Cells need to allow various molecules to pass through the plasma membrane on their surface. Some molecules have to enter the cell, whereas others have to leave. Cells rely on a process called endocytosis to move large molecules into the cell. This involves part of the membrane engulfing the molecule to form a ‘bubble’ around it. This bubble, which is called an endosome, then moves the molecule to final destination inside the cell.

A protein called Rab5 controls how a new endosome is produced. However, before this can happen, various other molecules—including two proteins called Rabex-5 and Rabaptin-5—must activate the Rab5 protein. Exactly how these three proteins interact with each other was unknown.

Zhang et al. used X-ray crystallography to examine the structures of the complexes formed when Rabex-5 and Rabaptin-5 bind to each other, both when Rab5 is present, and also when it is absent. Biochemical and biophysical experiments confirmed that the Rabex-5/Rabaptin-5 complex is able to activate Rab5.

Zhang et al. also found that Rabex-5, on its own, folds so that the site that normally binds to Rab5 instead binds to a different part of Rabex-5, thus preventing endocytosis. However, when Rabaptin-5 forms a complex with Rabex-5, the Rab5 binding site is freed up.

The Rabex-5/Rabaptin-5 complex can switch between a V shape and a linear structure. Binding to Rab5 stabilizes the linear form of the complex, which then helps activate Rab5, and subsequently the activated Rab5 can interact with other downstream molecules, triggering endocytosis.

Endocytosis is a major process which eukaryotic cells use to absorb extracellular materials (Doherty and McMahon, 2009; Grant and Donaldson, 2009; Huotari and Helenius, 2011). In this process, small GTPase Rab5 functions as a master regulator of the early endosomal biogenesis (Stenmark, 2009; Mizuno-Yamasaki et al., 2012; Zeigerer et al., 2012). Rab5 is localized to early endosomal membrane via its isoprenylated C-terminus and regulates early endosomal fusion through interactions with an array of effectors including Rabaptin-5 (Stenmark et al., 1995), Rabenosyn-5 (Nielsen et al., 2000), EEA1 (Mills et al., 1998; Simonsen et al., 1998), PI3Ks (Li et al., 1995; Christoforidis et al., 1999), and APPLs (Miaczynska et al., 2004). Like other small GTPases, Rab5 exists mainly in two states, the GTP-bound active state and the GDP-bound inactive state, and requires guanosine nucleotide exchange factor (GEF) for activation and GTPase-activating protein (GAP) for inactivation.

Rabex-5 is a specific GEF for Rab5, Rab17, and Rab21 (Horiuchi et al., 1997; Delprato et al., 2004; Delprato and Lambright, 2007; Mori et al., 2013). The GEF domain is located in the middle and consists of a helical bundle (HB) domain and a Vps9 domain (Figure 1—figure supplement 1). Besides, the N-terminal region comprises two distinct ubiquitin-binding domains, a zinc finger domain and a motif interacting with ubiquitin domain, which can interact with ubiquitinated cargoes or adaptors to recruit Rabex-5 to early endosomal membrane (Lee et al., 2006; Mattera et al., 2006; Penengo et al., 2006; Mattera and Bonifacino, 2008) and function as an E3 ligase for Ras ubiquitination to promote Ras endosomal localization (Xu et al., 2010; Yan et al., 2010). The following membrane binding motif domain and the HB domain together compose an early endosomal targeting domain that can direct Rabex-5 to early endosomal membrane (Zhu et al., 2007). The C-terminal region consists of a coiled-coil (CC) domain and a proline rich region; the CC domain is involved in autoinhibition of the GEF activity and binding of Rabaptin-5 (Lippe et al., 2001; Mattera et al., 2006; Delprato and Lambright, 2007). Rabaptin-5 is a key effector of Rab5 and plays an important role in both homotypic and heterotypic fusions of early endosomes (Stenmark et al., 1995; Stenmark, 2009). It is a scaffold protein consisting of primarily four coiled-coil domains, namely C1-1, C1-2, C2-1, and C2-2 domains (Figure 1—figure supplement 1). The C2-1 domain is responsible for interaction with and recruitment of Rabex-5 to early endosomal membrane to activate Rab5 (Lippe et al., 2001; Mattera et al., 2006; Delprato and Lambright, 2007). Besides, the N-terminal region can mediate interactions with Rab4 and Rab8 (Vitale et al., 1998; Omori et al., 2008); the middle region can interact with the GAE and GAT domains of GGAs that function as effectors of the Arf family small GTPases in the tethering and fusion of trans Golgi network (TGN) (Mattera et al., 2003; Miller et al., 2003; Zhu et al., 2004a); and the C-terminal region can interact with the GTP-bound Rab5 that recruits Rabaptin-5 to early endosomal membrane (Vitale et al., 1998; Zhu et al., 2004b). In addition, Rabex-5 and Rabaptin-5 have been shown to function as neoplastic tumor suppressors and are implicated in human cancers (Magnusson et al., 2001; Wang et al., 2009; Christoforides et al., 2012; Thomas and Strutt, 2014), and Rabex-5 has also been shown to determine the neurite localization of its substrate Rab proteins and thus plays an important role in the development of hippocampal neurons (Mori and Fukuda, 2013; Mori et al., 2013).

