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1.  Putative GTP-binding protein, Gtr1, associated with the function of the Pho84 inorganic phosphate transporter in Saccharomyces cerevisiae. 
Molecular and Cellular Biology  1992;12(7):2958-2966.
We have found an open reading frame which is 1.1 kb upstream of PHO84 (which encodes a Pi transporter) and is transcribed from the opposite strand. In Saccharomyces cerevisiae, this gene is distal to the TUB3 locus on the left arm of chromosome XIII and is named GTR1. GTR1 encodes a protein consisting of 310 amino acid residues containing, in its N-terminal region, the characteristic tripartite consensus elements for binding GTP conserved in GTP-binding proteins, except for histidine in place of a widely conserved aspargine residue in element III. Disruption of the GTR1 gene resulted in slow growth at 30 degrees C and no growth at 15 degrees C; other phenotypes resembled those of pho84 mutants and included constitutive synthesis of repressible acid phosphatase, reduced Pi transport activity, and resistance to arsenate. The latter phenotypes were shown to be due to a defect in Pi uptake, and the Gtr1 protein was found to be functionally associated with the Pho84 Pi transporter. Recombination between chromosome V (at the URA3 locus) and chromosome XIII (in the GTR1-PHO84-TUB3 region) by using a plasmid-encoded site-specific recombination system indicated that the order of these genes was telomere-TUB3-PHO84-GTR1-CENXIII.
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PMCID: PMC364509  PMID: 1620108
2.  SEACing the GAP that nEGOCiates TORC1 activation 
Cell Cycle  2013;12(18):2948-2952.
The target of rapamycin complex 1 (TORC1) regulates eukaryotic cell growth in response to a variety of input signals. In S. cerevisiae, amino acids activate TORC1 through the Rag guanosine triphosphatase (GTPase) heterodimer composed of Gtr1 and Gtr2 found together with Ego1 and Ego3 in the EGO complex (EGOC). The GTPase activity of Gtr1 is regulated by the SEA complex (SEAC). Specifically, SEACIT, a SEAC subcomplex containing Iml1, Npr2, and Npr3 functions as a GTPase activator (GAP) for Gtr1 to decrease the activity of TORC1 and, consequently, growth, after amino acid deprivation. Here, we present genetic epistasis data, which show that SEACAT, the other SEAC subcomplex, containing Seh1, Sea2–4, and Sec13, antagonizes the GAP function of SEACIT. Orthologs of EGOC (Ragulator), SEACIT (GATOR1), and SEACAT (GATOR2) are present in higher eukaryotes, highlighting the remarkable conservation, from yeast to man, of Rag GTPase and TORC1 regulation.
doi:10.4161/cc.26000
PMCID: PMC3875668  PMID: 23974112
Rag GTPases; TOR complex 1; EGO complex; SEA complex; amino acid signaling; Iml1-Npr2-Npr3 Rag GTPase GAP complex; GATOR1; GATOR2; Seh1; Sec13
3.  Topological characterisation and identification of critical domains within glucosyltransferase IV (GtrIV) of Shigella flexneri 
BMC Biochemistry  2011;12:67.
Background
The three bacteriophage genes gtrA, gtrB and gtr(type) are responsible for O-antigen glucosylation in Shigella flexneri. Both gtrA and gtrB have been demonstrated to be highly conserved and interchangeable among serotypes while gtr(type) was found to be specific to each serotype, leading to the hypothesis that the Gtr(type) proteins are responsible for attaching glucosyl groups to the O-antigen in a site- and serotype- specific manner. Based on the confirmed topologies of GtrI, GtrII and GtrV, such interaction and attachment of the glucosyl groups to the O-antigen has been postulated to occur in the periplasm.
Results
In this study, the topology of GtrIV was experimentally determined by creating different fusions between GtrIV and a dual-reporter protein, PhoA/LacZ. This study shows that GtrIV consists of 8 transmembrane helices, 2 large periplasmic loops, 2 small cytoplasmic N- and C- terminal ends and a re-entrant loop that occurs between transmembrane helices III and IV. Though this topology differs from that of GtrI, GtrII, GtrV and GtrX, it is very similar to that of GtrIc. Furthermore, both the N-terminal periplasmic and the C-terminal periplasmic loops are important for GtrIV function as shown via a series of loop deletion experiments and the creation of chimeric proteins between GtrIV and its closest structural homologue, GtrIc.
Conclusion
The current study provides the basis for elucidating the structure and mechanism of action of this important O-antigen modifying glucosyltransferase.
doi:10.1186/1471-2091-12-67
PMCID: PMC3259042  PMID: 22188643
4.  Glutamyl-tRNA Reductase of Chlorobium vibrioforme Is a Dissociable Homodimer That Contains One Tightly Bound Heme per Subunit 
Journal of Bacteriology  2005;187(13):4444-4450.
δ-Aminolevulinic acid, the biosynthetic precursor of tetrapyrroles, is synthesized from glutamate via the tRNA-dependent five-carbon pathway in the green sulfur bacterium Chlorobium vibrioforme. The enzyme glutamyl-tRNA reductase (GTR), encoded by the hemA gene, catalyzes the first committed step in this pathway, which is the reduction of tRNA-bound glutamate to produce glutamate 1-semialdehyde. To characterize the GTR protein, the hemA gene from C. vibrioforme was cloned into expression plasmids that added an N-terminal His6 tag to the expressed protein. The His-tagged GTR protein was purified using Ni affinity column chromatography. GTR was observable as a 49-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. The native molecular mass, as determined by gel filtration chromatography, appeared to be approximately 40 kDa, indicating that native GTR is a monomer. However, when the protein was mixed with 5% (vol/vol) glycerol, the product had an apparent molecular mass of 95 kDa, indicating that the protein is a dimer under these conditions. Purified His6-GTR was catalytically active in vitro when it was incubated with Escherichia coli glutamyl-tRNAGlu and purified recombinant Chlamydomonas reinhardtii glutamate-1-semialdehyde aminotransferase. The expressed GTR contained 1 mol of tightly bound heme per mol of pep tide subunit. The heme remained bound to the protein throughout purification and was not removed by anion- or cation-exchange column chromatography. However, the bound heme was released during SDS-PAGE if the protein was denatured in the presence of β-mercaptoethanol. Added heme did not inhibit the activity of purified expressed GTR in vitro. However, when the GTR was expressed in the presence of 3-amino-2,3- dihydrobenzoic acid (gabaculine), an inhibitor of heme synthesis, the purified GTR had 60 to 70% less bound heme than control GTR, and it was inhibited by hemin in vitro.
