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Membrane-enclosed transport carriers sort biological molecules between stations in the cell in a dynamic process that is fundamental to the physiology of eukaryotic organisms. While much is known about the formation and release of carriers from specific intracellular membranes, the mechanism of carrier formation from the recycling endosome, a compartment central to cellular signaling, remains to be resolved. In Caenorhabditis elegans, formation of transport carriers from the recycling endosome requires the dynamin-like, Eps15-homology domain (EHD) protein, RME-1, functioning with the Bin/Amphiphysin/Rvs (N-BAR) domain protein, AMPH-1. Here we show, using a free-solution single-particle technique known as burst analysis spectroscopy (BAS), that AMPH-1 alone creates small, tubular-vesicular products from large, unilamellar vesicles by membrane fission. Membrane fission requires the amphipathic H0 helix of AMPH-1 and is slowed in the presence of RME-1. Unexpectedly, AMPH-1-induced membrane fission is stimulated in the presence of GTP. Furthermore, the GTP-stimulated membrane fission activity seen for AMPH-1 is recapitulated by the heterodimeric N-BAR amphiphysin protein from yeast, Rvs161/167p, strongly suggesting that GTP-stimulated membrane fission is a general property of this important class of N-BAR proteins.

The interconversion of guanosine triphosphate (GTP) and guanosine diphosphate (GDP) is known to be integral to a wide variety of biological cellular activities, yet to date there are no analytical methods available to directly detect the ratio of intracellular GTP to GDP. Herein, we report GRISerHR, a genetically encoded fluorescent biosensor to monitor the GTP : GDP ratio in multiple cell types and in various organelles under metabolic perturbation. Additionally, we characterized the differential mitochondrial GTP : GDP ratios resulting from genetic modulation of two isoforms of a tricarboxylic acid (TCA) cycle enzyme (succinyl-CoA synthetase; SCS-ATP and SCS-GTP) and of a phosphoenolpyruvate (PEP) cycle enzyme (PEPCK-M). Thus, our GRISerHR sensor achieves spatiotemporally precise detection of dynamic changes in the endogenous GTP : GDP ratio in living cells and can help deepen our understanding about the energy metabolic contributions of guanosine nucleotides in biology.

IM30, the inner membrane-associated protein of 30 kDa, is conserved in cyanobacteria and chloroplasts. Although its exact physiological function is still mysterious, IM30 is clearly essential for thylakoid membrane biogenesis and/or dynamics. Recently, a cryptic IM30 GTPase activity has been reported, albeit thus far no physiological function has been attributed to this. Yet, it is still possible that GTP binding/hydrolysis affects formation of the prototypical large homo-oligomeric IM30 ring and rod structures. Here, we show that the Synechocystis sp. PCC 6803 IM30 protein in fact is an NTPase that hydrolyzes GTP and ATP, but not CTP or UTP, with about identical rates. While IM30 forms large oligomeric ring complexes, nucleotide binding and/or hydrolysis are clearly not required for ring formation.

IMP dehydrogenase(IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the guanine nucleotide pathway. In eukaryotic cells, GTP binding to the regulatory domain allosterically controls the activity of IMPDH by a mechanism that is fine-tuned by post-translational modifications and enzyme polymerization. Nonetheless, the mechanisms of regulation of IMPDH in bacterial cells remain unclear. Using biochemical, structural, and evolutionary analyses, we demonstrate that, in most bacterial phyla, (p)ppGpp compete with ATP to allosterically modulate IMPDH activity by binding to a, previously unrecognized, conserved high affinity pocket within the regulatory domain. This pocket was lost during the evolution of Proteobacteria, making their IMPDHs insensitive to these alarmones. Instead, most proteobacterial IMPDHs evolved to be directly modulated by the balance between ATP and GTP that compete for the same allosteric binding site. Altogether, we demonstrate that the activity of bacterial IMPDHs is allosterically modulated by a universally conserved nucleotide-controlled conformational switch that has divergently evolved to adapt to the specific particularities of each organism. These results reconcile the reported data on the crosstalk between (p)ppGpp signaling and the guanine nucleotide biosynthetic pathway and reinforce the essential role of IMPDH allosteric regulation on bacterial GTP homeostasis.

