The RAB-5 and RAB-7 GTPases regulate endosome to lysosome trafficking. Here, we show that Caenorhabditis elegans TBC-2 functions as a RAB-5 GAP. TBC-2 colocalizes with RAB-7 on late endosomes, and requires RAB-7 for membrane localization where TBC-2 could function to antagonize RAB-5 activity during early to late endosome maturation.

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Regulation of endosomal trafficking by Rab GTPases depends on selective interactions with multivalent effectors, including EEA1 and Rabenosyn-5, which facilitate endosome tethering, sorting, and fusion. Both EEA1 and Rabenosyn-5 contain a distinctive N-terminal C 2 H 2 zinc finger that binds Rab5. How these C 2 H 2 zinc fingers recognize Rab GTPases remains unknown. Here, we report the crystal structure of Rab5A in complex with the EEA1 C 2 H 2 zinc finger. The binding interface involves all elements of the zinc finger as well as a short N-terminal extension but is restricted to the switch and interswitch regions of Rab5. High selectivity for Rab5 and, to a lesser extent Rab22, is observed in quantitative profiles of binding to Rab family GTPases. Although critical determinants are identified in both switch regions, Rab4-to-Rab5 conversionof-specificity mutants reveal an essential requirement for additional substitutions in the proximal protein core that are predicted to indirectly influence recognition through affects on the structure and conformational stability of the switch regions.Rab5 | effector | Rabenosyn-5 | endosome | structure R ab GTPases regulate organelle biogenesis and vesicular transport by cycling between inactive (GDP-bound) and active (GTP-bound) states (1-4). After membrane targeting mediated by Rab GDI and conversion to the active form by GDP/GTP exchange factors (GEFs), Rab GTPases interact with effectors implicated in vesicular transport, tethering, and fusion (5-7). Rab5 controls endosome biogenesis, maturation, and fusion through multiple effectors (8-11). The Rab5 effector Early Endosomal Autoantigen 1 (EEA1) enhances endosome fusion and in combination with other soluble factors, including the Rab5 effector complex Rabenosyn-5·hVps45 and the Rab5 effector/GEF complex Rabaptin-5·Rabex-5, can substitute for cytosol in assays that reconstitute endosome fusion in vitro (12-15).EEA1 is a long coiled coil homodimer with an N-terminal C 2 H 2 zinc finger (ZF) and a C-terminal FYVE domain (16). The FYVE domain recognizes phosphatidyl inositol 3-phosphate (PI3P) and mediates PI3P-dependent recruitment to early endosomes (17-23). Low affinity Rab5 binding to the FYVE domain and proximal coiled coil is thought to enhance targeting to endosomes containing both Rab5 and PI3P (21). Higher affinity binding to the C 2 H 2 ZF is proposed to facilitate long range tethering preceding formation of SNARE complexes required for membrane docking and fusion (1,12,24). Rabenosyn-5 also contains an N-terminal C 2 H 2 ZF, which binds Rab5 with similar affinity, in addition to helical hairpin domains with distinct binding specificities for Rab4/Rab14 and Rab5/Rab22/Rab24 located within the central and C-terminal regions, respectively (24, 25).Rab-effector recognition is considered a key factor with respect to the functional specificity of trafficking events and family-wide analyses indicate that the binding domains in effector proteins have evolved the capacity for highly selective recognition of small subsets of ...

Summary Widespread utilization of small GTPases as major regulatory hubs in many different biological systems derives from a conserved conformational switch mechanism that facilitates cycling between GTP-bound active and GDP-bound inactive states under control of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), which accelerate slow intrinsic rates of activation by nucleotide exchange and deactivation by GTP hydrolysis, respectively. Here we review developments leading to current understanding of intrinsic and GAP catalyzed GTP hydrolytic reactions in small GTPases from structural, molecular and chemical mechanistic perspectives. Despite the apparent simplicity of the GTPase cycle, the structural bases underlying the hallmark hydrolytic reaction and catalytic acceleration by GAPs are considerably more diverse than originally anticipated. Even the most fundamental aspects of the reaction mechanism have been challenging to decipher. Through a combination of experimental and in silico approaches, the outlines of a consensus view have begun to emerge for the best studied paradigms. Nevertheless, recent observations indicate that there is still much to be learned.

In all eukaryotic organisms, Rab GTPases function as critical regulators of membrane traffic, organelle biogenesis and maturation, and related cellular processes. The numerous Rab proteins have distinctive yet overlapping sub-cellular distributions throughout the endomembrane system. Intensive investigation has clarified the underlying molecular and structural mechanisms for several ubiquitous Rab proteins that control membrane traffic between tubular-vesicular organelles in the exocytic, endocytic and recycling pathways. In this review, we focus on structural insights that inform our current understanding of the organization of the Rab family as well as the mechanisms for membrane targeting and activation, interaction with effectors, deactivation, and specificity determination.

SUMMARY Recruitment of the Legionella pneumophila effector DrrA to the Legionella-containing vacuole, where it activates and AMPylates Rab1, is mediated by a P4M domain that binds phosphatidylinositol 4-phosphate [PI(4)P] with high affinity and specificity. Despite the importance of PI(4)P in Golgi trafficking and its manipulation by pathogens, the structural bases for PI(4)P-dependent membrane recruitment remain poorly defined. Here, we determined the crystal structure of a DrrA fragment including the P4M domain in complex with dibutyl PI(4)P and investigated the determinants of phosphoinositide recognition and membrane targeting. Headgroup recognition involves an elaborate network of direct and water-mediated interactions with basic and polar residues in the context of a deep, constrictive binding pocket. An adjacent hydrophobic helical element packs against the acyl chains and inserts robustly into PI(4)P-containing monolayers. The structural, biochemical, and biophysical data reported here support a detailed structural mechanism for PI(4)P-dependent membrane targeting by DrrA.

Eukaryotic genomes encode a zinc finger protein (ZPR1) with tandem ZPR1 domains. In response to growth stimuli, ZPR1 assembles into complexes with eukaryotic translation elongation factor 1A (eEF1A) and the survival motor neurons protein. To gain insight into the structural mechanisms underlying the essential function of ZPR1 in diverse organisms, we determined the crystal structure of a ZPR1 domain tandem and characterized the interaction with eEF1A. The ZPR1 domain consists of an elongation initiation factor 2-like zinc finger and a double-stranded ␤ helix with a helical hairpin insertion. ZPR1 binds preferentially to GDP-bound eEF1A but does not directly influence the kinetics of nucleotide exchange or GTP hydrolysis. However, ZPR1 efficiently displaces the exchange factor eEF1B␣ from preformed nucleotide-free complexes, suggesting that it may function as a negative regulator of eEF1A activation. Structure-based mutational and complementation analyses reveal a conserved binding epitope for eEF1A that is required for normal cell growth, proliferation, and cell cycle progression. Structural differences between the ZPR1 domains contribute to the observed functional divergence and provide evidence for distinct modalities of interaction with eEF1A and survival motor neuron complexes.growth factor receptor ͉ structure ͉ neurodegeneration ͉ spinal muscular atrophy ͉ cell cycle