doi:10.1128/JB.187.13.4444-4450.2005
PMCID: PMC1151790  PMID: 15968053
5.  A P-loop Mutation in Gα Subunits Prevents Transition to the Active State: Implications for G-protein Signaling in Fungal Pathogenesis 
PLoS Pathogens  2012;8(2):e1002553.
Heterotrimeric G-proteins are molecular switches integral to a panoply of different physiological responses that many organisms make to environmental cues. The switch from inactive to active Gαβγ heterotrimer relies on nucleotide cycling by the Gα subunit: exchange of GTP for GDP activates Gα, whereas its intrinsic enzymatic activity catalyzes GTP hydrolysis to GDP and inorganic phosphate, thereby reverting Gα to its inactive state. In several genetic studies of filamentous fungi, such as the rice blast fungus Magnaporthe oryzae, a G42R mutation in the phosphate-binding loop of Gα subunits is assumed to be GTPase-deficient and thus constitutively active. Here, we demonstrate that Gα(G42R) mutants are not GTPase deficient, but rather incapable of achieving the activated conformation. Two crystal structure models suggest that Arg-42 prevents a typical switch region conformational change upon Gαi1(G42R) binding to GDP·AlF4− or GTP, but rotameric flexibility at this locus allows for unperturbed GTP hydrolysis. Gα(G42R) mutants do not engage the active state-selective peptide KB-1753 nor RGS domains with high affinity, but instead favor interaction with Gβγ and GoLoco motifs in any nucleotide state. The corresponding Gαq(G48R) mutant is not constitutively active in cells and responds poorly to aluminum tetrafluoride activation. Comparative analyses of M. oryzae strains harboring either G42R or GTPase-deficient Q/L mutations in the Gα subunits MagA or MagB illustrate functional differences in environmental cue processing and intracellular signaling outcomes between these two Gα mutants, thus demonstrating the in vivo functional divergence of G42R and activating G-protein mutants.
Author Summary
Heterotrimeric G-proteins function as molecular switches to convey cellular signals. When a G-protein coupled receptor encounters its ligand at the cellular membrane, it catalyzes guanine nucleotide exchange on the Gα subunit, resulting in a shift from an inactive to an active conformation. G-protein signaling pathways are conserved from mammals to plants and fungi, including the rice blast fungus Magnaporthe oryzae. A mutation in the Gα subunit (G42R), previously thought to eliminate its GTPase activity, leading to constitutive activation, has been utilized to investigate roles of heterotrimeric G-protein signaling pathways in multiple species of filamentous fungi. Here, we demonstrate through structural, biochemical, and cellular approaches that G42R mutants are neither GTPase deficient nor constitutively active, but rather are unable to transition to the activated conformation. A direct comparison of M. oryzae fungal strains harboring either G42R or truly constitutively activating mutations in two Gα subunits, MagA and MagB, revealed markedly different phenotypes. Our results suggest that activation of MagB is critical for pathogenic development of M. oryzae in response to hydrophobic surfaces, such as plant leaves. Furthermore, the lack of constitutive activity by Gα(G42R) mutants prompts a re-evaluation of its use in previous genetic experiments in multiple fungal species.
doi:10.1371/journal.ppat.1002553
PMCID: PMC3285607  PMID: 22383884
6.  Diversity and plasticity in Rab GTPase nucleotide release mechanism has consequences for Rab activation and inactivation 
eLife  2014;3:e01623.
Ras superfamily GTPase activation and inactivation occur by canonical nucleotide exchange and GTP hydrolysis mechanisms. Despite conservation of active-site residues, the Ras-related Rab GTPase activation pathway differs from Ras and between different Rabs. Analysis of DENND1-Rab35, Rabex-Rab5, TRAPP-Rab1 and DrrA-Rab1 suggests Rabs have the potential for activation by distinct GDP-release pathways. Conserved active-site residues in the Rab switch II region stabilising the nucleotide-free form differentiate these pathways. For DENND1-Rab35 and DrrA-Rab1 the Rab active-site glutamine, often mutated to create constitutively active forms, is involved in GEF mediated GDP-release. By contrast, in Rab5 the switch II aspartate is required for Rabex mediated GDP-release. Furthermore, Rab1 switch II glutamine mutants refractory to activation by DrrA can be activated by TRAPP, showing that a single Rab can be activated by more than one mechanistically distinct GDP-release pathway. These findings highlight plasticity in the activation mechanisms of closely related Rab GTPases.
DOI: http://dx.doi.org/10.7554/eLife.01623.001
eLife digest
The 70 or so members of the Rab subfamily of proteins perform a wide range of important tasks inside cells. A Rab protein is always bound to another molecule, which determines whether it is inactive or active. Binding to a molecule called GDP makes the Rab protein inactive, while binding to GTP makes it active. Proteins called guanine nucleotide exchange factors, or GEFs for short, activate the Rab protein by promoting the release of GDP and the binding of GTP. Other proteins—known as GAPs—lead to the inactivation of the Rab protein. Together these proteins form a molecular switch that can be turned on and off.
The Rab subfamily of proteins is part of the large Ras superfamily, and all members of this superfamily are activated and inactivated in a similar way, with the binding and unbinding of GDP and GTP taking place at a structure called the G-domain. The fact that the detailed structure of this domain (at the level of individual amino acids) has been conserved over evolution is often taken as an indication that its mechanism has also been conserved. Langemeyer et al. have now tested this assumption with four different types of GEFs—three from humans and one from the bacteria that cause Listeria—and found that the story is more complicated than expected.
