Catalytic efficiency (

评估针对Rab18的全长或核心Rab3GAP活性的膜调节。催化效率(

cat

猫

) was calculated using reactions with full-length Rab3GAP or core Rab3GAP (0–15 nM), 4 µM Mant-GDP loaded Rab18, 0.2 mg mL

)使用与全长Rab3GAP或核心Rab3GAP（0-15nm）的反应进行计算，4µM Mant GDP负载的Rab18，0.2毫克毫升

−1

liposomes (75% PC, 20% PE, 5% 18:1 DGS-NTA(Ni), 100 nm) and 100 µM GTPγS. Reactions were conducted in technical triplicate (

脂质体（75%PC，20%PE，5%18:1 DGS-NTA（Ni），100 nm）和100µM GTPγS。反应一式三份进行(

= 3). Data are presented as mean with error bars showing SEM and

数据以平均值表示，误差线显示SEM和

cat

猫

was calculated as described previously

如前所述进行计算

Comparison between various membrane compositions with respect to regulation of Rab3GAP activity. Nucleotide exchange was detected by measuring fluorescent decrease in reactions containing 300 nM GEF, 4 µM Mant-GDP loaded Rab18, 100 µM GTPγS and 100 nm extruded liposomes with various lipid composition (Supplementary Table .

关于Rab3GAP活性调节的各种膜组合物之间的比较。通过测量含有300 nM GEF的反应中的荧光减少来检测核苷酸交换，4µM Mant GDP负载的Rab18，具有各种脂质组成的100μMGTPγS和100 nm挤出脂质体（补充表）。

) at 0.2 mg mL

)0.2

−1

. Data are presented as mean with error bars showing SEM for assays performed in technical triplicate (

.数据以平均值表示，误差线显示SEM，用于一式三份进行的分析(

= 3).

f级

Data from

数据来自

was fit to a non-linear one-phase exponential decay model to determine the rate of nucleotide exchange (

拟合非线性单相指数衰减模型以确定核苷酸交换速率(

obs

obs公司

). Data are presented as mean with error bars showing SEM.

)。数据以平均值表示，误差线显示SEM。

values were generated using an ordinary one-way Anova (ns indicates

数值是使用普通的单向方差分析（ns表示

> 0.05). Source data are provided as a Source Data file.

>0.05）。源数据作为源数据文件提供。

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Cryo-EM structure of human core Rab3GAP

人核心Rab3GAP的低温电磁结构

Knowing that core Rab3GAP retains Rab18 GEF activity and is structurally homogeneous, we next pursued high-resolution structural analysis of this complex using cryo-EM. By performing PEGylation on core Rab3GAP, we were able to minimize aggregation at high protein concentrations which we observed in our initial vitrification trials.

知道核心Rab3GAP保留了Rab18 GEF活性并且结构均一，我们接下来使用cryo-EM对该复合物进行了高分辨率结构分析。通过对核心Rab3GAP进行PEG化，我们能够最大程度地减少高蛋白浓度下的聚集，这是我们在最初的玻璃化试验中观察到的。

. We subsequently acquired a cryo-EM dataset of 19,822 movies. The class averages obtained from the 2D analysis showed that the tadpole-shaped core Rab3GAP contains a knob-shaped region for the head connected to a central body density that carries an extended curved tail (Supplementary Fig.

。我们随后获得了19822部电影的cryo-EM数据集。从2D分析获得的类平均值表明，蝌蚪形核心Rab3GAP包含一个旋钮形区域，头部连接到一个中央身体密度，该密度带有一个延伸的弯曲尾巴（Supplementary Fig.）。

). The curved tail is blurry, suggesting that core Rab3GAP contains another conformationally flexible region. Local refinement to this flexible tail region however did not improve the resolution of the tail region. Following multiple rounds of 3D classification and local refinement to improve the rigid region of the core, we obtained a final density map with an average resolution of 3.37 Å from a final stack of 170,636 particles (Fig. .

)。弯曲的尾巴模糊不清，表明核心Rab3GAP包含另一个构象灵活的区域。然而，对该柔性尾部区域的局部细化并没有提高尾部区域的分辨率。经过多轮3D分类和局部细化以改善核心的刚性区域后，我们从最终的170636个粒子堆栈中获得了平均分辨率为3.37Å的最终密度图（图）。

4a级

and Supplementary Fig.

和补充图。

). We could visualize side chain densities in the central core but the map region proximal to the tail is smeared and has a lower local resolution (Fig.

)。我们可以看到中央核心的侧链密度，但靠近尾部的map区域被涂抹并且具有较低的局部分辨率（图）。

4b, c

4b，c

). We next iteratively fit the AlphaFold2-predicted models of the Rab3GAP1 and Rab3GAP2 subunits into the density map and then further optimized the fit and refined the structural model (Fig.

)。接下来，我们将Rab3GAP1和Rab3GAP2亚基的AlphaFold2预测模型迭代拟合到密度图中，然后进一步优化拟合并改进结构模型（图）。

4d级

). The flexible tail, which was excluded in local refinement, comprised residues 332 to 781 of Rab3GAP1 and could not be accurately modeled. Our final structural model consists of residues 19 to 331 and 782 to 981 for Rab3GAP1 and residues 2 to 544 for Rab3GAP2. We were unable to model the first N-terminal 18 residues, residues 242 to 263, 902 to 929 of Rab3GAP1, and the N-terminal residue, residues 27 to 60, 328 to 363, and 437 to 456 of Rab3GAP2 due to the lack of or poorly defined densities (Fig. .

)。在局部细化中被排除的柔性尾巴包含Rab3GAP1的残基332至781，无法准确建模。我们的最终结构模型由Rab3GAP1的残基19至331和782至981以及Rab3GAP2的残基2至544组成。由于缺乏或定义不明确的密度，我们无法模拟Rab3GAP1的第一个N端18个残基，残基242至263、902至929，以及Rab3GAP2的N端残基，残基27至60、328至363和437至456（图。

4b, d

4b，d

and Supplementary Fig.

和补充图。

Fig. 4: Cryo-EM structure of core Rab3GAP.

图4:Rab3GAP核心的低温电磁结构。

一

Side views of the locally refined cryo-EM density map of the core Rab3GAP solved to 3.39 Å resolution from 170,636 particles. Map was processed using Phenix.

核心Rab3GAP的局部精炼低温电磁密度图的侧视图从170636个粒子解析为3.39Å分辨率。使用Phenix处理Map。

b类

Local resolution of the cryo-EM map estimated in cryoSPARC.

cryoSPARC中估计的cryo-EM图的局部分辨率。

c级

Cryo-EM density and model for selected regions near the interface of the obtained map.

获得的地图界面附近选定区域的低温电磁密度和模型。

Atomic model and structural architecture of the core Rab3GAP complex. Rab3GAP1 and Rab3GAP2(1–544) are displayed in blue and pink, respectively.

核心Rab3GAP复合体的原子模型和结构体系结构。Rab3GAP1和Rab3GAP2（1-544）分别以蓝色和粉红色显示。

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Our refined structure revealed the Rab3GAP1 consists of two structural domains (Fig.

我们的精细结构揭示了Rab3GAP1由两个结构域组成（图）。

4d级

). Domain 1, which formed the central body density seen in the 2D class averages, comprises an αβ domain. Interestingly, Domain 1 forms a curved β-sheet from both the Rab3GAP1 N-terminal (58–316) and C-terminal region (880–981). This curved β-sheet is composed of β1 to β6, followed by the last 3 β-strands of the C-terminal region and then β7 to β9.

)。构成2D类平均值中所见的中心体密度的域1包含一个αβ域。有趣的是，结构域1从Rab3GAP1 N端（58-316）和C端区域（880-981）形成了一个弯曲的β折叠。该弯曲的β-折叠由β1至β6组成，然后是C末端区域的最后3条β-链，然后是β7至β9。

Aside from the β3-β4 pair, the extended β-strands of Domain 1 are antiparallel. Domain 2 contains a group of five mapped α-helices that form the extended stalk that leads to the unmapped tail density. Foldseek analysis of our model revealed that Rab3GAP1 shows structural similarities to Zwilch, a subunit of the ROD–Zwilch–ZW10 (RZZ) complex that is a component of the outer kinetochore and mediates recruitment of the dynein and Mad1-Mad2.

除了β3-β4对之外，结构域1的延伸β链是反平行的。域2包含一组五个映射的α螺旋，它们形成延伸的茎，从而导致未映射的尾部密度。对我们模型的Foldseek分析表明，Rab3GAP1与Zwilch具有结构相似性，Zwilch是杆-Zwilch-ZW10（RZZ）复合物的一个亚基，是外动粒的组成部分，介导动力蛋白和Mad1-Mad2的募集。

. On the other hand, Rab3GAP2 N-terminal domain represents the head of the tadpole-shaped complex and forms a canonical seven-bladed β-propeller with each blade having four antiparallel β-strands. This subunit also contains three α-helices (residues 15 to 21, 74 to 78 and 221 to 241 of Rab3GAP2) that are located distal to the interfacial region of the complex.

另一方面，Rab3GAP2 N末端结构域代表蝌蚪形复合体的头部，并形成一个典型的七叶β螺旋桨，每个叶片具有四个反平行的β链。该亚基还包含位于复合物界面区域远端的三个α螺旋（Rab3GAP2的残基15至21、74至78和221至241）。

Relative to Rab3GAP1, the β-propeller is perched onto the curved β-sheet of Rab3GAP1 along its edge. Sandwiched between the β-propeller and the curved β-sheet in the subunit interaction region are α-helices from each subunit (residues 142 to 159 and 22 to 37 of Rab3GAP1 and 277 to 287 of Rab3GAP2)..

相对于Rab3GAP1，β螺旋桨沿着其边缘固定在Rab3GAP1的弯曲β片上。在亚基相互作用区域的β螺旋桨和弯曲的β折叠之间夹着来自每个亚基的α螺旋（Rab3GAP1的残基142至159和22至37以及Rab3GAP2的277至287）。。

The Rab3GAP1-Rab3GAP2N subunit interaction interface

Rab3GAP1-Rab3GAP2N亚基相互作用界面

The binding of Rab3GAP1 to Rab3GAP2N results in the burial of 2063 Å

Rab3GAP1与Rab3GAP2N的结合导致2063的埋葬

of surface area. The interaction between Rab3GAP1 and Rab3GAP2N is held together by hydrophobic packing, hydrogen bonds from 8 residue pairs (T19 to K423, E24 to K423, H170 to N286, E191 to K282, V193 to S276, K215 to D279, P950 & G457, and Y951 to R197) (Supplementary Fig.

表面积。Rab3GAP1和Rab3GAP2N之间的相互作用通过疏水堆积，8个残基对（T19至K423，E24至K423，H170至N286，E191至K282，V193至S276，K215至D279，P950＆G457和Y951至R197）的氢键结合在一起（补充图）。

4a级

) and electrostatic interactions (Supplementary Fig.

)和静电相互作用（补充图）。

4b级

). The negative electrostatic potential of Rab3GAP1’s interface is composed of E24, E142, E180, D189 and the C-term has complementary charges to the positive electrostatic potential of Rab3GAP2’s R197, R377, R415, and K423 (Supplementary Fig.

)。Rab3GAP1界面的负静电势由E24，E142，E180，D189组成，C项与Rab3GAP2的R197，R377，R415和K423的正静电势具有互补电荷（补充图）。

4b级

). To determine if the observed interface in our cryo-EM-derived structure of core Rab3GAP is consistent with that of full-length Rab3GAP, we conducted HDX-MS analysis. HDX-MS measures the exchange of protein backbone amide hydrogens with deuterated solvent, with amide exchange rates acting as a useful surrogate for the stability of protein secondary structure.