Previous structural, biochemical, and biological data have demonstrated that Rabex-5 and Rabaptin-5 function together to activate Rab5 in endocytosis; the GEF activity of Rabex-5 could be autoinhibited by its CC domain and activated by binding of the Rabaptin-5 C2-1 domain (Rabaptin-5C21) to the CC domain (Lippe et al., 2001; Delprato and Lambright, 2007; Zhu et al., 2007, 2010). However, the underlying molecular mechanism is unclear. We report here the crystal structures of a Rabex-5 variant in complex with the dimeric Rabaptin-5C21 (Rabaptin-5C21₂) and in complex with Rabaptin-5C21₂ and Rab5. The structural data together with the in vitro functional data reveal the molecular mechanism for the regulation of the Rabex-5 GEF activity.

The previous biochemical and biological data showed that Rabex-5 functions together with Rabaptin-5 to activate Rab5 and then to promote the fusion of early endosomes in endocytosis (Lippe et al., 2001; Delprato and Lambright, 2007; Zhu et al., 2007). The GEF activity of Rabex-5 is autoinhibited by its CC domain and is activated by the binding of Rabaptin-5 via its C2-1 domain. However, the molecular mechanism is unknown. In this work, we determined the crystal structures of Rabex-5Δ in complex with Rabaptin-5C21 and in complex with Rabaptin-5C21 and Rab5, which are validated by biophysical and biochemical analyses.

Our structural data show that at the substrate-binding site of the Rabex-5 GEF domain, there is a surface groove composed of largely nonpolar residues. Rabex-5CC forms a stable amphipathic α-helix that tends to bury its nonpolar surface via oligomerization or interaction with the C-terminal regions of Rabaptin-5C21₂. The nonpolar surface of Rabex-5CC has good chemical and geometrical complementarities with the nonpolar surface groove of the GEF domain and thus might be able to bind there to block the substrate binding and hence autoinhibit the GEF activity as proposed by Delprato and Lambright (2007). Nonetheless, our structural and biochemical data show that although Rabex-5CC alone exists as a stable helix in both solution and crystal structure, it cannot form a stable complex with the GEF domain as shown by both GST pull-down assay and ITC analysis (data not shown). In addition, Rabex-5 and Rabex-5Δ cannot be crystallized alone and Rabex-5 can be easily proteolyzed in the linker region (Figure 1—figure supplement 2). These results suggest that the CC domain and the linker have high flexibility. Moreover, Rabex-5 itself has a basal GEF activity which is slightly weaker (about 1/3) than that of the constitutively active GEF domain (Figure 4A,B) but is not so weak compared with some other GEFs including DSS4 (a GEF for Ypt1p) (Esters et al., 2001) and MSS4 (a GEF for Rab8) (Zhu et al., 2001; Itzen et al., 2006). Mutations of the residues on the nonpolar surface of Rabex-5CC can enhance the GEF activity by 1.4–1.9 folds (Figure 4—figure supplement 1A). These results together indicate that the binding of Rabex5CC to the GEF domain is not tight, and Rabex-5 alone is not completely autoinhibited.