The experiments showed that different amino acids in the active site of the Rab protein are involved when the GEFs mediate the release of the GDP during the activation process. For example, the amino acid glutamine is involved when the Listeria GEF and one of the human GEFs activate the protein, whereas a different amino acid—aspartate—is involved when one of the other human GEFs is responsible for the activation. Using this information, Langemeyer et al. create a human Rab protein that cannot be activated by the GEF from the bacteria that cause Listeria, but can still be activated by its normal human GEF.
By showing that different Rab proteins are activated by different mechanisms, and that a single Rab protein can be activated by more than one mechanism, the work of Langemeyer et al. clearly illustrates the on-going ability of evolution to surprise researchers.
DOI: http://dx.doi.org/10.7554/eLife.01623.002
doi:10.7554/eLife.01623
PMCID: PMC3919270  PMID: 24520163
membrane traffic; Rab GTPase; nucleotide exchange factor; human
7.  The Folliculin tumor suppressor is a GAP for RagC/D GTPases that signal amino acid levels to mTORC1 
Molecular cell  2013;52(4):10.1016/j.molcel.2013.09.016.
SUMMARY
The mTORC1 kinase is a master growth regulator that senses numerous environmental cues, including amino acids. The Rag GTPases interact with mTORC1 and signal amino acid sufficiency by promoting the translocation of mTORC1 to the lysosomal surface, its site of activation. The Rags are unusual GTPases in that they function as obligate heterodimers, which consist of RagA or B bound to RagC or D. While the loading of RagA/B with GTP initiates amino acid signaling to mTORC1, the role of RagC/D is unknown. Here, we show that RagC/D is a key regulator of the interaction of mTORC1 with the Rag heterodimer and that, unexpectedly, RagC/D must be GDP-bound for the interaction to occur. We identify FLCN and its binding partners, FNIP1/2, as Rag-interacting proteins with GAP activity for RagC/D, but not RagA/B. Thus, we reveal a role for RagC/D in mTORC1 activation and a molecular function for the FLCN tumor suppressor.
doi:10.1016/j.molcel.2013.09.016
PMCID: PMC3867817  PMID: 24095279
8.  Molecular Modeling Study for Interaction between Bacillus subtilis Obg and Nucleotides 
PLoS ONE  2010;5(9):e12597.
The bacterial Obg proteins (Spo0B-associated GTP-binding protein) belong to the subfamily of P-loop GTPase proteins that contain two equally and highly conserved domains, a C-terminal GTP binding domain and an N-terminal glycine-rich domain which is referred as the “Obg fold” and now it is considered as one of the new targets for antibacterial drug. When the Obg protein is associated with GTP, it becomes activated, because conformation of Obg fold changes due to the structural changes of GTPase switch elements in GTP binding site. In order to investigate the effects and structural changes in GTP bound to Obg and GTPase switch elements for activation, four different molecular dynamics (MD) simulations were performed with/without the three different nucleotides (GTP, GDP, and GDP + Pi) using the Bacillus subtilis Obg (BsObg) structure. The protein structures generated from the four different systems were compared using their representative structures. The pattern of Cα-Cα distance plot and angle between the two Obg fold domains of simulated apo form and each system (GTP, GDP, and GDP+Pi) were significantly different in the GTP-bound system from the others. The switch 2 element was significantly changed in GTP-bound system. Also root-mean-square fluctuation (RMSF) analysis revealed that the flexibility of the switch 2 element region was much higher than the others. This was caused by the characteristic binding mode of the nucleotides. When GTP was bound to Obg, its γ-phosphate oxygen was found to interact with the key residue (D212) of the switch 2 element, on the contrary there was no such interaction found in other systems. Based on the results, we were able to predict the possible binding conformation of the activated form of Obg with L13, which is essential for the assembly with ribosome.
doi:10.1371/journal.pone.0012597
PMCID: PMC2935376  PMID: 20830302
9.  Nucleotide Binding Switches the Information Flow in Ras GTPases 
PLoS Computational Biology  2011;7(3):e1001098.
The Ras superfamily comprises many guanine nucleotide-binding proteins (G proteins) that are essential to intracellular signal transduction. The guanine nucleotide-dependent intrinsic flexibility patterns of five G proteins were investigated in atomic detail through Molecular Dynamics simulations of the GDP- and GTP-bound states (SGDP and SGTP, respectively). For all the considered systems, the intrinsic flexibility of SGDP was higher than that of SGTP, suggesting that Guanine Exchange Factor (GEF) recognition and nucleotide switch require higher amplitude motions than effector recognition or GTP hydrolysis. Functional mode, dynamic domain, and interaction energy correlation analyses highlighted significant differences in the dynamics of small G proteins and Gα proteins, especially in the inactive state. Indeed, SGDP of Gαt, is characterized by a more extensive energy coupling between nucleotide binding site and distal regions involved in GEF recognition compared to small G proteins, which attenuates in the active state. Moreover, mechanically distinct domains implicated in nucleotide switch could be detected in the presence of GDP but not in the presence of GTP. Finally, in small G proteins, functional modes are more detectable in the inactive state than in the active one and involve changes in solvent exposure of two highly conserved amino acids in switches I and II involved in GEF recognition. The average solvent exposure of these amino acids correlates in turn with the rate of GDP release, suggesting for them either direct or indirect roles in the process of nucleotide switch. Collectively, nucleotide binding changes the information flow through the conserved Ras-like domain, where GDP enhances the flexibility of mechanically distinct portions involved in nucleotide switch, and favors long distance allosteric communication (in Gα proteins), compared to GTP.