)。为了确定核心Rab3GAP的低温电磁衍生结构中观察到的界面是否与全长Rab3GAP的界面一致，我们进行了HDX-MS分析。HDX-MS测量蛋白质骨架酰胺氢与氘代溶剂的交换，酰胺交换速率可作为蛋白质二级结构稳定性的有用替代指标。

HDX-MS therefore is useful in mapping both direct protein-protein interactions, and allosteric changes in protein conformation.

因此，HDX-MS可用于绘制直接的蛋白质-蛋白质相互作用和蛋白质构象的变构变化。

As we could only purify the Rab3GAP1 subunit but not the Rab3GAP2 subunit, we designed HDX-MS experiments to measure conformational differences between Rab3GAP1 alone and Rab3GAP1 in the presence of Rab3GAP2. In brief, we incubated Rab3GAP1 and full-length Rab3GAP in deuterated buffer and measured deuterium incorporation over 5 time points (3, 30, 300, 3000 s at 18 °C and 3 s at 0 °C, which is referred to as 0.3 s).

由于我们只能纯化Rab3GAP1亚基，而不能纯化Rab3GAP2亚基，因此我们设计了HDX-MS实验来测量在Rab3GAP2存在下单独的Rab3GAP1和Rab3GAP1之间的构象差异。简而言之，我们将Rab3GAP1和全长Rab3GAP在氘代缓冲液中孵育，并在5个时间点（18°C下3、30、300、3000 s和0°C下3 s）测量氘掺入，称为0.3 s）。

For Rab3GAP1 the HDX-MS coverage spanned 83.6% of the primary sequence, with the full raw deuterium incorporation data for all generated peptides provided in the source data. The HDX-MS experiment comparing Rab3GAP1 alone and Rab3GAP1 in the fully assembled Rab3GAP complex had multiple significant decreases in H/D exchange (defined as having >5%, >0.4 Da and .

对于Rab3GAP1，HDX-MS覆盖率跨越了一级序列的83.6%，源数据中提供了所有生成肽的完整原始氘掺入数据。比较完全组装的Rab3GAP复合物中单独的Rab3GAP1和Rab3GAP1的HDX-MS实验在H/D交换中具有多重显着降低（定义为具有>5%，>0.4Da和。

< 0.01 in an unpaired two-tail t test at any time point), with the largest changes occurring at the N-terminus of Rab3GAP1 (residues 25–32, 37–53, 129–166, 169–191, 193–208, and 211–229) and the C-terminus of Rab3GAP1 (948–966, 973–977, and 978–987) (Fig.

在任何时间点的未配对双尾t检验中均小于0.01），最大变化发生在Rab3GAP1的N端（残基25-32、37-53129-166169-191193-208和211-229）和Rab3GAP1的C端（948-966973-977和978-987）（图）。

5a–c

). Although some of these regions were not modeled in our cryo-EM-derived structural model, this finding agrees with the observed Rab3GAP2N interaction interface, which is made up of both terminal regions of Rab3GAP1 according to our structure. We also observed small significant decreases in deuterium incorporation in regions distal to the subunit interaction interface (Fig. .

)。尽管在我们的低温电磁衍生结构模型中未对其中一些区域进行建模，但这一发现与观察到的Rab3GAP2N相互作用界面一致，该界面根据我们的结构由Rab3GAP1的两个末端区域组成。我们还观察到亚基相互作用界面远端区域的氘掺入量略有显着降低（图。

5b条

, Supplementary Fig.

，补充图。

and Source Data). These changes could potentially result from conformational changes that Rab3GAP1 undergoes after binding Rab3GAP2 or from obstruction from the C-terminal domain of Rab3GAP2.

和源数据）。这些变化可能是由于Rab3GAP1在结合Rab3GAP2后发生的构象变化或Rab3GAP2 C末端结构域的阻塞所致。

Fig. 5: HDX-MS differences between the free Rab3GAP1 and the Rab3GAP complex.

图5：游离Rab3GAP1和Rab3GAP复合物之间的HDX-MS差异。

一

Rab3GAP1 regions showing significant differences in deuterium exchange (defined as >5%, >0.4 Da, and

Rab3GAP1区域在氘交换方面显示出显着差异（定义为>5%，>0.4Da和

< 0.01 in an unpaired two-tail

不成对的两条尾巴中的<0.01

test at any time point) upon complex formation with Rab3GAP2 are highlighted on the cryo-EM structure. The differences in deuterium exchange are indicated by the legend in (

在低温电磁结构上突出显示了与Rab3GAP2形成复合物后的任何时间点的测试）。氘交换的差异由中的图例表示(

一

b类

Sum of the number of deuteron difference of Rab3GAP1 upon complex formation with Rab3GAP2, analyzed over the entire deuterium exchange time course for Rab3GAP1. Each point is representative of the center residue of an individual peptide. Peptides that met the significance criteria described in (

在与Rab3GAP2形成复合物后，Rab3GAP1的氘核差异数之和，在Rab3GAP1的整个氘交换时间过程中进行了分析。每个点代表单个肽的中心残基。符合中描述的重要性标准的肽(

一

) are colored red. Experiments were performed in technical triplicate (

)实验一式三份进行(

= 3). Each point represents a single peptide, and data are presented as the sum of the mean number of deuteron difference across all 5 time points (

= 3）。每个点代表一个肽，数据表示为所有5个时间点氘核差异平均数的总和(

= 3) and error bars represent the sum of standard deviations across all 5 time points (

3）和误差线表示所有5个时间点的标准偏差之和(

= 3 for each time point). Domain schematic above depicts Rab3GAP1 architecture, with the thin box representing unmodelled residues in (

每个时间点=3）。上面的域示意图描绘了Rab3GAP1体系结构，细框表示未建模的残基(

一

c级

Selected deuterium exchange time courses of Rab3GAP1 peptides that showed significant decreases and increases in exchange. Data are presented as mean values with error bars representing SD from experiments performed in technical triplicate (

Rab3GAP1肽的选定氘交换时间过程显示交换显着减少和增加。数据以平均值表示，误差线表示来自一式三份技术实验的SD(

= 3). A full list of all peptides and their deuterium incorporation are provided as a Source Data file.

= 3）。所有肽及其氘掺入的完整列表作为源数据文件提供。

Full size image

全尺寸图像

The Rab18 binding site is distal to the switch and interswitch regions

Rab18结合位点位于开关和开关间区域的远端

We next investigated how Rab3GAP interacts with its cognate substrate Rab18. Despite numerous efforts, we were unable to reconstitute a core Rab3GAP-Rab18 complex. Unlike other previously characterized Rab-GEF pairs, Rab3GAP does not appear to form a high-affinity complex with nucleotide-free Rab18.

接下来，我们研究了Rab3GAP如何与其同源底物Rab18相互作用。尽管付出了许多努力，我们仍无法重建核心Rab3GAP-Rab18复合物。与其他先前表征的Rab-GEF对不同，Rab3GAP似乎不会与无核苷酸的Rab18形成高亲和力复合物。

We thus turned the computational approach of AlphaFold3 to predict the structure of core Rab3GAP in a complex with Rab18 (Fig. .

因此，我们转向AlphaFold3的计算方法来预测与Rab18复合物中核心Rab3GAP的结构（图）。

and Supplementary Fig.

和补充图。

6a, b

6a，b

). All five models generated show Rab18 positioned at the edge of the subunit interaction interface of Rab3GAP. Each model has an ipTM score of 0.8, indicating confident high-quality predictions

)。生成的所有五个模型均显示Rab18位于Rab3GAP亚基相互作用界面的边缘。每个模型的ipTM得分为0.8，表明有信心进行高质量的预测

. Critically, the PAE of Rab18 relative to the core Rab3GAP interface was 5 Å or less, further highlighting the confidence of these models (Supplementary Fig.

至关重要的是，Rab18相对于核心Rab3GAP界面的PAE为5 或更小，进一步突出了这些模型的可信度（Supplementary Fig.）。

). Surprisingly, these models show that Rab3GAP does not appear to directly engage the switch I, switch II, and interswitch regions of Rab18 as had been observed for other Rab GEFs

)。令人惊讶的是，这些模型表明，Rab3GAP似乎不像其他Rab GEF所观察到的那样直接接合Rab18的开关I，开关II和开关间区域

. Instead, the unstructured N-terminus and α1 of Rab3GAP1 and the loops connecting adjacent β-sheets from the β-propeller of Rab3GAP2 contact the α5, α6 and β6 located on the opposite side of the switch regions of Rab18 (Fig.

相反，Rab3GAP1的非结构化N末端和α1以及连接来自Rab3GAP2的β螺旋桨的相邻β-折叠的环与位于Rab18开关区域相对侧的α5，α6和β6接触（图。

6a, b

6a，b

). A cluster of nonpolar residues on Rab18 (L137, L146, F147 and I148) and along the N-terminal region of Rab3GAP1 (F12, I14 and F17) are conserved and may be involved in mediating hydrophobic interaction between Rab3GAP1 and its substrate (Supplementary Fig.

)。Rab18（L137，L146，F147和I148）上以及Rab3GAP1的N端区域（F12，I14和F17）上的一簇非极性残基是保守的，可能参与介导Rab3GAP1与其底物之间的疏水相互作用（补充图）。

7a–c

Fig. 6: AlphaFold3, targeted mutagenesis and in vitro GEF assays reveal that the Rab3GAP subunit interface mediates an interaction with Rab18 through a platform opposite of the switch regions.

图6:AlphaFold3，靶向诱变和体外GEF分析表明，Rab3GAP亚基界面通过与开关区域相对的平台介导与Rab18的相互作用。

一

AlphaFold3 prediction of the interaction between Rab18 and core Rab3GAP. Rab3GAP1 in light blue, Rab3GAP2(1–544) in pink, Rab18 in cyan with switch I, switch II and p-loop colored in red, orange, and yellow, respectively.

AlphaFold3预测Rab18和核心Rab3GAP之间的相互作用。Rab3GAP1为浅蓝色，Rab3GAP2（1-544）为粉红色，Rab18为青色，开关I，开关II和p环分别为红色，橙色和黄色。

b类

Zoomed in view of AlphaFold3 model showing predicted electrostatic interactions at the Rab18-Rab3GAP binding interface. Residues predicted to form salt bridges (Rab3GAP2 R426 with Rab18 E164, Rab3GAP1 E13 and E31 with Rab18 R133 and R141 respectively) are shown as sticks and labeled on the structure.

放大AlphaFold3模型的视图，显示在Rab18-Rab3GAP结合界面处预测的静电相互作用。预计会形成盐桥的残基（分别带有Rab18 E164的Rab3GAP2 R426，带有Rab18 R133和R141的Rab3GAP1 E13和E31）显示为棒状并标记在结构上。

c级

In vitro GEF assays with full-length Rab3GAP to detect activity against Rab18 with mutations suspected of disrupting the Rab3GAP binding interface. Nucleotide exchange was detected by measuring fluorescent decrease in reactions containing 0 nM GEF (Mock) or 400 nM GEF with 4 µM Mant-GDP loaded Rab18 (WT, R133A, R141A or E164A) and 100 µM GTPγS.

使用全长Rab3GAP进行体外GEF分析，以检测针对Rab18的活性，这些突变被怀疑会破坏Rab3GAP结合界面。通过测量含有0 nM GEF（模拟物）或400 nM GEF与4µM Mant-GDP负载的Rab18（WT，R133A，R141A或E164A）和100µM GTPγS的反应中的荧光减少来检测核苷酸交换。

Data are presented as mean with error bars showing SEM for assays performed in technical triplicate (.