The millisecond timescale dynamics of activated Ras transiently sample a low-populated conformational state that has distinct surface property from the major state and represents a promising target for binding of small-molecule compounds. To avoid the complications of hydrolysis, dynamics and other properties of active Ras have so far been routinely investigated by using non-hydrolyzable GTP analogues, which, however, were previously reported to alter both the kinetics and distribution of the conformational exchange. In this study, we quantitatively measured and validated the internal dynamics of Ras complexed with a slowly hydrolyzable GTP analogue, GTPγS, which increases the lifetime of active Ras by 23 times relative to that of native GTP. It was found that GTPγS, in addition to its better mimicking of the exchange kinetics than the commonly used non-hydrolyzable analogues GppNHp and GppCH2 p, can rigorously reproduce the natural dynamics network in active Ras, thus indicating its fitness for use in the development of allosteric inhibitors.

Interferon-inducible large GTPases are critical for innate immunity. The distinctive feature of a large GTPase, human guanylate binding protein-1 (hGBP1), is the sequential hydrolysis of GTP into GMP via GDP. Despite several structural and biochemical studies, the underlying mechanism of assembly-stimulated GMP formation by hGBP1 and its role in immunity are not fully clarified. Using a series of biochemical, biophysical, and in silico experiments, we studied four tryptophan residues, located near switch I-II (in and around the active site) to understand the conformational changes near these regions and also to investigate their effect on enhanced GMP formation. The W79A mutation showed significantly reduced GMP formation, whereas the W81A and W180A substitutions exhibited only a marginal defect. The W114A mutation showed a long-range effect of further enhanced GMP formation, which was mediated through W79. We also observed that after first phosphate cleavage, the W79-containing region undergoes a conformational change, which is essential for stimulated GMP formation. We suggest that this conformational change helps to reposition the active site for the next cleavage step, which occurs through a stable contact between the indole moiety of W79 and the main chain carbonyl of K76. We also showed that stimulated GMP formation is crucial for antiviral activity against hepatitis C. Thus, the present study not only provides new insight for the stimulation of GMP formation in hGBP1, but also highlights the importance of the enhanced second phosphate cleavage product in the antiviral activity.

Overwhelming inflammation in the setting of acute critical illness induces capillary leak resulting in hypovolemia, edema, tissue dysoxia, organ failure and even death. The tight junction (TJ)-dependent capillary barrier is regulated by small GTPases, but the specific regulatory molecules most active in this vascular segment under such circumstances are not well described. We set out to identify GTPase regulatory molecules specific to endothelial cells (EC) that form TJs. Transcriptional profiling of confluent monolayers of TJ-forming human dermal microvascular ECs (HDMECs) and adherens junction only forming-human umbilical vein EC (HUVECs) demonstrate ARHGEF12 is basally expressed at higher levels and is only downregulated in HDMECs by junction-disrupting tumor necrosis factor (TNF). HDMECs depleted of ArhGEF12 by siRNA demonstrate a significantly exacerbated TNF-induced decrease in trans-endothelial electrical resistance and disruption of TJ continuous staining. ArhGEF12 is established as a RhoA-GEF in HUVECs and its knock down would be expected to reduce RhoA activity and barrier disruption. Pulldown of active GEFs from HDMECs depleted of ArhGEF12 and treated with TNF show decreased GTP-bound Rap1A after four hours but increased GTP-bound RhoA after 12 hours. In cell-free assays, ArhGEF12 immunoprecipitated from HDMECs is able to activate both Rap1A and RhoA, but not act on Rap2A-C, RhoB-C or even Rap1B which shares 95% sequence identity with Rap1A. We conclude that in TJ-forming HDMECs, ArhGEF12 selectively activates Rap1A to limit capillary barrier disruption in a mechanism independent of cAMP-mediated Epac1 activation.

Spinal cord injury (SCI) induces both motor and sensory dysfunctions. We wondered whether miR‐30b could promote primary sensory neuron (PSN) axon growth in inhibitory microenvironment. The neurite growth was promoted by miR‐30b agomir and inhibited by antagomir. MiR‐30b targeted and degraded sema3A mRNA. MiR‐30b regulated the formation of sema3A‐NRP‐1‐PlexinA1 complex via targeting sema3A. The neurite length was induced by the miR‐30b agomir, and the application of sema3A protein could reverse the effect of agomir. GTP‐RhoA and ROCK expression were down‐regulated by miR‐30b. Neurite outgrowth that inhibited by sema3A and the miR‐30b antagomir was increased by Y‐27632. Agomir promoted neurite growth in NogoA inhibitory conditions, which indicated miR‐30b could both enhance neuronal intrinsic regenerative ability and promote neurite growth against inhibitory microenvironment via Sema3A/NRP‐1/PlexinA1/RhoA/ROCK axis. The agomir could also regulate Sema3A/NRP‐1/PlexinA1/RhoA/ROCK axis in vivo and restore spinal cord sensory conductive function. In conclusion, miR‐30b could be a novel target for sensation recovery after SCI.