On the other hand, in the structure of the R2Δ complex, Rabaptin-5C21₂ forms 2 two-helix bundles with a V-shaped conformation, and Rabex-5CC interacts via the nonpolar surface with the C-terminal regions of Rabaptin-5C21₂ to form a tight three-helix bundle. Meanwhile, the GEF domain folds along the three-helix bundle with a partially occupied substrate-binding site, suggesting that further conformational change is required to completely expose the substrate-binding site for Rab5 binding and activation. Indeed, in the structure of the R3Δ complex, although Rabex-5CC still forms a tight three-helix bundle with the C-terminal regions of Rabaptin-5C21₂, Rabaptin-5C21₂ forms a linear two-helix bundle which is different from the V-shaped conformation but similar to that in the Rabex-5CC-Rabaptin-5C21₂ complex and the GAT-Rabaptin-5C21₂ complex (Zhu et al., 2004a). The GEF domain is dislodged from the three-helix bundle and interacts with the N-terminal regions of Rabaptin-5C21₂, and the substrate-binding site is completely exposed to bind Rab5. Meanwhile, our SAXS analysis results indicate that the R2 and R2Δ complexes mainly assume the V-shaped conformation as observed in the R2Δ structure but have some flexibility in solution, and our biochemical data show that the R2 and R2Δ complexes have full GEF activities as the constitutively active GEF domain and the N-terminal regions of Rabaptin-5C21₂ are required for the function of the R2 complex in the activation of Rab5.

Based on the structural and biological data in this work and those reported previously, we can propose the molecular mechanism for the regulation of the Rabex-5 GEF activity despite the lack of an intact Rabex-5 structure (Figure 5). In the free-form Rabex-5, Rabex-5CC may bind weakly via the nonpolar surface to the substrate-binding site of the GEF domain, leading to the blockage of the substrate-binding site and thus a weak autoinhibition of the GEF activity (Figure 5, State I). As the binding of Rabex-5CC to the GEF domain is not tight, it might assume alternative conformations. The linker between the GEF and CC domains might help to modulate the conformational flexibility and/or the relative conformations of the two domains and thus plays a minor role in the autoinhibition. One plausible alternative conformation of Rabex-5CC might be similar to that observed in the R2Δ structure with a largely exposed substrate-binding site of the GEF domain. As the interaction of Rabex-5CC with the GEF domain via the nonpolar surface is much tighter than that via the adjacent surface, Rabex-5CC might exist mainly in the autoinhibitory conformational state and partially in the alternative conformational state. This may explain why the free-form Rabex5 exhibits some basal GEF activity. Rabaptin-5C21 might bind to the C-terminal region of the nonpolar surface of Rabex-5CC in the autoinhibitory conformational state and induce conformational change of the α-helix to transform into the conformational state as observed in the R2Δ structure, or directly to the exposed nonpolar surface of Rabex-5CC in the alternative conformational state, leading to the relief of the autoinhibition and the release of the GEF activity (Figure 5, State II).

Figure 5

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When the Rabex-5-Rabaptin-5 complex is recruited to the early endosomal membrane via the interaction of the C-terminal region of Rabaptin-5 with the GTP-bound Rab5, Rabex-5 can activate Rab5 locally in a very efficient way. The binding of the GDP-bound Rab5 to Rabex-5 induces further conformational changes of the Rabex-5-Rabaptin-5 complex such that Rabaptin-5C21₂ transforms from the V-shaped to the linear conformation, the GEF domain is dislodged from Rabex-5CC and the C-terminal regions of Rabaptin-5C21₂, and the substrate-binding site is completely exposed to the solvent to bind Rab5 as observed in the R3Δ structure (Figure 5, State III). In this conformational state, the Rabex-5-Rabaptin-5 complex can facilitate the exchange of GDP- to GTP-bound Rab5. The not-so-tight interaction of the Rabex-5 GEF domain with Rabex-5CC and Rabaptin-5C21₂ as observed in the R2Δ structure allows the conformational changes easily during the substrate binding. The conformational changes of the Rabex-5-Rabaptin-5 complex induced by the binding of Rab5 leading to the full activation of the Rabex-5 GEF activity may provide another leverage to ensure the high substrate specificity.

In the context of the early endosomal membrane, Rabex-5 can directly target to the early endosomes either through the interaction of the N-terminal ubiquitin binding domain (UBD) with the ubiquitinated cargoes or through the early endosomal targeting domain (EET) (Zhu et al., 2007; Mattera and Bonifacino, 2008). In this case, as the GEF activity of Rabex-5 is autoinhibited by Rabex-5CC, Rabex-5 can only activate Rab5 at the basal level. In the cells, most Rabex-5 forms a stable complex with Rabaptin-5 which can be recruited to early endosomes via the binding of Rabaptin-5 through the C-terminal region to GTP-bound Rab5 (Lippe et al., 2001; Zhu et al., 2010). In this case, Rabaptin-5 not only assists the recruitment of Rabex-5 to the early endosomal membrane, but also activates the GEF activity of Rabex-5. The positive feedback loop among Rab5, its effector Rabaptin-5, and its GEF Rabex-5 can lead to a robust activation of Rab5, which then promotes the fusion of early endosomes efficiently.