数据以平均值表示，误差线显示SEM用于技术一式三份进行的测定（。

= 3).

Data from Fig.

来自Fig.的数据。

4c级

was fit to a non-linear one-phase exponential decay model to determine the rate of nucleotide exchange (

拟合非线性单相指数衰减模型以确定核苷酸交换速率(

obs

obs公司

). Data are presented as mean with error bars showing SEM.

)。数据以平均值表示，误差线显示SEM。

values were generated using a two-tailed Student’s

值是使用两尾学生的

tests (***

测试(***

< 0.01).

Nucleotid

核苷酸

exchange was detected by measuring fluorescent decrease in reactions containing 0 nM GEF (Mock) or 300 nM GEF with 4 µM Mant-GDP loaded Rab18 and 100 µM GTPγS. Data are presented as mean with error bars showing SEM for assays performed in technical triplicate (

通过测量含有0 nM GEF（模拟物）或300 nM GEF与4µM Mant-GDP负载的Rab18和100µM GTPγS的反应中的荧光减少来检测交换。数据以平均值表示，误差线显示SEM，用于一式三份的技术分析(

= 3).

f级

GTPase activity was measured using the Promega GTPase-Glo assay with reactions containing 0 nM GAP (Mock) or 500 nM GAP, 5 µM GTP and 8 µM GST-Rab3a. Data are presented as mean with error bars showing SEM for assays performed in technical triplicate (

使用Promega GTPase-Glo测定法测量GTPase活性，反应包含0 nM GAP（模拟）或500 nM GAP，5µM GTP和8µM GST-Rab3a。数据以平均值表示，误差线显示SEM，用于一式三份的技术分析(

= 3), with

=3），其中

values generated using a two-tailed Student’s

使用两尾学生的

tests (*

测试(*

< 0.05). Source data are provided as a Source Data file.

<0.05）。源数据作为源数据文件提供。

Full size image

全尺寸图像

According to the predicted models, residues R133, R141, E164 of Rab18, which are distal to the switch and interswitch regions, form salt bridges with Rab3GAP1 E13 and E31 and Rab3GAP2 R426 (Fig.

根据预测模型，Rab18的残基R133，R141，E164位于开关和开关间区域的远端，与Rab3GAP1 E13和E31以及Rab3GAP2 R426形成盐桥（图）。

6b条

). To validate the predicted interaction interface, we generated site-specific mutants targeting three Rab18 residues (R133A, R141A, E164A) that are predicted to mediate interaction with Rab3GAP. We loaded these three mutant Rab18 with Mant-GDP and then used them for in vitro GEF assays with full-length Rab3GAP.

)。为了验证预测的相互作用界面，我们生成了针对三个Rab18残基（R133A，R141A，E164A）的位点特异性突变体，这些残基预计会介导与Rab3GAP的相互作用。我们用Mant GDP加载了这三个突变Rab18，然后将其用于全长Rab3GAP的体外GEF测定。

Consistent with the AlphaFold3 generated structural models of Rab3GAP-Rab18, we found that the Rab18 R133A and R141A mutations significantly impair the ability of Rab3GAP to promote nucleotide exchange, while the Rab18 E164A mutation almost completely abolished nucleotide exchange (Fig. .

与AlphaFold3生成的Rab3GAP-Rab18结构模型一致，我们发现Rab18 R133A和R141A突变显着削弱了Rab3GAP促进核苷酸交换的能力，而Rab18 E164A突变几乎完全消除了核苷酸交换（图。

6c, d

6c，d

). Previous studies showed that Rab3GAP regulates Rab18 localization

)。先前的研究表明，Rab3GAP调节Rab18的定位

. To further examine the effects of the E164A mutation on Rab18’s localization, we transfected HeLa cells with construct encoding wild-type or E164A mutant GFP-Rab18 and then monitored their localization by fluorescent microscopy. The Rab18 E164A mutant showed a more diffuse and disorganized localization pattern compared to WT Rab18, supporting the hypothesis that Rab3GAP binds Rab18 through an interface opposite of the nucleotide-binding domain (Supplementary Fig. .

为了进一步检查E164A突变对Rab18定位的影响，我们用编码野生型或E164A突变GFP-Rab18的构建体转染了HeLa细胞，然后通过荧光显微镜监测了它们的定位。与WT Rab18相比，Rab18 E164A突变体显示出更弥散和无序的定位模式，支持了Rab3GAP通过核苷酸结合域相反的界面结合Rab18的假设（补充图）。

). Unlike the highly conserved switch and interswitch regions, α5, α6 and β6 are substantially less conserved between Rabs. Furthermore, multiple sequence alignment showed that the residues critical to Rab18 activation by Rab3GAP (R133, R141 and E164) are not conserved among Rab family members (Supplementary Fig. .

)。与高度保守的开关和开关间区域不同，Rabs之间的α5，α6和β6保守性要低得多。此外，多序列比对表明，Rab3GAP激活Rab18的关键残基（R133，R141和E164）在Rab家族成员中并不保守（补充图）。

), explaining how Rab3GAP might selectively regulate Rab18 despite significant structural homology within this family of small GTPases.

)，解释了Rab3GAP如何选择性调节Rab18，尽管在这个小GTP酶家族中具有显着的结构同源性。

As GEFs typically remodel the Rab switch regions to induce nucleotide exchange, we explored the possibility that another region of Rab3GAP may be responsible for engaging the Rab18 switches. Rab3GAP1 contains a putative GAP domain that is located in a conformationally flexible tail region and was previously shown to bind the switch regions of Rab3.

由于GEF通常会重塑Rab开关区域以诱导核苷酸交换，因此我们探索了Rab3GAP的另一个区域可能负责接合Rab18开关的可能性。Rab3GAP1包含一个假定的GAP结构域，该结构域位于构象灵活的尾部区域，先前已显示可结合Rab3的开关区域。

. To determine if this domain participates in Rab18 nucleotide exchange, we generated a core Rab3GAP construct that encodes Rab3GAP1 devoid of the GAP domain (residues 618–748) (Supplementary Fig.

为了确定该结构域是否参与Rab18核苷酸交换，我们生成了一个核心Rab3GAP构建体，该构建体编码没有GAP结构域的Rab3GAP1（残基618-748）（补充图）。

10a

10安

) and purified this truncated complex, which we will refer to as Rab3GAPΔGAP. In vitro GEF assays demonstrated that Rab3GAPΔGAP still retains Rab18 GEF activity (Fig.

)并纯化了这种截短的复合物，我们将其称为Rab3GAPΔGAP。体外GEF测定表明，Rab3GAPΔGAP仍保留Rab18 GEF活性（图）。

6e型

). By contrast, in vitro GAP assays showed that this Rab3GAPΔGAP is unable to catalyze GTP hydrolysis of Rab3a above basal levels (Fig.

)。相比之下，体外GAP分析表明，这种Rab3GAPΔGAP不能催化Rab3a的GTP水解高于基础水平（图）。

6f级

). These results suggest that the Rab3GAP1 GAP domain is not involved in Rab18 nucleotide exchange and that the GAP and GEF activities of Rab3GAP are mapped to distinct locations of the complex.

)。这些结果表明，Rab3GAP1 GAP结构域不参与Rab18核苷酸交换，并且Rab3GAP的GAP和GEF活性被映射到复合物的不同位置。

Warburg micro syndrome mutations likely affect substrate binding

Warburg micro综合征突变可能影响底物结合

Previous studies showed that three WMS-associated missense mutations (Rab3GAP1 T18P, Rab3GAP1 E24V, Rab3GAP2 R426C) disrupt Rab3GAP’s Rab18 GEF and membrane-targeting activities

先前的研究表明，三个与WMS相关的错义突变（Rab3GAP1 T18P，Rab3GAP1 E24V，Rab3GAP2 R426C）破坏了Rab3GAP的Rab18 GEF和膜靶向活性

. We generated expression constructs encoding these mutations and purified the three full-length Rab3GAP complexes carrying these mutations. We conducted negative stain EM analysis on these Rab3GAP mutants and our 2D analysis showed that the three WMS mutations did not alter the overall architecture of Rab3GAP (Fig. .

我们生成了编码这些突变的表达构建体，并纯化了携带这些突变的三个全长Rab3GAP复合物。我们对这些Rab3GAP突变体进行了负染色EM分析，我们的2D分析表明，这三个WMS突变并没有改变Rab3GAP的整体结构（图）。

7a个

). Using in vitro GEF assays, we validated that three mutant Rab3GAP complexes show dramatically decreased rates of Rab18 nucleotide exchange compared to WT (Fig.

)。使用体外GEF测定，我们验证了三种突变的Rab3GAP复合物与WT相比，Rab18核苷酸交换率显着降低（图）。

7b条

Fig. 7: Clinical Rab3GAP WMS mutants have impaired activity due to disruptions at the Rab18 binding interface as revealed by in vitro GEF assays and mutation mapping.

图7：通过体外GEF测定和突变作图揭示，由于Rab18结合界面的破坏，临床Rab3GAP WMS突变体的活性受损。

一

SDS-PAGE gel of purified Rab3GAP complex containing Rab3GAP1 T18P, Rab3GAP1 E24V or Rab3GAP2 R426C mutations stained with Coomassie Blue. 2D class averages comparing the general architecture of WT Rab3GAP to complexes containing T18P, E24V or R426C point mutations. SDS-PAGE gel were performed in biological triplicate (.

含有用考马斯蓝染色的Rab3GAP1 T18P，Rab3GAP1 E24V或Rab3GAP2 R426C突变的纯化Rab3GAP复合物的SDS-PAGE凝胶。2D类平均值将WT Rab3GAP的一般结构与包含T18P，E24V或R426C点突变的复合物进行比较。SDS-PAGE凝胶以生物学一式三份进行（。

= 3).

b类

In vitro GEF assays with clinical Rab3GAP WMS mutants to detect activity towards Rab18. Nucleotide exchange was detected by measuring fluorescent decrease in reactions containing 0 nM GEF (Mock) or 300 nM GEF with 4 µM Mant-GDP loaded Rab18 and 100 µM GTPγS. Data are presented as mean with error bars showing SEM for assays performed in technical triplicate (.

用临床Rab3GAP WMS突变体进行体外GEF测定，以检测对Rab18的活性。通过测量含有0 nM GEF（模拟物）或300 nM GEF与4µM Mant-GDP负载的Rab18和100µM GTPγS的反应中的荧光减少来检测核苷酸交换。数据以平均值表示，误差线显示SEM用于技术一式三份进行的测定（。

= 3).

c级

Zoomed in view showing Rab3GAP WMS mutants mapped onto the cryo-EM structure of core Rab3GAP. Residues mutated in WMS are shown as sticks and the predicted position of the T18P mutation is circled in black.

放大视图显示Rab3GAP WMS突变体映射到核心Rab3GAP的低温EM结构上。WMS中突变的残基显示为棒状，T18P突变的预测位置以黑色圈出。

Zoomed in view of the AlphaFold3 generated model showing the WMS mutants mapped to the predicted Rab18 binding interface. Residues mutated in WMS and residues predicted to interact electrostatically with WMS residues are shown as sticks and labeled. Rab3GAP1 in light blue, Rab3GAP2(1–544) in pink and Rab18 in cyan.

放大AlphaFold3生成的模型，显示映射到预测的Rab18结合界面的WMS突变体。在WMS中突变的残基和预测与WMS残基静电相互作用的残基显示为棒状并标记。Rab3GAP1为浅蓝色，Rab3GAP2（1-544）为粉红色，Rab18为青色。

Source data are provided as a Source Data file..