Author Summary
The Ras superfamily comprises many guanine nucleotide-binding proteins (G proteins) that are essential to intracellular signal transduction. These proteins act biologically as molecular switches cycling between ON and OFF states, thereby controlling a variety of processes ranging from cell growth and differentiation to vesicular and nuclear transport. In spite of the extremely high biological and medical relevance of the Ras GTPase superfamily, a comprehensive structural/dynamic view of the trans-family and family-specific functioning mechanisms is still lacking. In this study, we gained insights into the functional dynamics of Ras GTPases by deciphering the dynamic information encrypted in the topology of these proteins depending on the nucleotide-bound state, i.e. GDP- or GTP-bound (SGDP and SGTP, respectively). Collectively, nucleotide binding changes the information flow through the conserved Ras-like domain, where GDP enhances the flexibility of mechanically distinct portions involved in nucleotide switch, and favors long distance allosteric communication (in Gα proteins), compared to GTP. Functional dynamics is instrumental in GDP switch, which for the members of the Gα family, different from small G proteins, requires allosteric communication between nucleotide and Guanine Exchange Factor binding sites.
doi:10.1371/journal.pcbi.1001098
PMCID: PMC3048383  PMID: 21390270
10.  Amino Acids Activate Mammalian Target of Rapamycin (mTOR) Complex 1 without Changing Rag GTPase Guanyl Nucleotide Charging* 
The Journal of Biological Chemistry  2013;289(5):2658-2674.
Background: Signaling by mTOR complex 1 requires its amino acid-stimulated binding to Rag GTPase heterodimers.
Results: mTOR complex 1-Rag binding in vitro is independent of Rag guanyl nucleotide charging, and withdrawal of amino acids from cells does not alter Rag GTP charging.
Conclusion: Amino acids promote Rag binding to mTOR complex 1 without changing Rag GTP charging.
Significance: Amino acids promote Rag-mTORC1 binding by undefined mechanisms.
Activation of mammalian target of rapamycin complex 1 (mTORC1) by amino acids is mediated in part by the Rag GTPases, which bind the raptor subunit of mTORC1 in an amino acid-stimulated manner and promote mTORC1 interaction with Rheb-GTP, the immediate activator. Here we examine whether the ability of amino acids to regulate mTORC1 binding to Rag and mTORC1 activation is due to the regulation of Rag guanyl nucleotide charging. Rag heterodimers in vitro exhibit a very rapid, spontaneous exchange of guanyl nucleotides and an inability to hydrolyze GTP. Mutation of the Rag P-loop corresponding to RasSer-17 abolishes guanyl nucleotide binding. Such a mutation in RagA or RagB inhibits, whereas in RagC or RagD it enhances, Rag heterodimer binding to mTORC1. The binding of wild-type and mutant Rag heterodimers to mTORC1 in vitro parallels that seen with transient expression, but binding to mTORC1 in vitro is entirely independent of Rag guanyl nucleotide charging. HeLa cells stably overexpressing wild-type or P-loop mutant RagC exhibit unaltered amino acid regulation of mTORC1. Despite amino acid-independent raptor binding to Rag, mTORC1 is inhibited by amino acid withdrawal as in parental cells. Rag heterodimers extracted from 32P-labeled whole cells, or just from the pool associated with the lysosomal membrane, exhibit constitutive [32P]GTP charging that is unaltered by amino acid withdrawal. Thus, amino acids promote mTORC1 activation without altering Rag GTP charging. Raptor binding to Rag, although necessary, is not sufficient for mTORC1 activation. Additional amino acid-dependent steps couple Rag-mTORC1 to Rheb-GTP.
doi:10.1074/jbc.M113.528505
PMCID: PMC3908400  PMID: 24337580
Cell Signaling; GTPase; Insulin; Lysosomes; mTOR Complex (mTORC); Rag GTPase; Amino Acids; Raptor
11.  Disease mutations in Rab7 result in unregulated nucleotide exchange and inappropriate activation 
Human Molecular Genetics  2009;19(6):1033-1047.
Rab GTPases are molecular switches that orchestrate vesicular trafficking, maturation and fusion by cycling between an active, GTP-bound form, and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7 cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth type 2B through an unknown mechanism. We present the 2.8 Å crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. Through extensive biochemical analysis, we demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that the increased activation of mutant Rab7 is counterbalanced by unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease mutations are able to rescue the membrane cycling of a GTPase-deficient mutant. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease not by a gain of novel toxic function, but by misregulation of native Rab7 activity.
doi:10.1093/hmg/ddp567
PMCID: PMC2830827  PMID: 20028791
12.  Distinct Subclasses of Small GTPases Interact with Guanine Nucleotide Exchange Factors in a Similar Manner 
Molecular and Cellular Biology  1998;18(12):7444-7454.
The Ras-related GTPases are small, 20- to 25-kDa proteins which cycle between an inactive GDP-bound form and an active GTP-bound state. The Ras superfamily includes the Ras, Rho, Ran, Arf, and Rab/YPT1 families, each of which controls distinct cellular functions. The crystal structures of Ras, Rac, Arf, and Ran reveal a nearly superimposible structure surrounding the GTP-binding pocket, and it is generally presumed that the Rab/YPT1 family shares this core structure. The Ras, Rac, Ran, Arf, and Rab/YPT1 families are activated by interaction with family-specific guanine nucleotide exchange factors (GEFs). The structural determinants of GTPases required for interaction with family-specific GEFs have begun to emerge. We sought to determine the sites on YPT1 which interact with GEFs. We found that mutations of YPT1 at position 42, 43, or 49 (effector loop; switch I), position 69, 71, 73, or 75 (switch II), and position 107, 109, or 115 (alpha-helix 3–loop 7 [α3-L7]) are intragenic suppressors of dominant interfering YPT1 mutant N22 (YPT1-N22), suggesting these mutations prevent YPT1-N22 from binding to and sequestering an endogenous GEF. Mutations at these positions prevent interaction with the DSS4 GEF in vitro. Mutations in the switch II and α3-L7 regions do not prevent downstream signaling in yeast when combined with a GTPase-defective (activating) mutation. Together, these results show that the YPT1 GTPase interacts with GEFs in a manner reminiscent of that for Ras and Arf in that these GTPases use divergent sequences corresponding to the switch I and II regions and α3-L7 of Ras to interact with family-specific GEFs. This finding suggests that GTPases of the Ras superfamily each may share common features of GEF-mediated guanine nucleotide exchange even though the GEFs for each of the Ras subfamilies appear evolutionarily unrelated.