Two small Ras-like GTPases, MglA and SofG, work in synchrony to drive cell polarity and motility in the soil bacterium, Myxococcus xanthus. While MglA regulates two types of motility in Myxococcus and drives cell polarity reversals, SofG regulates social motility enabled by the type IV pili (T4P) machinery. In order to understand the molecular basis of how multiple GTPases act concertedly, we initiated biochemical studies on SofG. A construct of SofG (SofG∆60 ) was purified as a homogenous monomer and could bind to GDP and GTP. Intrinsic GTP hydrolysis by SofG∆60 was negligible. Earlier work from the laboratory revealed that MglB functions both as a GTPase-activating protein (GAP) and a guanine nucleotide exchange factor (GEF) for MglA. Biochemical assays of SofG∆60 established that MglB interacts with GTP-bound SofG∆60 and acts as a GAP for SofG∆60 . Interaction of MglB with SofG∆60 in the GDP-bound conformation was not observed, thereby suggesting that MglB might not act as a GEF for SofG∆60 . The existence of a common GAP for both SofG and MglA could potentially contribute to concerted regulation of their GTPase activities, and mediate crosstalk between the two GTPases involved in motility of M. xanthus. Sequence analysis revealed the features for a SofG-like subclass of prokaryotic small Ras-like GTPases that enable MglB to act as a dual-specificity GAP.

Ribosome hibernation is a prominent cellular strategy to modulate protein synthesis during starvation and the stationary phase of bacterial cell growth. Translational suppression involves the formation of either factor-bound inactive 70S monomers or dimeric 100S hibernating ribosomal complexes, the biological significance of which is poorly understood. Here, we demonstrate that the Escherichia coli 70S ribosome associated with stationary phase factors hibernation promoting factor or protein Y or ribosome-associated inhibitor A and the 100S ribosome isolated from both Gram-negative and Gram-positive bacteria are resistant to unfolded protein-mediated subunit dissociation and subsequent degradation by cellular ribonucleases. Considering that the increase in cellular stress is accompanied by accumulation of unfolded proteins, such resistance of hibernating ribosomes towards dissociation might contribute to their maintenance during the stationary phase. Analysis of existing structures provided clues on the mechanism of inhibition of the unfolded protein-mediated disassembly in case of hibernating factor-bound ribosome. Further, the factor-bound 70S and 100S ribosomes can suppress protein aggregation and assist in protein folding. The chaperoning activity of these ribosomes is the first evidence of a potential biological activity of the hibernating ribosome that might be crucial for cell survival under stress conditions.

The Rab family of small GTPases regulates intracellular membrane trafficking by orchestrating the biogenesis, transport, tethering, and fusion of membrane‐bound organelles and vesicles. Like other small GTPases, Rabs cycle between two states, an active (GTP‐loaded) state and an inactive (GDP‐loaded) state, and their cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase‐activating proteins (GAPs). Because an active form of each Rab localizes on a specific organelle (or vesicle) and recruits various effector proteins to facilitate each step of membrane trafficking, knowing when and where Rabs are activated and what effectors Rabs recruit is crucial to understand their functions. Since the discovery of Rabs, they have been regarded as one of the central hubs for membrane trafficking, and numerous biochemical and genetic studies have revealed the mechanisms of Rab functions in recent years. The results of these studies have included the identification and characterization of novel GEFs, GAPs, and effectors, as well as post‐translational modifications, for example, phosphorylation, of Rabs. Rab functions beyond the simple effector‐recruiting model are also emerging. Furthermore, the recently developed CRISPR/Cas technology has enabled acceleration of knockout analyses in both animals and cultured cells and revealed previously unknown physiological roles of many Rabs. In this review article, we provide the most up‐to‐date and comprehensive lists of GEFs, GAPs, effectors, and knockout phenotypes of mammalian Rabs and discuss recent findings in regard to their regulation and functions., The Rab family of small GTPases regulates intracellular membrane trafficking by localizing on specific organelles (or vesicles) and recruiting various effector proteins to facilitate each step of membrane trafficking. In this review article, we provide the most up‐to‐date and comprehensive lists of guanine nucleotide exchange factors, GTPase‐activating proteins, effectors, and knockout phenotypes of mammalian Rabs and discuss recent findings in regard to their regulation and functions.