源数据作为源数据文件提供。。

Full size image

全尺寸图像

Using our cryo-EM-derived structure, we mapped the locations of these mutation sites. Although Rab3GAP1 T18 was not modeled in our structure due to lack of density, the other two mutation sites (Rab3GAP1 E24 and Rab3GAP2 R426) are located near the edge of the intersubunit interaction interface (Fig. .

使用我们的cryo-EM衍生结构，我们绘制了这些突变位点的位置。尽管由于缺乏密度，我们的结构中未对Rab3GAP1 T18进行建模，但其他两个突变位点（Rab3GAP1 E24和Rab3GAP2 R426）位于亚基间相互作用界面的边缘附近（图。

7摄氏度

). Our structure shows that RabGAP1 E24 engages in electrostatic interaction with R407 of Rab3GAP2 to stabilize the Rab3GAP1 α1 helix. On the other hand, Rab3GAP2 R426 is located in an accessible region proximal to the intersubunit interaction interface. When we examined the AlphaFold3-predicted model of the Rab3GAP-Rab18 complex, we observed that the three WMS mutation sites are located within the Rab18 binding pocket and that these missense mutations could potentially disrupt interactions with Rab18 (Fig. .

)。我们的结构表明，RabGAP1 E24与Rab3GAP2的R407发生静电相互作用，以稳定Rab3GAP1α1螺旋。另一方面，Rab3GAP2 R426位于亚基间相互作用界面附近的可访问区域。当我们检查Rab3GAP-Rab18复合物的AlphaFold3预测模型时，我们观察到三个WMS突变位点位于Rab18结合口袋内，这些错义突变可能会破坏与Rab18的相互作用（图。

7天

). In particular, T18 is part of the Rab3GAP1 unstructured N-terminal region located at the center of the substrate binding interface. Substitution with a proline residue at this site would induce a kink in this region and may disrupt the flexibility needed to accommodate substrate binding. On the other hand, the Rab3GAP1 E24 is located in the α1 helix of this subunit that forms part of the Rab18 binding site.

)。特别是，T18是位于底物结合界面中心的Rab3GAP1非结构化N端区域的一部分。在该位点用脯氨酸残基取代会在该区域引起扭结，并可能破坏适应底物结合所需的灵活性。另一方面，Rab3GAP1 E24位于该亚基的α1螺旋中，该亚基形成Rab18结合位点的一部分。

As this residue is predicted to engage in an ionic interaction with R407 of Rab3GAP2 to stabilize the α1 helix, substitution with a valine residue may disrupt this local structure critical to substrate binding. Lastly, Rab3GAP2 R426 is predicted to engage in an electrostatic interaction with E164 of Rab18 and replacement with a cysteine interferes with substrate binding.

由于预计该残基会与Rab3GAP2的R407发生离子相互作用以稳定α1螺旋，因此用缬氨酸残基取代可能会破坏这种对底物结合至关重要的局部结构。最后，预计Rab3GAP2 R426与Rab18的E164发生静电相互作用，用半胱氨酸替代会干扰底物结合。

Indeed, as shown earlier, an E164A mutation to Rab18 prevented nucleotide exchange by Rab3GAP..

实际上，如前所述，Rab18的E164A突变阻止了Rab3GAP的核苷酸交换。。

Discussion

讨论

The biochemical and structural data on human Rab3GAP reported here contribute to building the knowledge base of the divergent and enigmatic family of Rab GEF regulators. Our cryo-EM structure of the core human Rab3GAP confirmed that this Rab18 GEF shares no structural homology with other Rab GEFs with known structures.

这里报道的人类Rab3GAP的生化和结构数据有助于建立Rab GEF监管机构的分歧和神秘家族的知识库。我们的核心人类Rab3GAP的低温EM结构证实，该Rab18 GEF与具有已知结构的其他Rab GEF没有结构同源性。

. Instead, we found that the Rab3GAP1 subunit of this heterodimeric complex shares structural similarity to Zwilch, a component of the mitotic RZZ complex that is involved in spindle assembly. Similar to Rab3GAP1, which binds the N-terminal β-propeller of Rab3GAP2 via its β-sheet structure, the recent cryo-EM structure of the RZZ complex showed that Zwilch similarly interacts with Rod within this complex.

相反，我们发现该异二聚体复合物的Rab3GAP1亚基与Zwilch具有结构相似性，Zwilch是有丝分裂RZZ复合物的一个组成部分，参与纺锤体组装。与Rab3GAP1类似，Rab3GAP1通过其β-折叠结构结合Rab3GAP2的N端β-螺旋桨，RZZ复合物最近的低温EM结构表明，Zwilch类似地与该复合物中的杆相互作用。

. Notably, the β-propeller from Rod associates with the curved β-sheet structure of Zwilch. Zwilch and Rod have been proposed to be Rab18 effectors but the molecular basis of how Zwilch and Rod binds Rab18 requires further investigation

值得注意的是，来自杆的β螺旋桨与Zwilch的弯曲β片结构相关联。Zwilch和Rod被认为是Rab18效应子，但Zwilch和Rod如何结合Rab18的分子基础需要进一步研究

Although human Rab3GAP is structurally unique compared to other Rab GEFs, this heterodimeric GEF appears to share two features found in several Rab GEFs. The first feature is the membrane-dependent enhancement of GEF activity. For example, it has been shown that the GEF activity of SH3BP5 is dependent on its substrate Rab11 being anchored to a membrane.

尽管与其他Rab GEF相比，人类Rab3GAP在结构上是独特的，但这种异二聚体GEF似乎具有在几个Rab GEF中发现的两个特征。第一个特征是GEF活性的膜依赖性增强。例如，已经表明，SH3BP5的GEF活性取决于其底物Rab11锚定在膜上。

. In the same vein, we demonstrated that membrane presentation of Rab18 resulted in a greater than 10-fold increase in GEF activity for both full-length and core Rab3GAP. The second feature is fine-tuning GEF activity by autoinhibition. The catalytic efficiency of core Rab3GAP is more than twice that of full-length Rab3GAP, leading us to speculate that the C-terminus of Rab3GAP2 is an autoinhibitory domain.

同样，我们证明了Rab18的膜呈递导致全长和核心Rab3GAP的GEF活性增加了10倍以上。第二个功能是通过自动抑制来微调GEF活性。核心Rab3GAP的催化效率是全长Rab3GAP的两倍以上，这使我们推测Rab3GAP2的C末端是一个自抑制结构域。

In comparison, the catalytic activity of the Rab5 GEF Rabex-5, which is autoinhibited and has low inherent GEF activity over its substrate, is increased 2- to 3-fold through binding the Rab5 effector Rabaptin-5.

相比之下，Rab5-GEF Rabex-5的催化活性通过结合Rab5效应子Rabaptin-5而增加了2至3倍，Rab5-GEF Rabex-5是自动抑制的，并且在其底物上具有较低的固有GEF活性。

or through truncations

或通过截断

. Based on our structural data showing the conformational flexibility of full-length Rab3GAP, we speculate that the Rab3GAP2 C-terminal domain suppresses Rab3GAP’s GEF activity by obstructing the substrate binding site. Future studies should focus on determining the basis of the enhanced GEF activity in the presence of membrane-anchored Rab18 and to delineate the potential autoinhibitory mechanism of Rab3GAP..

根据我们显示全长Rab3GAP构象灵活性的结构数据，我们推测Rab3GAP2 C末端结构域通过阻断底物结合位点来抑制Rab3GAP的GEF活性。未来的研究应侧重于确定在膜锚定Rab18存在下增强GEF活性的基础，并描述Rab3GAP的潜在自抑制机制。。

A major question concerning the function of Rab3GAP is why both subunits are required for its GEF activity. Our cryo-EM structure revealed that the two subunits form a stable complex by engaging in an extensive interaction interface. Our subsequent AlphaFold3 modeling predicted that the edge of this intersubunit interaction interface forms the substrate site where Rab18 binds through a platform composed of its .

关于Rab3GAP功能的一个主要问题是，为什么它的GEF活动需要两个亚基。我们的低温电磁结构表明，这两个亚基通过参与广泛的相互作用界面形成了稳定的复合物。我们随后的AlphaFold3建模预测，该亚基间相互作用界面的边缘形成了Rab18通过由其组成的平台结合的底物位点。

啊

5 and

5和

啊

6 helices and

6个螺旋和

6 strand. This predicted model was validated by targeted mutagenesis in conjunction with in vitro GEF assays and localization studies. Consistent with this in silico-generated model, our multiple sequence alignment (MSA) analysis showed that the Rab3GAP and Rab18 residues predicted to interact with one another are found to be conserved (Supplementary Fig. .

6股。通过靶向诱变以及体外GEF测定和定位研究验证了该预测模型。与此计算机生成的模型一致，我们的多序列比对（MSA）分析表明，预测相互作用的Rab3GAP和Rab18残基被发现是保守的（补充图）。

7a–c

). However, as illustrated in a structural overlay of the AlphaFold3-predicted model of Rab3GAP-Rab18 with other representative solved structures of Rab GEFs (PDB code: 3TW8, 2OT3 and 6DJL) bound to their respective Rab substrate, this mode of GEF-Rab interaction has not been previously observed (Supplementary Fig. .

)。然而，如Rab3GAP-Rab18的AlphaFold3预测模型的结构覆盖图所示，Rab GEF的其他代表性解析结构（PDB代码：3TW8、2OT3和6DJL）与其各自的Rab底物结合，这种GEF-Rab相互作用的模式以前没有被观察到（补充图）。

). Other Rab GEFs engage their substrates at the switch I, II and interswitch regions to induce structural rearrangement, reducing the affinity for the bound nucleotide

)。其他Rab GEF在开关I，II和开关间区域与底物结合，以诱导结构重排，降低对结合核苷酸的亲和力

. Without directly engaging the switch regions, how does Rab3GAP promote Rab18 nucleotide exchange? We initially speculated that Rab3GAP1’s flexible tail region, which contains the GAP domain, may assist in promoting nucleotide exchange by engaging the switch regions of Rab18. However, this idea was refuted by our finding that a GAP domain deficient Rab3GAP retains GEF activity towards Rab18 (Fig. .

在不直接参与开关区域的情况下，Rab3GAP如何促进Rab18核苷酸交换？我们最初推测，Rab3GAP1的柔性尾部区域（包含GAP结构域）可能通过参与Rab18的开关区域来促进核苷酸交换。然而，我们的发现驳斥了这一观点，即GAP结构域缺陷的Rab3GAP保留了GEF对Rab18的活性（图）。

6e型

). A systematic mutagenesis study on the small GTPase Gsp1/Ran uncovered the presence of allosteric sites away from the switch region and found that disturbances at these sites induced a preference for GTP binding over GDP binding

)。对小GTP酶Gsp1/Ran的系统诱变研究发现，远离开关区的变构位点的存在，发现这些位点的干扰导致GTP结合优于GDP结合

. Interestingly, the structural alignment of Rab18 with Gsp1/Ran shows that some of the corresponding allosteric sites map to regions where Rab18 binds Rab3GAP, with multiple sites located in

有趣的是，Rab18与Gsp1/Ran的结构比对表明，一些相应的变构位点映射到Rab18与Rab3GAP结合的区域，其中多个位点位于

啊

6 of the small GTPase. These findings raise the possibility that Rab3GAP could promote nucleotide release by modulating one or more of these putative allosteric sites in Rab18. Nevertheless, resolving the mystery surrounding Rab3GAP’s GEF mechanism would necessitate a high-resolution structural analysis of Rab3GAP in complex with Rab18..

