GTPases and phosphoinositides Regulation of intracellular membrane trafficking The trafficking of lipids, integral membrane proteins and soluble cargo between membrane delimited organelles is regulated by GTPases of the Rab, Arf, Arl and Sar families as well as mono- and polyphosphorylated derivatives of phosphatidyl inositol (PIPs). 
The Rab GTPase Cycle As evolutionarily conserved molecular switches, Rab GTPases cycle between inactive (GDP-bound) and active (GTP-bound) states. In the active state, Rab GTPases interact with structurally and functionally diverse effectors including cargo sorting complexes on donor membranes, motor proteins involved in vesicular transport and tethering complexes that regulate vesicle fusion with acceptor membranes. Rab GTPases are activated by guanine nucleotide exchange factors (GEFs) and deactivated by GTPase activating proteins (GAPs), which accelerate the slow intrinsic rates of nucleotide exchange and GTP hydrolysis. Dual prenylation of C-terminal cysteine motifs allows Rab GTPases to partition with membranes. Transfer between membranes is facilitated by GDP dissociation inhibitor (GDI) and GDI displacement factors (GDFs). Targeting of Rab GTPases to specific organelles also depends on GEFs, effectors and GAPs. 
Membrane targeting mechanisms Lipid binding domains including those that recognize phosphoinositides utilize ligand-specific and/or non-specific mechanisms for partitioning with lipid bilayers. 
For additional information, see: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=14679290&query_hl=1&itool=pubmed_docsum http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16807090&query_hl=1&itool=pubmed_docsum Rab family-wide interaction analyses Quantitative profiling of interactions with the Rab GTPase family Quantitative high throughput microplate assays are used to profile interactions of GEFs, GAPs and effectors with Rab GTPases. Applications include determination of family-wide specificity profiles, identification of novel interaction partners and mutational analyses of specificity determinants. 
Activation of Rab GTPases by Vps9 domain GEFs Rab specificity profile for the Rabex-5 catalytic core Rab5 is an essential regulator of endosomal trafficking and endosome biogenesis. Rabex-5 is a Rab5 GEF with a Vps9 domain homologous to the yeast Vps9 protein implicated in vacuolar protein sorting. A profile of the Rab specificity of the catalytic core of Rabex-5 revealed equivalently high exchange activity for Rabs 5 and 21, weak activity for Rab22 and no detectable activity for 29 other Rab GTPases. Rabs 5, 21 and 22 comprise a small phylogenetic subfamily of endosomal Rab GTPases. 
For additional information, see: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15339665&query_hl=1&itool=pubmed_docsum Rabex-5 structure and mutational analyses of recognition determinants The crystal structure of the Rabex-5 catalytic core revealed a tandem architecture consisting of a Vps9 domain stabilized by a helical bundle. Conserved exchange determinants map to a common surface of the Vps9 domain, which recognizes invariant aromatic residues in the switch regions of Rab GTPases and selects for the Rab5 subfamily by requiring a small nonacidic residue preceding a critical phenylalanine in the switch I region. 
For additional information, see: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15339665&query_hl=1&itool=pubmed_docsum Structural basis for Rab GTPase activation by Vps9 domain GEFs The crystal structure of the RABEX-5 helical bundle-Vps9 tandem in complex with nucleotide free RAB21 (a key intermediate in the exchange reaction pathway) revealed how the VPS9 domain recognizes Rab5 subfamily GTPases (Rabs 5, 21 and 22), accelerates GDP release by destabilizing the magnesium binding site and subsequently stabilizes the high energy nucleotide free intermediate via an aspartic acid finger that simultaneously engages the P-loop lysine and switch II backbone. 
For additional information, see: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17450153&query_hl=1&itool=pubmed_docsum Rab-effector recognition Structural genomic survey of the Rab family To understand how structural similarity and diversity in the active conformation of Rab GTPases contributes to effector recognition, we conducted a structural genomic survey of the mammalian Rab GTPase family. The results revealed non-phylogenetic similarity and variability in the active conformations of Rab GTPases. 
For additional information, see: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16034420&query_hl=1&itool=pubmed_docsum Rab specificity profile of the multivalent effector Rabensoyn-5 Surface plasmon resonance (SPR) was used to profile the interaction of the central and C-terminal Rab binding domains (RBDs) of the multivalent endosomal effector Rabenosyn-5 with the active form of 33 Rab GTPases. Despite similar tertiary structures, the central and C-terminal RBDs recognize distinct subsets of Rab GTPases (Rabs 4 and 14 vs. Rabs 5, 22 and 24). 
For additional information, see: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16034420&query_hl=1&itool=pubmed_docsum Structural basis for Rab GTPase recognition by Rabenosyn-5 A truncation analysis of the multivalent Rab GTPase and PI3P effector Rabenosyn-5 mapped the central and C-terminal Rab binding domains (RBDs) to homologous regions. Structures of the RBDs in complex with Rab4 and Rab22 revealed a common binding modality in which a structurally similar helical hairpin core in the RBDs engages a structurally similar active conformation of the switch and interswitch regions in the respective Rab GTPases. Mutational analyses further revealed that the differential specificity of the RBDs is due in part to a non-conserved N-terminal extension (NTE) in RDB1 and in part to compositional differences within the conserved helical hairpin cores. 