Formation of clathrin-coated vesicles (CCVs) in receptor-mediated endocytosis is a mechanistically well-established process, in which clathrin, the adaptor protein complex AP-2, and the large GTPase dynamin play crucial roles. In order to obtain more mechanistic insight into this process, here we established a giant unilamellar vesicle (GUV)-based in vitro CCV reconstitution system with chemically defined components and the full-length recombinant proteins clathrin, AP-2, epsin-1, and dynamin-2. Our results support the predominant model in which hydrolysis of GTP by dynamin is a prerequisite to generate CCVs. Strikingly, in this system at near physiological concentrations of reagents, epsin-1 alone does not have the propensity for scission but is required for bud formation, whereas AP-2 and clathrin are not sufficient. Thus, our study reveals that epsin-1 is an important factor for the maturation of clathrin coated buds, a prerequisite for vesicle generation.

Human guanylate-binding protein 1 (hGBP-1) shows a dimer-induced acceleration of the GTPase activity yielding GDP as well as GMP. While the head-to-head dimerization of the large GTPase (LG) domain is well understood, the role of the rest of the protein, particularly of the GTPase effector domain (GED), in dimerization and GTP hydrolysis is still obscure. In this study, with truncations and point mutations on hGBP-1 and by means of biochemical and biophysical methods, we demonstrate that the intramolecular communication between the LG domain and the GED (LG:GED) is crucial for protein dimerization and dimer-stimulated GTP hydrolysis. In the course of GTP binding and γ-phosphate cleavage, conformational changes within hGBP-1 are controlled by a chain of amino acids ranging from the region near the nucleotide-binding pocket to the distant LG:GED interface and lead to the release of the GED from the LG domain. This opening of the structure allows the protein to form GED:GED contacts within the dimer, in addition to the established LG:LG interface. After releasing the cleaved γ-phosphate, the dimer either dissociates yielding GDP as the final product or it stays dimeric to further cleave the β-phosphate yielding GMP. The second phosphate cleavage step, that is, the formation of GMP, is even more strongly coupled to structural changes and thus more sensitive to structural restraints imposed by the GED. Altogether, we depict a comprehensive mechanism of GTP hydrolysis catalyzed by hGBP-1, which provides a detailed molecular understanding of the enzymatic activity connected to large structural rearrangements of the protein. DATABASE: Structural data are available in RCSB Protein Data Bank under the accession numbers: 1F5N, 1DG3, 2B92.

KRAS homo-dimerization has been implicated in the activation of RAF kinases, however, the mechanism and structural basis remain elusive. We developed a system to study KRAS dimerization on nanodiscs using paramagnetic relaxation enhancement (PRE) NMR, and determined distinct structures of membrane-anchored KRAS dimers in the active GTP- and inactive GDP-loaded states. Both dimerize through an α4-α5 interface, but the relative orientation of the protomers and their contacts differ substantially. Dimerization of KRAS-GTP, stabilized by electrostatic interactions between R135 and E168, favours an orientation on the membrane that promotes accessibility of the effector-binding site. Remarkably, ‘cross’-dimerization between GTP- and GDP-bound KRAS molecules is unfavorable. These models provide a vital platform to elucidate the structural basis of RAF activation by RAS and to develop newer inhibitors that can disrupt the KRAS dimerization. The methodology developed to specifically probe the intermolecular interactions within KRAS dimer is applicable to many other farnesylated small GTPases.

Parkinson's disease (PD) is one of the most common movement disorders with loss of dopaminergic neurons and the presence of Lewy bodies in certain brain areas. However, it is not clear how Lewy body (inclusion with protein aggregation) formation occurs. Mutations in leucine-rich repeat kinase 2 (LRRK2) can cause a genetic form of PD and contribute to sporadic PD with the typical Lewy body pathology. Here, we used our recently identified LRRK2 GTP-binding inhibitors as pharmacological probes to study the LRRK2-linked ubiquitination and protein aggregation. Pharmacological inhibition of GTP-binding by GTP-binding inhibitors (68 and Fx2149) increased LRRK2-linked ubiquitination predominantly via K27 linkage. Compound 68- or Fx2149 increased G2019S-LRRK2-linked ubiquitinated aggregates, which occurred through the atypical linkage types K27 and K63. Coexpression of K27R and K63R, which prevented ubiquitination via K27 and K63 linkages, reversed the effects of 68 and Fx2149. Moreover, 68 and Fx2149 also promoted G2019S-LRRK2-linked aggresome (Lewy body-like inclusion) formation via K27 and K63 linkages. These findings demonstrate that LRRK2 GTP-binding activity is critical in LRRK2-linked ubiquitination and aggregation formation. These studies provide novel insight into the LRRK2-linked Lewy body-like inclusion formation underlying PD pathogenesis.