6个小GTPase。这些发现提高了Rab3GAP通过调节Rab18中一个或多个这些假定的变构位点来促进核苷酸释放的可能性。然而，解决围绕Rab3GAP的GEF机制的谜团将需要对Rab3GAP与Rab18的复合物进行高分辨率的结构分析。。

Rab3GAP is the only known dual-function Rab regulator in eukaryotic cells. What allows Rab3GAP to serve both as a GAP for Rab3a and a GEF for Rab18 remains unclear. Previous studies demonstrated that Rab3GAP1 alone exhibits Rab3a GAP activity and identified a putative GAP domain located in the C-terminal region of this Rab3GAP subunit.

Rab3GAP是真核细胞中唯一已知的双功能Rab调节剂。什么使Rab3GAP既可以作为Rab3a的GAP，又可以作为Rab18的GEF尚不清楚。先前的研究表明，Rab3GAP1单独表现出Rab3a GAP活性，并鉴定了位于该Rab3GAP亚基C端区域的推定GAP结构域。

. Our cryo-EM structure of core Rab3GAP revealed that the GAP domain is located in the flexible tail of Rab3GAP1 and is not involved in the interaction with Rab3GAP2. We found that removing the GAP domain in core Rab3GAP abolished its GAP activity toward Rab3a but had no effect on its GEF activity towards Rab18 (Fig. .

我们的核心Rab3GAP的低温电磁结构表明，GAP结构域位于Rab3GAP1的柔性尾部，不参与与Rab3GAP2的相互作用。我们发现，去除核心Rab3GAP中的GAP结构域消除了其对Rab3a的GAP活性，但对其对Rab18的GEF活性没有影响（图。

6e, f

6e，f

). These findings suggest that the two catalytic activities are present in different locations of this complex. AlphaFold3 modeling of the Rab3GAP1-Rab3a complex showed that the GAP domain of Rab3GAP1 binds to the switch and interswitch region of its substrate (Supplementary Fig.

)。这些发现表明，这两种催化活性存在于该复合物的不同位置。Rab3GAP1-Rab3a复合物的AlphaFold3建模显示Rab3GAP1的GAP结构域与其底物的开关和开关间区域结合（Supplementary Fig.）。

10a

10安

). In agreement with mutational analysis of previous studies, AlphaFold predictions are consistent with a mechanism similar to that of Ras and Rho GAPs with R728 of Rab3GAP1 and Q81 of Rab3a providing critical arginine and glutamine fingers to stabilize the transition state during GTP hydrolysis (Supplementary Fig. .

)。与先前研究的突变分析一致，AlphaFold预测与Ras和Rho GAP的机制相似，Rab3GAP1的R728和Rab3a的Q81提供了关键的精氨酸和谷氨酰胺指，以稳定GTP水解过程中的过渡状态（补充图）。

10b

10亿

)

. It is not known if Rab3GAP has preferential substrate specificity in neuronal cells where both Rab3 and Rab18 are expressed. Future studies could focus on mechanistic investigation of the GAP activity of Rab3GAP and further delineate the functional relationships between these two distinct catalytic activities..

尚不清楚Rab3GAP在同时表达Rab3和Rab18的神经元细胞中是否具有优先的底物特异性。未来的研究可能集中在Rab3GAP GAP活性的机理研究上，并进一步描述这两种不同催化活性之间的功能关系。。

Mutations to Rab3GAP1 and Rab3GAP2 cause the multisystem disorder WMS

Rab3GAP1和Rab3GAP2突变导致多系统疾病WMS

. Data from our biochemical and structural studies show that three WMS-associated missense mutations do not affect the assembly or the overall architecture of Rab3GAP. Instead, these mutations disrupt Rab3GAP function by altering the Rab18 binding site. Our cryo-EM structure of core Rab3GAP, the Rab3GAP-Rab18 structural model predicted by AlphaFold3, and the biochemical platform to reconstitute human Rab3GAP offers experimental tools to predict and validate the potential impact of numerous variants of unknown significance of Rab3GAP1 and Rab3GAP2 subunits on the biochemical and structural properties of Rab3GAP..

我们的生化和结构研究数据表明，三个与WMS相关的错义突变不会影响Rab3GAP的组装或整体结构。相反，这些突变通过改变Rab18结合位点来破坏Rab3GAP功能。我们的核心Rab3GAP的低温电磁结构，AlphaFold3预测的Rab3GAP-Rab18结构模型以及重建人Rab3GAP的生化平台提供了实验工具，可以预测和验证Rab3GAP1和Rab3GAP2亚基的许多未知意义的变体对Rab3GAP的生化和结构特性的潜在影响。。

Methods

方法

Plasmids

质粒

Full-length human Rab3GAP1 (HsCD00860097) and Rab3GAP2 (HsCD00867180) genes were purchased from DNASU. Rab3GAP1 was subcloned into a pLIB vector containing an N-terminal TSP tag and Rab3GAP2 was subcloned into a pLIB vector containing a C-terminal 3x-FLAG tag. Site-directed mutagenesis was carried out on pLIB-TSP-Rab3GAP1 to generate T18P and E24V mutations and on pLIB-Rab3GAP2-3x-FLAG to generate R426C and 1–544 mutations.

全长人Rab3GAP1（HsCD00860097）和Rab3GAP2（HsCD00867180）基因购自DNASU。将Rab3GAP1亚克隆到含有N端TSP标签的pLIB载体中，并将Rab3GAP2亚克隆到含有C端3x FLAG标签的pLIB载体中。在pLIB-TSP-Rab3GAP1上进行定点突变以产生T18P和E24V突变，在pLIB-Rab3GAP2-3x-FLAG上进行定点突变以产生R426C和1-544突变。

The biGBac protocol was used to assemble various combinations of both Rab3GAP1 and Rab3GAP2 into pBIG1A.

biGBac协议用于将Rab3GAP1和Rab3GAP2的各种组合组装成pBIG1A。

. Rab3a, Rab11a and Rab18 were subcloned into pOPTGcH vectors containing a TEV-cleavable GST tag at the N-terminus and a 6x-His tag at the C-terminus.

将Rab3a，Rab11a和Rab18亚克隆到pOPTGcH载体中，该载体在N端含有TEV可切割的GST标签，在C端含有6x His标签。

Protein expression

蛋白质表达

Baculovirus containing Rab3GAP was produced from the appropriate vector (Supplementary Table

含有Rab3GAP的杆状病毒是从适当的载体中产生的（补充表

) using the Baculovirus Expression Vector System. Optimized amounts of baculovirus were used to infect Sf9 cells at a density between 1.5 and 4 × 10

)使用杆状病毒表达载体系统。使用优化量的杆状病毒以1.5至4×10之间的密度感染Sf9细胞

cells mL

细胞

−1

, and cells were harvested 72 to 96 h post infection. Cell pellets were flash frozen in liquid nitrogen and stored at −70 °C until use. Rabs were expressed in BL21 C41(DE3)

，感染后72至96小时收获细胞。将细胞沉淀在液氮中速冻，并保存在-70°C直至使用。Rabs在BL21 C41（DE3）中表达

E. coli

E、大肠杆菌

induced with 0.5 mM isopropyl B-d-1-thiogalacpyranoside [IPTG] (GoldBio) for 4 h at 37 °C. Cells were then harvested, flash frozen in liquid nitrogen and stored at −70 °C.

用0.5mM异丙基B-d-1-硫代半乳糖吡喃糖苷[IPTG]（GoldBio）在37℃诱导4小时。然后收获细胞，在液氮中快速冷冻并保存在-70℃。

Protein purification

蛋白质纯化

Sf9 cell pellets expressing Rab3GAP were resuspended in buffer A (50 mM Tris pH 7.4, 150 mM NaCl, 5% glycerol, 0.1% Tween-20, 2 mM phenylmethylsulfonyl fluoride [PMSF] and Complete ethylenediaminetetraacetic acid (EDTA) free protease inhibitor). Cells were lysed with four cycles of sonication for 30 s using the Branson Sonicator 450 set to duty cycle 40% and output control 4.

将表达Rab3GAP的Sf9细胞沉淀重悬于缓冲液A（50mM Tris pH 7.4，150 mM NaCl，5%甘油，0.1%吐温-20，2 mM苯甲基磺酰氟（PMSF）和完全不含乙二胺四乙酸（EDTA）的蛋白酶抑制剂）。使用设置为占空比40%和输出对照4的Branson超声仪450，通过四个超声处理循环裂解细胞30秒。

The lysate was centrifuged at 125,000 × .

将裂解物以125000×10离心。

克

for 40 min at 4 °C. Supernatant containing Rab3GAP1 was incubated with Strep-Tactin XT 4Flow resin (IBA) and supernatant containing all other Rab3GAP constructs was incubated with Anti-FLAG M2 affinity gel (Sigma-Aldrich) equilibrated with buffer B (50 mM Tris pH 7.4, 150 mM NaCl and 5% glycerol). The resin was washed with buffer B and eluted with 50 mM biotin in buffer B for Strep-Tactin purifications and 250 µg mL.

在4°C下放置40分钟。将含有Rab3GAP1的上清液与Strep-Tactin XT 4Flow树脂（IBA）孵育，并将含有所有其他Rab3GAP构建体的上清液与用缓冲液B（50mM Tris pH 7.4，150 mM NaCl和5%甘油）。用缓冲液B洗涤树脂，并用缓冲液B中的50mM生物素洗脱，用于链球菌-肌动蛋白纯化和250μg/mL。

−1

3x FLAG peptide in buffer B for Anti-FLAG M2 purifications. The eluate was concentrated using an Amicon 100 kDa concentrator (Millipore) and applied to an ENrich SEC650 10 × 300 column (Bio-Rad) equilibrated with buffer C (20 mM HEPES pH 7.5, 150 mM NaCl and 1 mM TCEP). Fractions containing desired proteins were pooled, concentrated, flash frozen and stored at −70 °C..

缓冲液B中的3x FLAG肽用于抗FLAG M2纯化。使用Amicon 100 kDa浓缩器（Millipore）浓缩洗脱液，并将其应用于用缓冲液C（20 mM HEPES pH 7.5，150 mM NaCl和1 mM TCEP）。将含有所需蛋白质的级分合并，浓缩，速冻并保存在-70℃。。

E. coli

E、大肠杆菌

C41(DE3) cell pellets expressing Rabs were resuspended in buffer D (20 mM Tris pH 8, 100 mM NaCl, 2 mM B-mercaptoethanol (BME) and 2 mM PMSF). Cells were lysed with four cycles of sonication for 60 s at duty cycle 50% and output control 5. The lysate was centrifuged at 20,000 ×

将表达Rabs的C41（DE3）细胞沉淀重悬于缓冲液D（20mM Tris pH 8，100 mM NaCl，2 mM B-巯基乙醇（BME）和2 mM PMSF）。在占空比为50%和输出对照为5的情况下，用四个超声处理循环裂解细胞60秒。裂解液在20000℃下离心

克

for 40 min at 4 °C. The supernatant was incubated with glutathione resin (GenScript) equilibrated with buffer E (20 mM Tris pH 8, 100 mM NaCl and 2 mM BME). The resin was washed with buffer E and eluted with 30 mM glutathione in buffer E. TEV protease was added to the eluate and dialyzed in buffer F (20 mM Tris pH 8, 100 mM NaCl, 10 mM BME and 5 mM EDTA) overnight at 4 °C.