For additional information, see: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16034420&query_hl=1&itool=pubmed_docsum Conceptual model for Rab-effector recognition The model shown here is based on known structures of Rab GTPases alone and in complex with effectors and takes into account the results of mutational analyses. Structural variability influences the spatial disposition and exposure of the conserved residues, sub-dividing the Rab family into non-phylogenetic subsets that satisfy the structural requirements for effector recognition. Compositional diversity within the switch/interswitch regions (and in certain cases CDRs) further refines the specificity through enhanced affinity for Rab GTPases with compatible compositions (positive selection) and/or reduced affinity for Rab GTPases with incompatible compositions (negative selection). The family-wide nature of the recognition process is underscored by the conservation of positive determinants in interacting subsets and negative determinants in non-interacting Rab GTPases. This model is also applicable to interactions with regulatory/accessory factors. 
Structural basis for recruitment of FIP3 to recycling endosomes Rab11 regulates recycling of internalized plasma membrane receptors and is essential for completion of cytokinesis. A family of Rab11 interacting proteins (FIPs) that conserve a C-terminal Rab-binding domain (RBD) selectively recognize the active form of Rab11. Normal completion of cytokinesis requires a complex between Rab11 and FIP3. Shown here is the structure of a heterotetrameric complex between constitutively active (GTP-bound) Rab11 and a FIP3 construct that includes the RBD. Two Rab11 molecules bind to dyad symmetric sites at the C terminus of FIP3, which forms a non-canonical coiled-coiled dimer with a flared C terminus and hook region. The RBD overlaps with the coiled coil and extends through the C-terminal hook. Although FIP3 engages the switch and interswitch regions of Rab11, the mode of interaction differs from that of other Rab-effector complexes. In particular, the switch II region undergoes a large structural rearrangement that facilitates the interaction with FIP3. 
For additional information, see: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=17007872&query_hl=1&itool=pubmed_docsum De-activation of Rab GTPases by TBC domain GAPs Rab specificity profile of the Gyp1p TBC domain GAP TBC (Tre-2, Bub2 and Cdc16) domains are broadly conserved in eukaryotes and function as GAPs for Rab GTPases as well as GTPases that control cytokinesis. A profile of the specificity of the Gyp1p TBC domain revealed high GAP activity for Rab33 in addition to Rab1 (the mammalian homologue of the in vivo Gyp1p substrate Ypt1p). The identification of Rab33 as a Gyp1p substrate facilitated determination of the structure of a TBC domain-Rab GTPase complex. 
For additional information, see: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16855591&query_hl=1&itool=pubmed_docsum TBC domains accelerate GTP hydrolysis by a dual finger mechanism In the crystal structure of the Gyp1p TBC-domain-Rab33-aluminium fluoride complex, which approximates the transition-state intermediate for GTP hydrolysis, the TBC domain supplies two catalytic residues in trans, an arginine finger analogous to Ras/Rho family GAPs and a glutamine finger that substitutes for the glutamine in the DxxGQ motif of the GTPase. The glutamine from the Rab GTPase does not stabilize the transition state as expected but instead interacts with the TBC domain. Strong conservation of both catalytic fingers suggests that most TBC-domain GAPs will accelerate GTP hydrolysis by a similar dual-finger mechanism. These conclusions are supported by mutational and complementation analyses. 
For additional information, see: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=16855591&query_hl=1&itool=pubmed_docsum Phosphoinositide recognition and membrane targeting Structural basis for 3-phosphoinositide recognition by PH domains The pleckstrin homology (PH) domain of Grp1, a PI 3-kinase-activated exchange factor for Arf GTPases, selectively binds PIP3 with high affinity. The structure of the Grp1 PH domain bound to the head group of PIP3 (IP4) revealed a novel mode of phosphoinositide recognition involving a 20-residue beta hairpin insertion within the beta6/beta7 loop. The observed mode of recognition involving residues from a conserved basic motif as well as the variable loops surrounding the phosphoinositide binding site explains the high affinity and specificity of the Grp1 PH domain and the promiscuous 3-phosphoinositide binding typical of several PH domains that lack the hairpin insertion. 
For additional information, see: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10983985&query_hl=1&itool=pubmed_DocSum Structural determinants of phosphoinositide selectivity in splice variants of Grp1 family PH domains The PH domains of the homologous proteins Grp1, ARNO and Cytohesin-1 bind PIP3 with unusually high selectivity. Remarkably, splice variants that differ only by the insertion of a single glycine residue in the beta1/beta2 loop exhibit dual specificity for PIP2 and PIP3. Crystal structures for the dual specificity 'triglycine' variant of the ARNO PH domain in complex with the head groups of PIP2 (IP3) and PIP3 (IP4) revealed the structural basis underlying this dramatic selectivity switch. Loss of contacts with the beta1/beta2 loop with no significant change in head group orientation accounts for the significant decrease in PIP3 affinity observed for the dual specificity variants. Conversely, a small increase rather than decrease in affinity for PIP2 is explained by a novel 'rotated' binding mode, in which the glycine insertion alleviates unfavorable interactions with the beta1/beta2 loop. These and other observations supported by systematic mutational analyses suggested a general model for phosphoinositide recognition by PH domains that conserve a 'signature' basic motif. 
For additional information, see: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15359279&query_hl=1&itool=pubmed_docsum Structural basis for PI3P recognition and multivalent endosome targeting by FYVE domains The localization of the early endosomal marker and tethering factor EEA1 is mediated by a C-terminal region that includes a calmodulin binding motif, a Rab5 binding site and a FYVE domain that selectively binds PI3P. The crystal structure of the C-terminal region bound to the head group of PI3P (IP2) revealed an organized quaternary assembly consisting of a parallel coiled coil and a dyad-symmetric FYVE domain homodimer. Structural and biochemical observations support a multivalent mechanism for endosomal targeting in which domain organization, dimerization and quaternary structure amplify the weak affinity and modest specificity of head group interactions with conserved residues. 
For additional information, see: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=11741531&query_hl=1&itool=pubmed_docsum |