During adhesion, cells develop filopodia to facilitate the attachment to the extracellular matrix. The small guanosine triphosphate (GTP)-binding protein, Cdc42, plays a central role in the formation of filopodia. It has been reported that Cdc42 activity is regulated by cholesterol (Chol). We examined Chol distribution in filopodia using Chol-binding domain 4 (D4) fragment of bacterial toxin, perfringolysin O that senses high membrane concentration of Chol. Our results indicate that fluorescent D4 was enriched at the tip of the outer leaflet of filopodia in the initiation phase of cell adhesion. This enrichment was accompanied by a defect of D4 labeling in the inner leaflet. Steady phase adhered cell experiment indicated that both Cdc42 and ATP-binding cassette transporter, ABCA1, were involved in the binding of D4 to the cell surface. Depletion of Chol activated Cdc42. Our results suggest that asymmetric distribution of Chol at the tip of filopodia induces activation of Cdc42, and thus, facilitates filopodia formation.

In this study, we assessed the importance of cytoskeleton organization in the mammalian cells used to produce therapeutic proteins. Two cytoskeletal genes, Actin alpha cardiac muscle 1 (ACTC1) and a guanosine triphosphate GTPase‐activating protein (TAGAP), were found to be upregulated in highly productive therapeutic protein‐expressing Chinese hamster ovary (CHO) cells selected by the deprivation of vitamin B5. We report here that the overexpression of the ACTC1 protein was able to improve significantly recombinant therapeutic production, as well as to decrease the levels of toxic lactate metabolic by‐products. ACTC1 overexpression was accompanied by altered as well as decreased polymerized actin, which was associated with high protein production by CHO cell cultured in suspension. We suggest that the depolymerization of actin and the possible modulation of integrin signaling, as well as changes in basal metabolism, may be driving the increase of protein secretion by CHO cells., Pourcel et al. observed that several cytoskeletal genes are upregulated in highly productive CHO cell clones. Actin alpha overexpression yielded altered cytoskeletal organization, which was accompanied by improved recombinant therapeutic production as well as decreased levels of toxic lactate metabolic by‐products. Cells spontaneously displaying low actin polymerization yielded higher protein production in suspension cultures, highlighting the importance of the cytoskeleton for the efficient production of easy‐ as well as difficult‐to‐express therapeutic proteins.

Here we report the design, synthesis, and characterization of bifunctional chemical ligands that induce the association of Ras with ubiquitously expressed immunophilin proteins such as FKBP12 and cyclophilin A. We show this approach is applicable to two distinct Ras ligand scaffolds, and that both the identity of the immunophilin ligand and the linker chemistry affect compound efficacy in biochemical and cellular contexts. These ligands bind to Ras in an immunophilin-dependent fashion and mediate the formation of tripartite complexes of Ras, immunophilin, and the ligand. The recruitment of cyclophilin A to GTP-bound Ras blocks its interaction with B-Raf in biochemical assays. Our study demonstrates the feasibility of ligand-induced association of Ras with intracellular proteins and suggests it as a promising therapeutic strategy for Ras-driven cancers.