PMCID: PMC109325  PMID: 9819430
13.  Signal recognition particle (SRP) and SRP receptor: A new paradigm for multi-state regulatory GTPases 
Biochemistry  2009;48(29):6696-6704.
The GTP-binding proteins or GTPases comprise a superfamily of proteins that provide molecular switches in numerous cellular processes. The ‘GTPase switch’ paradigm, in which a GTPase acts as a bimodal switch that is turned ‘on’ and ‘off’ by external regulatory factors, has been used to interpret the regulatory mechanism of many GTPases for over two decades. Nevertheless, recent work has unveiled an emerging class of ‘multi-state’ regulatory GTPases that do not adhere to this classical paradigm. Instead of relying on external nucleotide exchange factors or GTPase activating proteins to switch between the ‘on’ and ‘off’ states, these GTPases have the intrinsic ability to exchange nucleotides and to sense and respond to upstream and downstream factors. In contrast to the bimodal nature of the ‘GTPase switch’, these GTPases undergo multiple conformational rearrangements, allowing multiple regulatory points to be built into a complex biological process to ensure the efficiency and fidelity of the pathway. We suggest that these multi-state regulatory GTPases are uniquely suited to provide spatial and temporal control over complex cellular pathways that require multiple molecular events to occur in a highly coordinated fashion.
doi:10.1021/bi9006989
PMCID: PMC2883566  PMID: 19469550
14.  Ras-related GTPases and the cytoskeleton. 
Molecular Biology of the Cell  1992;3(5):475-479.
Incorporation of the available data on rac in neutrophils, CDC42 in yeast, and rho in fibroblasts suggests a general model for the function of rho-like GTPase (Figure 1). Conversion of an inactive cytoplasmic rho-related p21GDP/GDI complex to active p21. GTP occurs by inhibition of GAP and/or stimulation of exchange factors in response to cell signals. p21.GTP is then able to interact with its target at the plasma membrane. This could result in a conformational change in the target, enabling it to bind cytosolic protein(s). Alternatively, p21.GTP could be actively involved in transporting cytosolic protein(s) to the target. A GAP protein, perhaps intrinsic to the complex, would stimulate GTP hydrolysis allowing p21.GDP to dissociate. Solubilization of p21GDP by interaction with GDI would complete a cycle. What about the nature of the final complex? The rac-regulated NADPH oxidase complex in neutrophils is currently the best understood and most amenable to further biochemical analysis. Two plasma-membrane bound subunits encode the catalytic function necessary for producing superoxide, but the two cytosolic proteins, p47 and p67, are essential for activity. Why the complexity? Production of superoxide is tightly coordinated with phagocytosis, a membrane process driven by rearrangement of cortical actin. This is not unrelated to the membrane ruffling and macropinocytosis that we observe in fibroblasts microinjected with p21rac. It is tempting to speculate, therefore, that in neutrophils rac is involved not only in promoting the assembly of the NADPH oxidase but also in the coordinate reorganization of cortical actin leading to phagocytosis. For CDC42 controlled bud assembly in yeast, the components of the plasma-membrane complex are not so clear. By analogy with rac in neutrophils, it seems likely that CDC42 is involved in promoting the assembly of cytosolic components at the bud site on the plasma membrane. These putative cytosolic proteins have not yet been identified, but BEM1 and ABP1 are two possible candidates. The biochemical basis for the stimulation of adhesion plaques and actin stress fibers by p21rho in fibroblasts is also unclear. However, components of the adhesion plaque such as vinculin and talin are known to be cytosolic when not complexed with integrin receptors, and rho could be involved in regulating their assembly into the adhesion plaque. Several things are still difficult to incorporate into this model. First the target for CDC42, the bud site, although not yet structurally defined requires the activity of another small GTPase, BUD1. Similarly, in activated neutrophils, the NADPH oxidase is found in a complex with rap1, the mammalian homologue of BUD1 (BoKoch et al., 1989). It seems likely, therefore, that the target is not simply a plasma-membrane protein but may be a complex of proteins whose formation is under the control of the rap1/BUD1 GTPase. The other black box in this model is the actin connection: activation of bud assembly by CDC42 is followed by actin polymerization, activation of NADPH oxidase in neutrophils occurs concomitantly with phagocytosis, a cortical actin-dependent process, and p21rho in fibroblasts couples the formation of adhesion plaques to actin stress fibers. One possible link between the GTPase-driven assembly of a plasma-membrane complex and actin polymerization could involve the SH3 domain. Interestingly, both p47 and p67 and yeast ABP1 and BEM1 have SH3 domain. If rho-like GTPases recognize plasma-membrane targets already associated with cortical actin, then this could promote an interaction with a subset of SH3-containing proteins. The result of this would be a GTPase-regulated aggregation of a group of proteins at a single site in the plasma membrane. It is not too difficult to imagine biological processes where such a spatial integration of different biochemical activities would be essential: coupling the assembly of bud components to the formation of actin fibers in yeast; or the activation of NADPH oxidase to phagocytosis in neutrophils; or the assembly of adhesion plaques and the formation of actin stress fibers in fibroblasts are just three examples that have emerged so far. In conclusion, although rho-like GTPases clearly have distinct roles in different mammalian cell types and in yeast, their underlying mechanism of action appears to be strikingly similar. Whether this will remain so when there are some biochemical data to back up these initial observations, time will tell.
PMCID: PMC275601  PMID: 1611153
15.  An expanded Ragulator is a GEF for the Rag GTPases that signal amino acid levels to mTORC1 
Cell  2012;150(6):1196-1208.