在4°C下放置40分钟。将上清液与用缓冲液E（20mM Tris pH 8，100 mM NaCl和2 mM BME）。用缓冲液E洗涤树脂，并用缓冲液E中的30 mM谷胱甘肽洗脱。将TEV蛋白酶加入洗脱液中，并在缓冲液F（20 mM Tris pH 8，100 mM NaCl，10mM BME和5mM EDTA）在4℃过夜。

The protein was then applied to a HiPrepQ FF 16/10 column (GE Healthcare) equilibrated with buffer E and gradient elution with buffer G (20 mM Tris pH 8, 1 M NaCl and 2 mM BME) was performed to isolate target proteins. Glutathione resin in buffer E was used to remove GST, and cleaved Rabs were collected in the flowthrough.

然后将蛋白质应用于用缓冲液E平衡的HiPrepQ FF 16/10色谱柱（GE Healthcare），并用缓冲液G（20mM Tris pH 8，进行1M NaCl和2mM BME）以分离靶蛋白。使用缓冲液E中的谷胱甘肽树脂去除GST，并在流通液中收集裂解的Rabs。

The flowthrough was concentrated, flash frozen in liquid nitrogen and stored at −70 °C..

将流通液浓缩，在液氮中速冻，并保存在-70℃。。

Lipid vesicle preparation

脂质囊泡制剂

Nickelated lipid vesicles were prepared by combining chloroform stocks of phosphatidylcholine (egg yolk PC, Avanti), phosphatidylethanolamine (egg yolk PE, Sigma), phosphatidylserine (porcine brain PS, Avanti), L-α-phosphatidylinositol (soy PI, Avanti) and 18:1 DGS NTA(Ni) (Avanti) according to Supplementary Table .

根据补充表，通过将磷脂酰胆碱（蛋黄PC，Avanti），磷脂酰乙醇胺（蛋黄PE，Sigma），磷脂酰丝氨酸（猪脑PS，Avanti），L-α-磷脂酰肌醇（大豆PI，Avanti）和18:1 DGS NTA（Ni）（Avanti）的氯仿原液组合来制备镍化脂质囊泡。

. Argon gas was used to evaporate residual chloroform followed by overnight desiccation under vacuum. Liposome buffer (20 mM HEPES pH 7.5, 150 mM NaCl) was used to resuspend the lipid film at a final concentration of 1.5 mg mL

.使用氩气蒸发残留的氯仿，然后在真空下干燥过夜。脂质体缓冲液（20mM HEPES pH 7.5，150 mM NaCl）用于重悬脂质膜，终浓度为1.5 mg mL

−1

. The mixture was vortexed vigorously for 20 min then sonicated for 15 min with a Branson 1510 sonicator. Vesicles were flash frozen in liquid nitrogen then warmed in a water bath for a total of 10 freeze-thaw cycles. Vesicles were extruded 21 times through a 100 nm polycarbonate membrane (Avanti) and stored at −70 °C..

将混合物剧烈涡旋20分钟，然后用Branson 1510超声仪超声处理15分钟。将囊泡在液氮中速冻，然后在水浴中加热，共进行10次冻融循环。将囊泡通过100 nm聚碳酸酯膜（Avanti）挤出21次，并保存在-70℃。。

In vitro GEF assay

体外GEF测定

His-tagged Rabs were purified as described above in protein purification. EDTA was added at a final concentration of 5 mM and incubated at 25 °C for 30 min. Mant-GDP (Thermo Scientific) was added at a fivefold molar excess and incubated at 25 °C for 1 h. MgCl

如上所述，在蛋白质纯化中纯化了带有His标签的Rabs。加入终浓度为5mM的EDTA，并在25℃下孵育30分钟。以五倍摩尔过量加入Mant-GDP（Thermo Scientific），并在25℃下孵育1小时。MgCl

was added at a final concentration of 10 mM and incubated at 25 °C for 1 h. Rabs were loaded onto a Superdex 75 Increase column (GE Healthcare) equilibrated with buffer H (20 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, and 1 mM MgCl

以10mM的终浓度加入，并在25℃下孵育1h。将Rabs加载到用缓冲液h（20mM HEPES pH 7.5，150 mM NaCl，1毫米TCEP和1毫米MgCl

). Fractions containing Mant-GDP loaded Rabs were pooled, concentrated, flash frozen in liquid nitrogen at stored at −70 °C. Ten µL reactions were prepared with a final concentration of 4 µM Mant-GDP loaded Rab, 0–300 nM Rab3GAP complex and 100 µM GTPγS in buffer H. Rab and membrane (0–0.2 mg mL

)。将含有Mant-GDP负载的Rabs的级分合并，浓缩，在液氮中速冻，储存在-70℃。制备了10L反应，终浓度为4M Mant-GDP负载的Rab，在缓冲液H.Rab和膜（0–0.2 mg·mL）中，0–300 nM Rab3GAP复合物和100µM GTPγS

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) were aliquoted into a Corning 384-well low volume polystyrene microplate (4514). Rab3GAP complex and GTPγS were added to the plate and a BioTek Synergy HTX Multimode Reader was used to measure the fluorescent signal for 1 h at 25 °C (excitation

)将其等分到Corning 384孔低体积聚苯乙烯微孔板（4514）中。将Rab3GAP复合物和GTPγS添加到平板中，并使用BioTek Synergy HTX多模式读取器在25°C（激发）下测量1小时的荧光信号

小时

= 366 nm; emission

==同步，由elderman校正==同步==排放

小时

= 443 nm). GraphPad Prism 7 Software was used to analyze data and

=443nm）。GraphPad Prism 7软件用于分析数据和

cat

猫

analysis was performed as described by Delprato et al. Curves were fit to a non-linear one-phase exponential decay model according to the following equation,

如Delprato等人所述进行分析。根据以下等式将曲线拟合为非线性单相指数衰减模型，

我

(

) = (

)

我

−

我

f级

)*exp(−

)*exp（-）

obs

obs公司

) +

)

我

f级

(GraphPad Software).

（GraphPad软件）。

我

(

) is the emission intensity at time

)是当时的发射强度

我

is the initial intensity,

是初始强度，

我

f级

is the intensity at

是强度

= inf. Catalytic efficiency,

=inf.催化效率，

cat

猫

, was obtained by the slope of a linear least squares fit to

，是通过线性最小二乘拟合的斜率获得的

obs

obs公司

cat

猫

*[GEF] +

*[GEF]：

我

, with

，带有

我

being

存在

obs

obs公司

in the absence of GEF.

在没有全球环境基金的情况下。

In vitro GAP assays

体外GAP测定

GAP activity was measured using the Promega GTPase-Glo Assay with GST-tagged Rab3a and Rab3GAP1, core Rab3GAP or Rab3GAPΔGAP. Twenty µL reactions were prepared in a white CELLSTAR 96-well plate with a final concentration of 5 µM GTP, 0.5 mM DTT, 8 µM GST-Rab3a and 0 nM (Mock) or 500 nM GAP in Promega GTPase/GAP buffer and incubated at 25 °C for 1 h.

使用带有GST标签的Rab3a和Rab3GAP1，核心Rab3GAP或Rab3GAPΔGAP的Promega GTPase-Glo测定法测量GAP活性。在最终浓度为5µM GTP的白色CELLSTAR 96孔板中制备了20µL反应，0.5毫米DTT，在Promega GTPase/GAP缓冲液中加入8µM GST-Rab3a和0 nM（模拟）或500 nM GAP，并在25°C下孵育1小时。

Twenty µL of 1x GTPase-Glo reagent with 10 mM ADP in GTPase-Glo buffer was added to each reaction and incubated at 25 °C for 30 min, followed by 40 µL detection reagent and incubated at 25 °C for 10 min. Luminescence was measured using a BioTek Synergy HTX Multimode Reader. Reactions were performed in triplicate..

向每个反应中加入20μl1X GTPase-Glo试剂和GTPase-Glo缓冲液中的10mM ADP，并在25℃下孵育30分钟，然后加入40μL检测试剂并在25℃下孵育10分钟。使用BioTek Synergy HTX Multimode Reader测量发光。反应一式三份进行。。

HDX-MS sample preparation

HDX-MS样品制备

HDX reactions comparing Rab3GAP1 apo to Rab3GAP1 + Rab3GAP2 were carried out in 50 µL reaction volumes containing 8 pmol of Rab3GAP1 or Rab3GAP1 + Rab3GAP2. Prior to the exchange reactions, 1.5 mM EDTA was added to each sample and samples were incubated on ice for 1 h. Exchange reactions were initiated by the addition of 46 µL of D.

比较Rab3GAP1 apo与Rab3GAP1+ Rab3GAP2的HDX反应在含有8 pmol Rab3GAP1或Rab3GAP1+ Rab3GAP2的50µL反应体积中进行。在交换反应之前，向每个样品中加入1.5mM EDTA，并将样品在冰上孵育1小时。通过加入46μlD引发交换反应。

O buffer (20 mM HEPES pH 7.5, 100 mM NaCl, 94.34% D

O缓冲液（20mM HEPES pH 7.5，100mM NaCl，94.34%D

O (V/V)) to 4 µL of protein mixture (final D

O（V/V））至4l蛋白质混合物（最终D

O concentration of 86.79%). The reactions proceeded for 0.3 s (3 s on ice), 3, 30, 300, or 3000 s at room temperature, before being quenched with ice cold acidic quench buffer resulting in a final concentration of 0.6 M guanidine-HCl and 0.9% formic acid post quench. All conditions and timepoints were created and run in independent triplicate.

O浓度为86.79%）。反应在室温下进行0.3s（冰上3s），3,30300或3000 s，然后用冰冷的酸性淬灭缓冲液淬灭，最终浓度为0.6M盐酸胍和0.9%甲酸后淬灭。创建所有条件和时间点，并一式三份独立运行。

Samples were flash frozen immediately after quenching and stored at −80 °C..

淬火后立即将样品速冻并保存在-80℃。。

Protein digestion and MS/MS data collection

蛋白质消化和MS/MS数据收集

Protein samples were rapidly thawed and injected onto an integrated fluidics system containing a HDx-3 PAL liquid handling robot and climate-controlled (2 °C) chromatography system (LEAP Technologies), a Dionex Ultimate 3000 UHPLC system, as well as an Impact HD QTOF Mass spectrometer (Bruker). The full details of the automated LC system are described previously.

将蛋白质样品快速解冻并注射到集成流体系统中，该系统包含HDx-3 PAL液体处理机器人和气候控制（2℃）色谱系统（LEAP Technologies），Dionex Ultimate 3000 UHPLC系统以及Impact HD QTOF质谱仪（Bruker）。自动化LC系统的全部细节如前所述。

. The Rab3GAP1 ± Rab3GAP2 samples were run over one immobilized pepsin column (Trajan; ProDx protease column, 2.1 mm × 30 mm PDX.PP01-F32) at 200 µL min

将Rab3GAP1±Rab3GAP2样品在一个固定的胃蛋白酶柱（Trajan；ProDx蛋白酶柱，2.1mm×30mm PDX）上电泳。PP01-F32）在200微升/分钟

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for 3 min at 8 °C. The resulting peptides were collected and desalted on a C18 trap column (ACQUITY UPLC BEH C18 1.7 µm column, 2.1 mm × 5 mm; Waters 186004629). The trap was subsequently eluted in line with an ACQUITY 1.7 μm particle, 100 mm × 1 mm C18 UPLC column (Waters), using a gradient of 3–35% B (Buffer A 0.1% formic acid; Buffer B 100% acetonitrile) over 11 min immediately followed by a gradient of 35–80% over 5 min.