3-Fucosyllactose (3-FL) is one of the major fucosylated oligosaccharides in human milk. Along with 2'-fucosyllactose (2'-FL), it is known for its prebiotic, immunomodulator, neonatal brain development, and antimicrobial function. Whereas the biological production of 2'-FL has been widely studied and made significant progress over the years, the biological production of 3-FL has been hampered by the low activity and insoluble expression of α-1,3-fucosyltransferase (FutA), relatively low abundance in human milk oligosaccharides compared with 2'-FL, and lower digestibility of 3-FL than 2'-FL by bifidobacteria. In this study, we report the gram-scale production of 3-FL using E. coli BL21(DE3). We previously generated the FutA quadruple mutant (mFutA) with four site mutations at S46F, A128N, H129E, Y132I, and its specific activity was increased by nearly 15 times compared with that of wild-type FutA owing to the increase in kcat and the decrease in Km . We overexpressed mFutA in its maximum expression level, which was achieved by the optimization of yeast extract concentration in culture media. We also overexpressed L-fucokinase/GDP- L-fucose pyrophosphorylase to increase the supply of GDP-fucose in the cytoplasm. To increase the mass of recombinant whole-cell catalysts, the host E. coli BW25113 was switched to E. coli BL21(DE3) because of the lower acetate accumulation of E. coli BL21(DE3) than that of E. coli BW25113. Finally, the lactose operon was modified by partially deleting the sequence of LacZ (lacZΔm15) for better utilization of D-lactose. Production using the lacZΔm15 mutant yielded 3-FL concentration of 4.6 g/L with the productivity of 0.076 g·L-1 ·hr-1 and the specific 3-FL yield of 0.5 g/g dry cell weight.

Dynamin-related proteins are multidomain, mechanochemical GTPases that self-assemble and orchestrate a wide array of cellular processes. Over the past decade, structural insights from X-ray crystallography and cryo-electron microscopy have reshaped our mechanistic understanding of these proteins. Here we provide a historical perspective on these advances that highlights the structural attributes of different dynamin family members and explores how these characteristics affect GTP hydrolysis, conformational coupling, and oligomerization. We also discuss a number of lingering challenges remaining in the field that suggest future directions of study.

The FEBS journal 287(3), 496-514 (2019). doi:10.1111/febs.15015, Published by Wiley-Blackwell, Oxford [u.a.]

Although naturally occurring low-molecular-weight compounds have many known roles within the cell, these do not usually involve the direct inhibition of protein-protein interactions. Based on the results of high-throughput screening of a library of bioactive compounds and neurotransmitters, we report here that the four nucleoside triphosphates ATP, GTP, CTP and UTP inhibit the SH2 domain of the tumor-related transcription factor STAT5b. ATP and GTP are the most active nucleoside triphosphates and show specificity for STAT5b over STAT5a, STAT3, STAT6 and the p53-binding protein HDM2. As the inhibition constant of ATP against STAT5b is significantly lower than published values for the intracellular ATP concentration, our data suggest that ATP might inhibit the protein-protein interactions of STAT5b in living cells.

Sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) is an intrinsic antiviral restriction factor known to play a vital role in preventing multiple viral infections and in the control of the cellular deoxynucleoside triphosphate (dNTP) pool. Human and mouse SAMHD1 have both been extensively studied; however, our knowledge on porcine SAMHD1 is limited. Here, we report our findings from comprehensive structural and functional studies on porcine SAMHD1. We determined the crystal structure of porcine SAMHD1 and showed that it forms a symmetric tetramer. Moreover, we modified the deoxynucleotide triphosphohydrolase (dNTPase) activity of SAMHD1 by site-directed mutagenesis based on the crystal structure, and obtained an artificial dimeric enzyme possessing high dNTPase activity. Taken together, our results define the mechanism underlying dNTP regulation and provide a deeper understanding of the regulation of porcine SAMHD1 functions. Directed modification of key residues based on the protein structure enhances the activity of the enzyme, which will be beneficial in the search for new antiviral strategies and for future translational applications.

The interferon γ-inducible large GTPases, human guanylate-binding protein (hGBP)-1 and hGBP-2, mediate antipathogenic and antiproliferative effects in human cells. Both proteins hydrolyse GTP to GDP and GMP through successive cleavages of phosphate bonds, a property that functionally distinguishes them from other GTPases. However, it is unclear why hGBP-2 yields lower GMP than hGBP-1 despite sharing a high sequence identity (~ 78%). We previously reported that the hGBP-1 tetramer is crucial for enhanced GMP formation. We show here that the hGBP-2 tetramer has no role in GMP formation. Using truncated hGBP-2 variants, we found that its GTP-binding domain alone hydrolyses GTP only to GDP. However, this domain along with the intermediate region enabled dimerization and hydrolysed GTP further to GMP. We observed that unlike in hGBP-1, the helical domain of hGBP-2 has an insignificant role in the regulation of GTP hydrolysis, suggesting that the differences in GMP formation between hGBP-2 and hGBP-1 arise from differences in their GTP-binding domains. A large sequence variation seen in the guanine cap may be responsible for the lower GMP formation in hGBP-2. Moreover, we identified the sites in the hGBP-2 domains that are critical for both dimerization and tetramerization. We also found the existence of hGBP-2 tetramer in mammalian cells, which might have a role in the suppression of the carcinomas. Our study suggests that sequence variation near the active site in these two close homologues leads to differential second phosphate cleavage and highlights the role of individual hGBP-2 domains in the regulation of GTP hydrolysis.