The mTOR Complex 1 (mTORC1) pathway promotes cell growth in response to a diverse set of cues, including growth factors as well as energy and amino acid levels. Amino acids signal through the Rag GTPases to promote the translocation of mTORC1 to the lysosomal surface, its site of activation. The four mammalian Rag proteins form obligate heterodimers consisting of RagA or RagB bound to RagC or RagD. A key upstream component of the Rag GTPases is Ragulator, a trimeric complex that tethers them to the lysosome and also interacts with the v-ATPase, which is necessary for amino acid sensing by mTORC1. Amino acids stimulate the binding of GTP to RagB, a critical step in the sensing mechanism, but the factors that regulate Rag nucleotide loading are unknown. Here, we identify the proteins encoded by the HBXIP and C7orf59 genes as novel Ragulator components that are required for mTORC1 activation by amino acids. The pentameric Ragulator has nucleotide exchange activity towards RagA and RagB and interacts with the Rag heterodimers in an amino acid- and v-ATPase-dependent fashion. Thus, we provide mechanistic insight into how mTORC1 senses amino acids by revealing Ragulator to be a scaffold with guanine nucleotide exchange factor (GEF) activity for the Rag GTPases.
doi:10.1016/j.cell.2012.07.032
PMCID: PMC3517996  PMID: 22980980
16.  Intragenic Suppressor Mutations Restore GTPase and Translation Functions of a Eukaryotic Initiation Factor 5B Switch II Mutant▿  
Molecular and Cellular Biology  2006;27(5):1677-1685.
Structural studies of GTP-binding proteins identified the Switch I and Switch II elements as contacting the γ-phosphate of GTP and undergoing marked conformational changes upon GTP versus GDP binding. Movement of a universally conserved Gly at the N terminus of Switch II is thought to trigger the structural rearrangement of this element. Consistently, we found that mutation of this Gly in the Switch II element of the eukaryotic translation initiation factor 5B (eIF5B) from Saccharomyces cerevisiae impaired cell growth and the guanine nucleotide-binding, GTPase, and ribosomal subunit joining activities of eIF5B. In a screen for mutations that bypassed the critical requirement for this Switch II Gly in eIF5B, intragenic suppressors were identified in the Switch I element and at a residue in domain II of eIF5B that interacts with Switch II. The intragenic suppressors restored yeast cell growth and eIF5B nucleotide-binding, GTP hydrolysis, and subunit joining activities. We propose that the Switch II mutation distorts the geometry of the GTP-binding active site, impairing nucleotide binding and the eIF5B domain movements associated with GTP binding. Accordingly, the Switch I and domain II suppressor mutations induce Switch II to adopt a conformation favorable for nucleotide binding and hydrolysis and thereby reestablish coupling between GTP binding and eIF5B domain movements.
doi:10.1128/MCB.01258-06
PMCID: PMC1820465  PMID: 17189426
17.  Structural stabilization of GTP-binding domains in circularly permuted GTPases: Implications for RNA binding 
Nucleic Acids Research  2006;34(8):2196-2205.
GTP hydrolysis by GTPases requires crucial residues embedded in a conserved G-domain as sequence motifs G1–G5. However, in some of the recently identified GTPases, the motif order is circularly permuted. All possible circular permutations were identified after artificially permuting the classical GTPases and subjecting them to profile Hidden Markov Model searches. This revealed G4–G5–G1–G2–G3 as the only possible circular permutation that can exist in nature. It was also possible to recognize a structural rationale for the absence of other permutations, which either destabilize the invariant GTPase fold or disrupt regions that provide critical residues for GTP binding and hydrolysis, such as Switch-I and Switch-II. The circular permutation relocates Switch-II to the C-terminus and leaves it unfastened, thus affecting GTP binding and hydrolysis. Stabilizing this region would require the presence of an additional domain following Switch-II. Circularly permuted GTPases (cpGTPases) conform to such a requirement and always possess an ‘anchoring’ C-terminal domain. There are four sub-families of cpGTPases, of which three possess an additional domain N-terminal to the G-domain. The biochemical function of these domains, based on available experimental reports and domain recognition analysis carried out here, are suggestive of RNA binding. The features that dictate RNA binding are unique to each subfamily. It is possible that RNA-binding modulates GTP binding or vice versa. In addition, phylogenetic analysis indicates a closer evolutionary relationship between cpGTPases and a set of universally conserved bacterial GTPases that bind the ribosome. It appears that cpGTPases are RNA-binding proteins possessing a means to relate GTP binding to RNA binding.
doi:10.1093/nar/gkl178
PMCID: PMC1450330  PMID: 16648363
18.  Genomic analysis of bacteriophage ε34 of Salmonella enterica serovar Anatum (15+) 
BMC Microbiology  2008;8:227.
Background
The presence of prophages has been an important variable in genetic exchange and divergence in most bacteria. This study reports the determination of the genomic sequence of Salmonella phage ε34, a temperate bacteriophage that was important in the early study of prophages that modify their hosts' cell surface and is of a type (P22-like) that is common in Salmonella genomes.
Results
The sequence shows that ε34 is a mosaically related member of the P22 branch of the lambdoid phages. Its sequence is compared with the known P22-like phages and several related but previously unanalyzed prophage sequences in reported bacterial genome sequences.
Conclusion
These comparisons indicate that there has been little if any genetic exchange within the procapsid assembly gene cluster with P22-like E. coli/Shigella phages that are have orthologous but divergent genes in this region. Presumably this observation reflects the fact that virion assembly proteins interact intimately and divergent proteins can no longer interact. On the other hand, non-assembly genes in the "ant moron" appear to be in a state of rapid flux, and regulatory genes outside the assembly gene cluster have clearly enjoyed numerous and recent horizontal exchanges with phages outside the P22-like group. The present analysis also shows that ε34 harbors a gtrABC gene cluster which should encode the enzymatic machinery to chemically modify the host O antigen polysaccharide, thus explaining its ability to alter its host's serotype. A comprehensive comparative analysis of the known phage gtrABC gene clusters shows that they are highly mobile, having been exchanged even between phage types, and that most "bacterial" gtrABC genes lie in prophages that vary from being largely intact to highly degraded. Clearly, temperate phages are very major contributors to the O-antigen serotype of their Salmonella hosts.
doi:10.1186/1471-2180-8-227
PMCID: PMC2629481  PMID: 19091116
19.  Characterization of GTPase Activity of TrmE, a Member of a Novel GTPase Superfamily, from Thermotoga maritima 
Journal of Bacteriology  2000;182(24):7078-7082.