在8°C下放置3分钟。收集所得肽并在C18捕集柱（ACQUITY UPLC BEH C18 1.7µm柱，2.1毫米×5毫米；Waters 186004629）。随后按照ACQUITY 1.7μm颗粒洗脱陷阱，100毫米×1毫米C18超高效液相色谱柱（Waters），在11分钟内使用3-35%B（缓冲液a 0.1%甲酸；缓冲液B 100%乙腈）的梯度，然后在5分钟内梯度为35-80%。

Mass spectrometry experiments acquired over a mass range from 150 to 2200 m/z using an electrospray ionization source operated at a temperature of 200 °C and a spray voltage of 4.5 kV..

使用在200°C温度和4.5 kV喷雾电压下操作的电喷雾电离源，在150至2200 m/z的质量范围内获得质谱实验。。

Peptide identification

肽鉴定

For the Rab3GAP1 apo and Rab3Gap1 + Rab3GAP2 experiment, peptides were identified from the non-deuterated samples of Rab3GAP1 using data-dependent acquisition following tandem MS/MS experiments (0.5 s precursor scan from 150–2000 m/z; twelve 0.25 s fragment scans from 150–2000 m/z). MS/MS datasets were analyzed using PEAKS7 (PEAKS), and peptide identification was carried out by using a false discovery-based approach, with a threshold set to 0.1% using a database of purified proteins and known contaminants.

对于Rab3GAP1 apo和Rab3GAP1+ Rab3GAP2实验，在串联MS/MS实验后，使用数据依赖性采集从Rab3GAP1的非氘代样品中鉴定肽（150-2000m/z的0.5s前体扫描；从150–2000 m/z进行十二次0.25 s片段扫描）。使用PEAKS7（峰）分析MS/MS数据集，并使用基于错误发现的方法进行肽鉴定，使用纯化蛋白质和已知污染物的数据库将阈值设置为0.1%。

The search parameters were set with a precursor tolerance of 20 ppm, fragment mass error 0.02 Da, charge states from 1–8, leading to a selection criterion of peptides that had a −10logP score of 26.6..

搜索参数设置为前体耐受性为20 ppm，片段质量误差为0.02 Da，电荷状态为1-8，从而得出-10logP得分为26.6的肽的选择标准。。

Mass analysis and measurement of deuterium incorporation

氘掺入的质量分析和测量

HD-Examiner Software (Sierra Analytics) was used to automatically calculate the level of deuterium incorporation into each peptide. All peptides were manually inspected for correct charge state, correct retention time, appropriate selection of isotopic distribution, etc. Deuteration levels were calculated using the centroid of the experimental isotope clusters.

HD Examiner软件（Sierra Analytics）用于自动计算氘掺入每种肽的水平。手动检查所有肽的正确电荷状态，正确的保留时间，适当选择同位素分布等。使用实验同位素簇的质心计算氘化水平。

Results are presented as relative levels of deuterium incorporation and the only control for back exchange was the level of deuterium present in the buffer (86.79%). Differences in exchange in a peptide were considered significant if they met all three of the following criteria: >5% change in exchange, >0.4 Da difference in exchange, and a .

结果表示为氘掺入的相对水平，反向交换的唯一控制是缓冲液中存在的氘水平（86.79%）。如果肽的交换差异满足以下三个标准，则认为它们具有显着性：>交换变化>5%，>交换中的0.4 Da差异，以及a。

value < 0.01 using a two-tailed Student’s

使用两尾学生的

test. Samples were only compared within a single experiment and were never compared to experiments completed at a different time with a different final D

测试。样本仅在单个实验中进行比较，从未与在不同时间完成的具有不同最终D的实验进行比较

O level. The data analysis statistics for all HDX-MS experiments are in the source data according to the guidelines set out previously

O级。根据之前制定的指南，所有HDX-MS实验的数据分析统计数据都在源数据中

. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository

。质谱蛋白质组学数据已通过PRIDE合作伙伴存储库保存到ProteomeXchange Consortium

with the dataset identifier

使用数据集标识符

PXD033072

Description of %D and #D graphs

%D和#D图的描述

The raw HDX data are shown in two different formats. To allow for visualization of differences across all peptides, we utilized number of deuteron difference (#D) plots (Fig.

原始HDX数据以两种不同的格式显示。为了可视化所有肽的差异，我们利用了氘核差异数（#D）图（图）。

5b条

). The raw peptide deuterium incorporation graphs for a selection of peptides with significant differences are shown in Fig.

)。图1显示了用于选择具有显着差异的肽的原始肽氘掺入图。

5摄氏度

and Supplementary Fig.

和补充图。

, with the raw data for all analyzed peptides in the source data. These plots show the total difference in deuterium incorporation over the entire H/D exchange time course, with each point indicating a single peptide.

，以及源数据中所有分析肽的原始数据。这些图显示了在整个H/D交换时间过程中氘掺入的总差异，每个点表示单个肽。

Negative stain electron microscopy and image processing

负染电子显微镜和图像处理

Negative-stained specimens were prepared as previously described

如前所述制备阴性染色标本

. Briefly, FL Rab3GAP and core Rab3GAP from their respective peak fraction of size exclusion chromatography were adsorbed onto a carbon-coated copper grid for 0 s, washed with ddH2O and stained with 0.75% uranyl formate (Electron Microscopy Sciences) for 30 s. Using the Talos L120C Transmission Electron Microscope (Thermo Fisher Scientific) equipped with a Ceta camera, micrographs were collected with an accelerating voltage of 120 kV, nominal magnification of ×49,000 and a defocus of 2 µm.

简而言之，将来自尺寸排阻色谱各自峰分数的FL Rab3GAP和核心Rab3GAP吸附在碳涂覆的铜网格上0 s，用ddH2O洗涤并用0.75%甲酸铀酰（电子显微镜科学）染色30 s。使用配备有Ceta相机的Talos L120C透射电子显微镜（Thermo Fisher Scientific），以120 kV的加速电压，×49000的标称放大率和2µm的散焦收集显微照片。

The micrographs were imported and processed using CryoSPARC v4.0.3.

显微照片是使用CryoSPARC v4.0.3导入和处理的。

. A small subset of particles was manually picked and extracted with a box size of 100 pixels. 2D class averages were created from these particles to be used to generate templates for particle picking. FL Rab3GAP was subjected to multiple rounds of 2D classification to curate particle quality. A 2D classification was used to determine the overall architecture of core Rab3GAP..

。手动拾取并提取一小部分粒子，框大小为100像素。从这些粒子创建2D类平均值，以用于生成用于粒子拾取的模板。FL Rab3GAP经过多轮2D分类以确定颗粒质量。2D分类用于确定核心Rab3GAP的总体架构。。

Cryo-EM sample preparation and data collection

低温电磁样品制备和数据收集

C-Flat 2/1 grids were subjected to a 25 s glow discharge at 15 mA using a Pelco easiGlow glow-discharger. Following this, 3 µL of purified truncated Rab3GAP1/2, at a concentration of 0.3 mg mL

使用Pelco easiGlow辉光放电仪在15 mA下对C平面2/1网格进行25 s辉光放电。在此之后，3l纯化的截短Rab3GAP1/2，浓度为0.3mg/mL

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, was adsorbed onto the grids and quickly plunge frozen into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific), with a blot force of 5 and 2 s blot time at 100% humidity and 4 °C. To assess the quality of the ice and particle quality, grids were screened using the 200 kV Glacios transmission electron microscope, equipped with a Falcon 3EC direct electron detector (DED).

将其吸附在网格上，并使用Vitrobot Mark IV（Thermo Fisher Scientific）快速将其冷冻到液态乙烷中，在100%湿度和4°C下的印迹力为5和2 s印迹时间。为了评估冰的质量和颗粒质量，使用配备有Falcon 3EC直接电子检测器（DED）的200 kV Glacios透射电子显微镜筛选网格。

A total of 19,822 movies were collected using the Titan Krios transmission electron microscope (Thermo Fisher Scientific) operating at 300 kV, equipped with a Falcon IV DED and Selectris energy filter. The movies were obtained at a nominal magnification of ×215,000, corresponding to a pixel size of 0.59 Å, and with a total dose of 50 e- Å.

使用在300 kV下运行的Titan Krios透射电子显微镜（Thermo Fisher Scientific）收集了总共19822部电影，该显微镜配备了Falcon IV DED和Selectris能量过滤器。电影的标称放大倍数为215000，对应于0.59Å的像素大小，总剂量为50 e。

−2

over 59 frames.

超过59帧。

Cryo-EM data processing and model building

低温电磁数据处理与模型建立

The cryo-EM data in this study was processed using cryoSPARC v.4.0.3. The movies were subjected to patch motion correction with a Fourier cropping by a factor of 2 and the CTF parameters of the micrographs were generated using patch CTF estimation with default settings. Initially, a subset of movies was selected for template-free 2D class averaging to generate templates for particle picking.

本研究中的cryo-EM数据使用cryoSPARC v.4.0.3进行处理。电影经过傅里叶裁剪2倍的补丁运动校正，显微照片的CTF参数是使用默认设置的补丁CTF估计生成的。最初，选择电影的一个子集进行无模板2D类平均，以生成用于粒子拾取的模板。

These templates were employed to pick 15,244,538 particles from the whole dataset. Particles were extracted with a box size of 640 pixels, Fourier cropped to a box size of 160 pixels, and were subjected to multiple rounds of 2D classification to discard poorly defined particles, resulting in the selection of high-quality particles for further processing.

这些模板用于从整个数据集中挑选15244538个粒子。提取框大小为640像素的粒子，傅立叶裁剪为160像素的框大小，并进行多轮2D分类以丢弃定义不明确的粒子，从而选择高质量的粒子进行进一步处理。

Particles from the best classes were re-extracted with a 640-pixel box size to be utilized for multiclass ab initio reconstruction and heterogeneous refinement using two classes. These particles were used to carry out per-particle local-motion correction with a 1280-pixel box size, downsized to 640 pixels.

来自最佳类别的粒子以640像素的盒子大小重新提取，以用于使用两个类别的多类从头开始重建和异质细化。这些粒子用于执行每粒子局部运动校正，框大小为1280像素，缩小到640像素。

Particles were then subjected to additional rounds of 2D classification, ab initio reconstruction, homogeneous refinement, and non-uniform-refinement, which generated a global reconstruction with an overall resolution of 3.39 Å, as assessed by the Fourier shell correlation 0.143 criterion. Local refinements of the rigid region of the complex using masks were performed to further improve the map quality to be resolved at 3.37 Å.

然后对粒子进行额外的几轮2D分类，从头开始重建，均匀细化和非均匀细化，这产生了整体分辨率为3.39Å的全局重建，如傅立叶壳相关0.143标准所评估的。使用掩模对复合物的刚性区域进行局部细化，以进一步提高在3.37Å处解析的地图质量。

Local resolutions were estimated using the local resolution estimation tool in cryoSPARC and auto-sharpening was performed in PHENIX.