The m7 G cap is a unique nucleotide structure at the 5'-end of all eukaryotic mRNAs. The cap specifically interacts with numerous cellular proteins and participates in biological processes that are essential for cell growth and function. To provide small molecular probes to study important cap-recognizing proteins, we synthesized m7 G nucleotides labeled with fluorescent tags via the terminal phosph(on)ate group and studied how their emission properties changed upon protein binding or enzymatic cleavage. Only the pyrene-labeled compounds behaved as sensitive turn-on probes. A pyrene-labeled m7 GTP analogue showed up to eightfold enhanced fluorescence emission upon binding to eukaryotic translation initiation factor 4E (eIF4E) and over 30-fold enhancement upon cleavage by decapping scavenger (DcpS) enzyme. These observations served as the basis for developing binding- and hydrolytic-activity assays. The assay utility was validated with previously characterized libraries of eIF4E ligands and DcpS inhibitors. The DcpS assay was also applied to study hydrolytic activity and inhibition of endogenous enzyme in cytoplasmic extracts from HeLa and HEK cells.

Cell division control protein 42 homolog (Cdc42) influences a variety of cellular responses such as cell migration and polarity. Deregulation of Cdc42 has been associated with several human diseases and developmental disorders. Over-activation of Cdc42 through guanine nucleotide exchange factor (GEF) is a critical event for Cdc42 involved cancer metastasis. Members of DOCK family of GEF are important activators of Cdc42. However, this activation mechanism is still unknown. Molecular dynamics (MD) simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) calculations were employed to investigate the central step of the activation of Cdc42: the dissociation mechanism of GDP from Cdc42 via DOCK9. Simulation results show that Mg2+ ion has a remarkable influence on the conformational change of switch I of Cdc42 through residue Pro34 which functions as a "clasp" to control the flexibility of switch I. In the GDP dissociation process, the Mg2+ ion leave first to result in a suitable conformation of Cdc42 for following DOCK9 binding to. When DOCK9 binds to Cdc42, it changes the orientations of residues Lys16, Thr17, Cys18 and Phe28 of Cdc42 to weaken the interactions between Cdc42 and GDP to release GDP. This study first elucidates the dissociation mechanism of GDP from Cdc42 via DOCK9 and identifies the essential residues of Cdc42 in this process. These simulation results are consistent with the recent findings of biochemical and amino acid mutational studies, and the observations are beneficial to understand the activation mechanism of Cdc42 and to provide insights for designing compounds targeting on Cdc42 related cancer metastasis.

Characterization of native GTP-bound Ras is important for an appreciation of its cellular signaling and for the design of inhibitors, which however has been depressed by its intrinsic instability. Herein, an effective approach for extending the lifetime of Ras⋅GTP samples by exploiting the active role of Son of Sevenless (Sos) is demonstrated that sustains the activated state of Ras. This approach, combined with a postprocessing method that suppresses residual Ras⋅GDP signals, is applied to the site-resolved NMR measurement of the allosteric dynamics of Ras⋅GTP. The observed network of concerted motions well covers the recently identified allosteric inhibitor-binding pockets, but the motions are more confined than those of Ras⋅GppNHp, advocating the use of native GTP for development of allosteric inhibitors. The Sos-based approach is anticipated to generally facilitate experiments on active Ras when native GTP is preferred.

Wounds close by keratinocytes migrating from the edge of the wound and re-epithelializing the epidermis. It has been proposed that the major stimuli for wound closure are blood-derived growth factors, chemokines and cytokines. The small GTPase R-Ras, a known integrin activator, also regulates vascular permeability during angiogenesis, and blood vessels lacking R-Ras leak plasma proteins constantly. We explored whether the access to blood-derived proteins influences skin wound healing in R-Ras knockout (KO) mice. In skin wounds, R-Ras expression was mostly restricted to the vasculature in the granulation tissue. Angiogenic blood vessels in the R-Ras KO mice were significantly more permeable than in wild-type (WT) controls. Although the distances between epidermal tongues, and the panniculus carnosus muscles, were significantly longer in R-Ras KO than WT controls before the granulation tissue formation took place, there were no differences in the wound closure or re-epithelialization rates or granulation tissue formation. These findings were also corroborated in a special splint excision wound model. Our study shows that although R-Ras does not influence the skin wound healing itself, the blood vessels lacking R-Ras are leaky and thus could facilitate the access of blood-derived proteins to the wound.