A gene encoding a putative GTP-binding protein, a TrmE homologue that is highly conserved in both prokaryotes and eukaryotes, was cloned from Thermotoga maritima, a hyperthermophilic bacterium. T. maritima TrmE was overexpressed in Escherichia coli and purified. TrmE has a GTPase activity but no ATPase activity. The GTPase activity can be competed with GTP, GDP, and dGTP but not with GMP, ATP, CTP, or UTP. Km and kcat at 70°C were 833 μM and 9.3 min−1, respectively. Our results indicate that TrmE is a GTP-binding protein with a very high intrinsic GTP hydrolysis rate. We also propose that TrmE homologues constitute a novel subfamily of the GTPase superfamily.
PMCID: PMC94838  PMID: 11092873
20.  GxcDD, a putative RacGEF, is involved in Dictyostelium development 
BMC Cell Biology  2007;8:23.
Background
Rho subfamily GTPases are implicated in a large number of actin-related processes. They shuttle from an inactive GDP-bound form to an active GTP-bound form. This reaction is catalysed by Guanine nucleotide exchange factor (GEFs). GTPase activating proteins (GAPs) help the GTPase return to the inactive GDP-bound form. The social amoeba Dictyostelium discoideum lacks a Rho or Cdc42 ortholog but has several Rac related GTPases. Compared to our understanding of the downstream effects of Racs our understanding of upstream mechanisms that activate Rac GTPases is relatively poor.
Results
We report on GxcDD (Guanine exchange factor for Rac GTPases), a Dictyostelium RacGEF. GxcDD is a 180-kDa multidomain protein containing a type 3 CH domain, two IQ motifs, three PH domains, a RhoGEF domain and an ArfGAP domain. Inactivation of the gene results in defective streaming during development under different conditions and a delay in developmental timing. The characterization of single domains revealed that the CH domain of GxcDD functions as a membrane association domain, the RhoGEF domain can physically interact with a subset of Rac GTPases, and the ArfGAP-PH tandem accumulates in cortical regions of the cell and on phagosomes. Our results also suggest that a conformational change may be required for activation of GxcDD, which would be important for its downstream signaling.
Conclusion
The data indicate that GxcDD is involved in proper streaming and development. We propose that GxcDD is not only a component of the Rac signaling pathway in Dictyostelium, but is also involved in integrating different signals. We provide evidence for a Calponin Homology domain acting as a membrane association domain. GxcDD can bind to several Rac GTPases, but its function as a nucleotide exchange factor needs to be studied further.
doi:10.1186/1471-2121-8-23
PMCID: PMC1914345  PMID: 17584488
21.  Kissing G Domains of MnmE Monitored by X-Ray Crystallography and Pulse Electron Paramagnetic Resonance Spectroscopy 
PLoS Biology  2009;7(10):e1000212.
The authors of this research article demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle, and show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein. These movements are of crucial importance for understanding the mechanistic principles of this GAD.
MnmE, which is involved in the modification of the wobble position of certain tRNAs, belongs to the expanding class of G proteins activated by nucleotide-dependent dimerization (GADs). Previous models suggested the protein to be a multidomain protein whose G domains contact each other in a nucleotide dependent manner. Here we employ a combined approach of X-ray crystallography and pulse electron paramagnetic resonance (EPR) spectroscopy to show that large domain movements are coupled to the G protein cycle of MnmE. The X-ray structures show MnmE to be a constitutive homodimer where the highly mobile G domains face each other in various orientations but are not in close contact as suggested by the GDP-AlFx structure of the isolated domains. Distance measurements by pulse double electron-electron resonance (DEER) spectroscopy show that the G domains adopt an open conformation in the nucleotide free/GDP-bound and an open/closed two-state equilibrium in the GTP-bound state, with maximal distance variations of 18 Å. With GDP and AlFx, which mimic the transition state of the phosphoryl transfer reaction, only the closed conformation is observed. Dimerization of the active sites with GDP-AlFx requires the presence of specific monovalent cations, thus reflecting the requirements for the GTPase reaction of MnmE. Our results directly demonstrate the nature of the conformational changes MnmE was previously suggested to undergo during its GTPase cycle. They show the nucleotide-dependent dynamic movements of the G domains around two swivel positions relative to the rest of the protein, and they are of crucial importance for understanding the mechanistic principles of this GAD.
Author Summary
MnmE is an evolutionary conserved G protein that is involved in modification of the wobble U position of certain tRNAs to suppress translational wobbling. Despite high homology between its G domain and the small G protein Ras, MnmE displays entirely different regulatory properties to that of many molecular switch-type G proteins of the Ras superfamily, as its GTPase is activated by nucleotide-dependent homodimerization across the nucleotide-binding site. Here we explore the unusual G domain cycle of the MnmE protein by combining X-ray crystallography with pulse electron paramagnetic resonance (EPR) spectroscopy, which enables distance determinations between spin markers introduced at specific sites within the G domain. We determined the structures of the full-length MnmE dimer in the diphosphate and triphosphate states, which represent distinct steps of the G domain cycle, and demonstrate that the G domain cycle of MnmE comprises large conformational changes and domain movements of up to 18 Å, in which the G domains of the dimeric protein traverse from a GDP-bound open state through an open/closed equilibrium in the triphosphate state to a closed conformation in the transition state, so as to assemble the catalytic machinery.
doi:10.1371/journal.pbio.1000212
PMCID: PMC2749940  PMID: 19806182
22.  A Chemical Genetic Screen for Modulators of Exocytic Transport Identifies Inhibitors of a Transport Mechanism Linked to GTR2 Function▿  
Eukaryotic Cell  2009;9(1):116-126.