使用cryoSPARC中的局部分辨率估计工具估计局部分辨率，并在PHENIX中进行自动锐化。

. Structural models of Rab3GAP1 and Rab3GAP2 from the AlphaFold Protein Structure Database were next fit into the cryo-EM density map using Chimera

接下来，使用Chimera将来自AlphaFold蛋白质结构数据库的Rab3GAP1和Rab3GAP2的结构模型拟合到低温EM密度图中

. This was followed by iterative rounds of refinement in Phenix.real_space_refine and manual model building in COOT

随后在Phenix.real\u space\u refine中进行了迭代轮的优化，并在COOT中进行了手动模型构建

. Structural and refinement statistics is summarized in Supplementary Table

结构和改进统计数据总结在补充表中

Bioinformatics

生物信息学

Protein structure and binding interfaces were predicted for core Rab3GAP with and without Rab18 using AlphaFold3 with default settings except for minimization of the top-ranked structure

使用默认设置的AlphaFold3预测了有无Rab18的核心Rab3GAP的蛋白质结构和结合界面，但最小化了排名靠前的结构

. Prediction confidence metrics were reported as pTMscore, iPTMscore, PAE and pLDDT. The cryo-EM structure of core Rab3GAP was used as input for Foldseek to identify structurally related proteins

预测置信度指标报告为pTMscore，iPTMscore，PAE和pLDDT。核心Rab3GAP的cryo-EM结构被用作Foldseek的输入，以鉴定结构相关蛋白

. Clustal Omega Multiple Sequence Alignment

.Clustal Omega多序列比对

was used to align the sequences of human Rabs, followed by ESPript 3.0 analysis to identify conserved regions

用于比对人类Rabs的序列，然后进行ESPript 3.0分析以鉴定保守区域

Cell culture

细胞培养

HeLa cells were cultured in DMEM high glucose with L-glu Na-pyruvate (Sigma-Aldrich, D6429) supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma-Aldrich, F1051) at 37 °C and 5% CO

将HeLa细胞在DMEM高葡萄糖中培养，其中L-glu-Na丙酮酸（Sigma-Aldrich，D6429）补充有10%（v/v）胎牛血清（FBS）（Sigma-Aldrich，F1051），温度为37°C，CO为5%

. Cells are routinely checked for mycoplasma contamination.

.常规检查细胞的支原体污染。

Immunofluorescence

免疫荧光

HeLa cells were seeded at 2.5 × 10

HeLa细胞接种于2.5×10

cells per well onto #1.5 coverslips 24 h prior to co-transfection with 0.25 μg of pcDNA3.1-GFP-Rab18 (Genscript) using Lipofectamine 3000 (Invitrogen, L3000008) according to the manufacturer’s protocol. Transfection reagent was removed after 5 h and replaced with fresh media and allowed to incubate for another 19 h.

根据制造商的方案，使用Lipofectamine 3000（Invitrogen，L300008）与0.25μgpcDNA3.1-GFP-Rab18（Genscript）共转染前24小时，将每孔细胞置于1.5个盖玻片上。5小时后取出转染试剂，用新鲜培养基代替，再孵育19小时。

Cells were rinsed 2x with PBS and then fixed in 3% PFA + 0.2% Glutaraldehyde for 15 min at room temperature. Coverslips were washed 3x with PBS before permeabilization with 0.2% Triton X-100 for 5 min at room temperature. Coverslips were washed 3x with PBS before incubating with 1 mg mL.

将细胞用PBS冲洗2次，然后在室温下在3%PFA++0.2%戊二醛中固定15分钟。盖玻片用PBS洗涤3次，然后在室温下用0.2%Triton X-100透化5分钟。盖玻片用PBS洗涤3次，然后与1mg/mL孵育。

−1

sodium borohydride for 10 min at room temperature. Coverslips were washed 3x with PBS and then blocked using 2.5% Bovine serum albumin (Sigma-Aldrich) for 1 h at room temperature. Nuclei were stained with 0.5 mg mL

硼氢化钠在室温下放置10分钟。盖玻片用PBS洗涤3次，然后在室温下用2.5%牛血清白蛋白（Sigma-Aldrich）封闭1小时。细胞核用0.5毫克毫升染色

−1

DAPI (Invitrogen, D1306) for 5 min. Cells were washed 3x with PBS and then coverslips were mounted using ProLong Glass Antifade (Invitrogen, P36980). Fixed samples were imaged on a 63x/1.40 NA objective (oil immersion, HC PL APO Leica Microsystems) on a Leica STELLARIS 5 LIAchroic inverted scanning confocal microscope.

DAPI（Invitrogen，D1306）5分钟。将细胞用PBS洗涤3次，然后使用ProLong Glass Antifade（Invitrogen，P36980）固定盖玻片。固定样品在Leica STELLARIS 5 LIAchroic倒置扫描共聚焦显微镜上的63x/1.40 NA物镜（油浸，HC PL APO Leica Microsystems）上成像。

GFP signal was acquired using a 488 nM laser line and the power HyD S detector (Leica Microsystems). Brightness was adjusted in ImageJ/Fiji..

使用488nm激光线和power HyD S检测器（Leica Microsystems）采集GFP信号。亮度在ImageJ/Fiji中进行了调整。。

Statistical analysis

统计分析

For all GAP and GEF assays, experiments were performed in technical triplicate with mean ± SEM shown in figures. Statistical analysis between conditions was performed using a two-tailed Student’s

对于所有GAP和GEF测定，实验一式三份进行，平均SEM如图所示。使用两尾学生的

test or ordinary one-way ANOVA (GraphPad Prism). For HDX-MS assays, experiments were performed in technical triplicated with mean ± SD shown in figures. Statistical analysis between conditions was performed using a two-tailed Student’s

测试或普通单向方差分析（GraphPad Prism）。对于HDX-MS测定，实验以技术一式三份进行，平均SD如图所示。使用两尾学生的

test. The following legend is used for statistical significance: *

测试。以下图例用于统计显着性：*

< 0.05 and ns

<0.05和ns

> 0.05.

Reporting summary

报告摘要

Further information on research design is available in the

有关研究设计的更多信息，请参阅

Nature Portfolio Reporting Summary

自然投资组合报告摘要

linked to this article.

链接到本文。

Data availability

数据可用性

The 3D reconstruction of core Rab3GAP is available at the Electron Microscopy Data Bank under accession code

核心Rab3GAP的3D重建可在电子显微镜数据库中以登录号获得

EMD-43655

. The atomic coordinates for core Rab3GAP are available at the Protein Data Bank under accession code

。核心Rab3GAP的原子坐标可在蛋白质数据库中以登录号获得

8VYB

. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository

。质谱蛋白质组学数据已通过PRIDE合作伙伴存储库保存到ProteomeXchange Consortium

with the dataset identifier

使用数据集标识符

PXD033072

. All other data are available from the corresponding author upon request.

。所有其他数据可应要求从通讯作者处获得。

Source data

源数据

are provided with this paper.

随本文提供。

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Acknowledgements

致谢

This work was supported by a Project Grant from the Canadian Institutes of Health Research to C.K.Y. (PJT-168907) and a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to C.K.Y. (AWD-007855). Grids were prepared and data collected at the High Resolution Macromolecular Electron Microscopy (HRMEM) facility at the University of British Columbia (.

这项工作得到了加拿大卫生研究院对C.K.Y.（PJT-168907）的项目资助和加拿大自然科学与工程研究委员会对C.K.Y.（AWD-007855）的发现资助。在不列颠哥伦比亚大学（University of British Columbia）的高分辨率大分子电子显微镜（HRMEM）设施中制备了网格并收集了数据。

https://cryoem.med.ubc.ca

). We thank Claire Atkinson, Joeseph Felt, Liam Worrall and Natalie Strynadka. HRMEM is funded by the Canadian Foundation for Innovation and the British Columbia Knowledge Development Fund. Fluorescent images were collected and/or image analysis for this work was performed in the University of British Columbia Life Sciences Institute Imaging Core Facility, RRID: SCR_023783.

)。我们感谢克莱尔·阿特金森、乔·塞夫·费尔特、利亚姆·沃拉尔和娜塔莉·斯特里纳德卡。HRMEM由加拿大创新基金会和不列颠哥伦比亚知识发展基金资助。收集荧光图像和/或在不列颠哥伦比亚大学生命科学研究所成像核心设施RRID中进行这项工作的图像分析：SCR\U 023783。

We thank Guang Guo for support with image analysis. Molecular graphics and analyses performed with UCSF Chimera (developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311), ChimeraX (developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases), and PyMOL Molecular Graphics System (Schrödinger, LLC)..

我们感谢Guang Guo对图像分析的支持。使用UCSF Chimera（由加州大学旧金山分校生物计算，可视化和信息学资源开发，NIH P41-GM103311支持），ChimeraX（由加州大学旧金山分校生物计算，可视化和信息学资源开发，由美国国立卫生研究院R01-GM129325和美国国家过敏和传染病研究所网络基础设施和计算生物学办公室支持）和PyMOL分子图形系统（Schrödinger，LLC）进行分子图形和分析。。

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These authors contributed equally: Gage M. J. Fairlie, Kha M. Nguyen.

这些作者做出了同样的贡献：Gage M.J.Fairlie，Kha M.Nguyen。

Authors and Affiliations

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Life Sciences Institute, Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, BC, V6T 1Z3, Canada

不列颠哥伦比亚大学生物化学与分子生物学系生命科学研究所，温哥华，不列颠哥伦比亚省，V6T 1Z3，加拿大

Gage M. J. Fairlie, Kha M. Nguyen, Sung-Eun Nam, Alexandria L. Shaw, Hannah R. Shariati, Xinyin Wang, Michael Gong, John E. Burke & Calvin K. Yip

Gage M.J.Fairlie、Kha M.Nguyen、Sung Eun Nam、Alexandria L.Shaw、Hannah R.Shariati、Xinyin Wang、Michael Gong、John E.Burke和Calvin K.Yep

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 2Y2, Canada

维多利亚大学生物化学与微生物学系，不列颠哥伦比亚省维多利亚州，V8W 2Y2，加拿大

Alexandria L. Shaw, Matthew A. H. Parson, Meredith L. Jenkins & John E. Burke

亚历山大·L·肖、马修·A·H·帕森、梅雷迪思·L·詹金斯和约翰·E·伯克

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Contributions

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G.M.J.F., K.M.N., H.R.S., S.N., A.L.S., J.E.B., and C.K.Y. designed research; G.M.J.F., K.M.N., H.R.S., S.N., A.L.S., M.A.H.P., X.W., M.L.J., and M.G. performed research; G.M.J.F., K.M.N., H.R.S., S.N., A.L.S., M.A.H.P., J.E.B., and C.K.Y. analyzed data; and G.M.J.F., K.M.N., and C.K.Y. wrote the paper..

G、 M.J.F.，K.M.N.，H.R.S.，S.N.，A.L.S.，J.E.B。和C.K.Y.设计的研究；G、 M.J.F.，K.M.N.，H.R.S.，S.N.，A.L.S.，M.A.H.P.，X.W.，M.L.J。和M.G.进行了研究；G、 M.J.F.，K.M.N.，H.R.S.，S.N.，A.L.S.，M.A.H.P.，J.E.B。和C.K.Y.分析的数据；和G.M.J.F.，K.M.N。和C.K.Y.写了这篇论文。。

Corresponding author

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Calvin K. Yip

叶嘉文

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Competing interests

相互竞争的利益

J.E.B. reports personal fees from Scorpion Therapeutics and Reactive Therapeutics and research contracts from Novartis and Calico Life Sciences. All other authors declare no competing interests.

J、 E.B.报告了Scorpion Therapeutics和Reactive Therapeutics的个人费用，以及诺华和Calico Life Sciences的研究合同。所有其他作者都声明没有利益冲突。

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thanks Daniel Deredge who co-reviewed with Juliet Obi; and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

感谢与朱丽叶·欧比共同评论的丹尼尔·德里奇；另一位匿名审稿人对这项工作的同行评审做出了贡献。同行评审文件可用。

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Fairlie, G.M.J., Nguyen, K.M., Nam, SE.

费尔利，G.M.J.，阮，K.M.，南，东南。

et al.