As a scaffold protein, Rabaptin-5 comprises several coiled-coil regions which can mediate interactions with different proteins to exert different functions. In addition to acting as the effector of Rab5 to function in the fusion of early endosomes, Rabaptin-5 can interact with Rab4 via the N-terminal region and thus may serve as the effector of Rab4 to function in the endocytic recyling process (Vitale et al., 1998). As the C2-1 domain of Rabaptin-5 can interact with the CC domain of Rabex5 and the GAT domain of GGA1, and Rabaptin-5 is the effector of Rab5 and GGA1 is the effector of Arf1, this dual interaction might mediate the crosstalk between Rab and Arf GTPases to promote the tethering and fusion of early endosomes and trans-Golgi network-derived vesicles (Mattera et al., 2003; Zhu et al., 2004a; Kawasaki et al., 2005).

In the GAT-Rabaptin-5C21₂ structure, Rabaptin-5C21₂ assumes a linear conformation, the N-terminal regions of Rabaptin-5C21₂ bind one GAT, and two symmetry-related Rabaptin-5C21₂ dimerize through the C-terminal regions (Zhu et al., 2004a), which are the binding site for Rabex-5CC. Interestingly, Rabaptin-5C21₂ assumes a linear conformation in the Rabex-5CC-Rabaptin-5C21₂ structure but a V-shaped conformation in the R2Δ structure, and in both structures, the C-terminal regions of Rabaptin-5C21₂ bind one Rabex-5CC and two symmetry-related Rabaptin-5C21₂ dimerize through the N-terminal regions which are the binding site for GAT. Moreover, in the R3Δ structure, Rabaptin-5C21₂ also assumes a linear conformation but two Rabaptin-5C21₂ dimerize through the middle regions (residues 590–600) between the GAT-binding site and the Rabex-5CC-binding site. As the GAT-binding site is unoccupied, Rabaptin-5 may bind Rabex-5 and GGA1 simultaneously. These results demonstrate that Rabaptin-5C21 always forms a dimer which can assume either a V-shaped or a linear conformation and can bind other proteins via different regions. In addition, the dimeric Rabaptin-5C21₂ prefers to further dimerize to form a dimer of dimers via the regions that are not involved in the interactions with other proteins. In the context of full-length Rabaptin-5, it is possible that Rabaptin-5C21₂ might exist in either the V-shaped or the linear conformation depending on the functional state. The dimerization of Rabaptin-5C21₂ might not only avoid exposure of the hydrophobic surface and minimize the overall energy of the protein in aqueous environment, but also play some functional roles in the tethering and fusion of early endosomes and/or with other vesicles.

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Author details

1. Zhe Zhang

   State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

   Contribution

   ZZ, Carried out the cloning, protein purification, crystallization, structure determination and analyses, biophysical and biochemical studies, and drafted and revised the manuscript

   Competing interests

   The authors declare that no competing interests exist.

2. Tianlong Zhang

   State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

   Contribution

   TZ, Participated in the structure determination and analyses

   Competing interests

   The authors declare that no competing interests exist.

3. Shanshan Wang

   State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

   Contribution

   SW, Participated in the cloning and protein purification

   Competing interests

   The authors declare that no competing interests exist.

4. Zhou Gong

   State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, China

   Contribution

   ZG, Participated in the design, analysis and discussion of the SAXS study

   Competing interests

   The authors declare that no competing interests exist.

5. Chun Tang

   State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan, China

   Contribution

   CT, Participated in the design, analysis and discussion of the SAXS study, and revised the manuscript

   Competing interests

   The authors declare that no competing interests exist.

6. Jiangye Chen

   State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

   Contribution

   JC, Participated in the design, analysis and discussion of the biochemical studies

   Competing interests

   The authors declare that no competing interests exist.

7. Jianping Ding

   State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

   Contribution

   JD, Conceived the study, participated in the designs and data analyses of all experiments, and wrote and revised the manuscript

   For correspondence

   jpding@sibcb.ac.cn

   Competing interests

   The authors declare that no competing interests exist.

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