Membrane and protein traffic to the cell surface is mediated by partially redundant pathways that are difficult to perturb in ways that yield a strong phenotype. Such robustness is expected in a fine-tuned process, regulated by environmental cues, that is required for controlled cell surface growth and cell proliferation. Synthetic genetic interaction screens are especially valuable for investigating complex processes involving partially redundant pathways or mechanisms. In a previous study, we used a triple-synthetic-lethal yeast mutant screen to identify a novel component of the late exocytic transport machinery, Avl9. In a chemical-genetic version of the successful mutant screen, we have now identified small molecules that cause a rapid (within 15 min) accumulation of secretory cargo and abnormal Golgi compartment-like membranes at low concentration (<2 μM), indicating that the compounds likely target the exocytic transport machinery at the Golgi. We screened for genes that, when overexpressed, suppress the drug effects, and found that the Ras-like small GTPase, Gtr2, but not its homolog and binding partner, Gtr1, efficiently suppresses the toxic effects of the compounds. Furthermore, assays for suppression of the secretory defect caused by the compounds suggest that Gtr proteins can regulate a pathway that is perturbed by the compounds. Because avl9Δ and gtr mutants share some phenotypes, our results indicate that the small molecules identified by our chemical-genetic strategy are promising tools for understanding Avl9 function and the mechanisms that control late exocytic transport.
doi:10.1128/EC.00184-09
PMCID: PMC2805290  PMID: 19897736
23.  Structural basis of family-wide Rab GTPase recognition by Rabenosyn-5 
Nature  2005;436(7049):415-419.
Rab GTPases regulate all stages of membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion1, 2. In the inactive (GDP-bound) conformation, accessory factors facilitate the targeting of Rab GTPases to intracellular compartments3–8. Following nucleotide exchange to the active (GTP-bound) conformation, Rab GTPases interact with functionally diverse effectors including lipid kinases, motor proteins, and tethering complexes. How effectors distinguish between homologous Rab GTPases represents an unresolved problem with respect to the specificity of vesicular trafficking. Using a structural proteomic approach, we have determined the specificity and structural basis underlying the interaction of the multivalent effector Rabenosyn-5 with the Rab family. The results demonstrate that even structurally similar effectors can achieve highly selective recognition of distinct subsets of Rab GTPases exclusively through interactions with the switch and interswitch regions. The observed specificity is determined at a family-wide level by structural diversity in the active conformation, which governs the spatial disposition of critical conserved recognition determinants, and by a small number of both positive and negative sequence determinants that allow further discrimination between Rab GTPases with similar switch conformations.
doi:10.1038/nature03798
PMCID: PMC1360218  PMID: 16034420
24.  Rag GTPases are cardioprotective by regulating lysosomal function 
Nature communications  2014;5:4241.
The Rag family proteins are Ras-like small GTPases that play a critical role in amino acid-stimulated mTORC1 activation by recruiting mTORC1 to lysosome. Despite progress in the mechanistic understanding of Rag GTPases in mTORC1 activation, little is known about the physiological function of Rag GTPases in vivo. Here, we show that loss of RagA and RagB (RagA/B) in cardiomyocytes results in hypertrophic cardiomyopathy and phenocopies lysosomal storage diseases although mTORC1 activity is not substantially impaired in vivo. We demonstrate that despite upregulation of lysosomal protein expression by constitutive activation of the transcription factor EB (TFEB) in RagA/B knockout mouse embryonic fibroblasts, lysosomal acidification is compromised due to decreased v-ATPase level in the lysosome fraction. Our study uncovers RagA/B GTPases as key regulators of lysosomal function and cardiac protection.
doi:10.1038/ncomms5241
PMCID: PMC4100214  PMID: 24980141
25.  Phosphorylation of the effector complex HOPS by the vacuolar kinase Yck3p confers Rab nucleotide specificity for vacuole docking and fusion 
Molecular Biology of the Cell  2012;23(17):3429-3437.
The Rab GTPase Ypt7p and its effector complex HOPS participate in catalyzing the fusion of yeast vacuoles. The role of the vacuolar kinase Yck3p in this relation is examined. It is shown how the regulatory ability of the Rab GTPase cycle is enforced only by posttranslational modification of the effector complex HOPS.
The homotypic fusion of yeast vacuoles requires the Rab-family GTPase Ypt7p and its effector complex, homotypic fusion and vacuole protein sorting complex (HOPS). Although the vacuolar kinase Yck3p is required for the sensitivity of vacuole fusion to proteins that regulate the Rab GTPase cycle—Gdi1p (GDP-dissociation inhibitor [GDI]) or Gyp1p/Gyp7p (GTPase-activating protein)—this kinase phosphorylates HOPS rather than Ypt7p. We addressed this puzzle in reconstituted proteoliposome fusion reactions with all-purified components. In the presence of HOPS and Sec17p/Sec18p, there is comparable fusion of 4-SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor) proteoliposomes when they have Ypt7p bearing either GDP or GTP, a striking exception to the rule that only GTP-bound forms of Ras-superfamily GTPases have active conformations. However, the phosphorylation of HOPS by recombinant Yck3p confers a strict requirement for GTP-bound Ypt7p for binding phosphorylated HOPS, for optimal membrane tethering, and for proteoliposome fusion. Added GTPase-activating protein promotes GTP hydrolysis by Ypt7p, and added GDI captures Ypt7p in its GDP-bound state during nucleotide cycling. In either case, the net conversion of Ypt7:GTP to Ypt7:GDP has no effect on HOPS binding or activity but blocks fusion mediated by phosphorylated HOPS. Thus guanine nucleotide specificity of the vacuolar fusion Rab Ypt7p is conferred through downstream posttranslational modification of its effector complex.
doi:10.1091/mbc.E12-04-0279
PMCID: PMC3431944  PMID: 22787280

Results 1-25 (848451)