Drug development is an arduous procedure, necessitating testing the interaction of a large number of potential candidates with potential interacting (macro)molecules. Therefore, any method which could provide an initial screening of potential candidate drugs might be of interest for the acceleration of the procedure, by highlighting interesting compounds, prior to in vitro and in vivo validation. In this line, we present a method which may identify potential hits, with agonistic and/or antagonistic properties on GPCR receptors, integrating the knowledge on signaling events triggered by receptor activation (GPCRs binding to Gα,β,γ proteins, and activating Gα, exchanging GDP for GTP, leading to a decreased affinity of the Gα for the GPCR). We show that, by integrating GPCR‐ligand and Gα‐GDP or ‐GTP binding in docking simulation, which correctly predicts crystallographic data, we can discriminate agonists, partial agonists, and antagonists, through a linear function, based on the ΔG (Gibbs‐free energy) of liganded‐GPCR/Gα‐GDP. We built our model using two Gαs (β2‐adrenergic and prostaglandin‐D2), four Gαi (μ‐opioid, dopamine‐D3, adenosine‐A1, rhodopsin), and one Gαo (serotonin) receptors and validated it with a series of ligands on a recently deorphanized Gαi receptor (OXER1). This approach could be a valuable tool for initial in silico validation and design of GPRC‐interacting ligands.

Luminogenic materials with aggregation-induced emission (AIE) have attracted considerable interest for applications. If these systems aggregate, the free rotation of their scaffold is restricted, and as a consequence the photoluminescence increases. Herein, the first experimental observation of a "dynameric effect" on AIE is described. A comparison is made of the AIE of molecular and dynameric sensors that exhibit non-linear turn-on switching of fluorescence upon their interaction with adenosine triphosphate (ATP). Confirmation was obtained from the enhanced ATP detection with multivalent dynameric networks compared with a molecular sensor. The dynamic, reversible behaviour of the imine linkages is critical to produce this enhancement, as a static, imine-reduced, polymeric sensor showed decreased AIE activity. The dynameric frameworks showed selectivity for ATP over adenosine diphosphate, and adenosine monophosphate over guanosine triphosphate or cytidine triphosphate. Together, these results will accelerate the systematic discovery of efficient adaptive biomimetic sensors.

The increase in cAMP levels in endothelial cells triggers cellular signaling to alter vascular permeability. It is generally considered that cAMP signaling stabilizes the endothelial barrier function and reduces permeability. However, previous studies have only examined the permeability shortly after cAMP elevation and thus have only investigated acute responses. Because cAMP is a key regulator of gene expression, elevated cAMP may have a delayed but profound impact on the endothelial permeability by altering the expression of the genes that are vital for the vessel wall stability. The small guanosine triphosphate hydrolase Ras-related protein (R-Ras) stabilizes VE-cadherin clustering and enhances endothelial barrier function, thereby stabilizing the integrity of blood vessel wall. Here we show that cAMP controls endothelial permeability through RRAS gene regulation. The prolonged cAMP elevation transcriptionally repressed RRAS in endothelial cells via a cAMP response element-binding protein (CREB) 3-dependent mechanism and significantly disrupted the adherens junction. These effects resulted in a marked increase of endothelial permeability that was reversed by R-Ras transduction. Furthermore, cAMP elevation in the endothelium by prostaglandin E2 or phosphodiesterase type 4 inhibition caused plasma leakage from intact microvessels in mouse skin. Our study demonstrated that, contrary to the widely accepted notion, cAMP elevation in endothelial cells ultimately increases vascular permeability, and the cAMP-dependent RRAS repression critically contributes to this effect.-Perrot, C. Y., Sawada, J., Komatsu, M. Prolonged activation of cyclic AMP signaling leads to endothelial barrier disruption via transcriptional repression of RRAS.

Ras homolog enriched in brain (Rheb) is a small GTPase that regulates mammalian/mechanistic target of rapamycin complex 1 (mTORC1) and, thereby, cell growth and metabolism. Here we show that cycling between the inactive GDP- and the active GTP-bound state modulates the backbone dynamics of a C-terminal truncated form, RhebΔCT, which is suggested to influence its interactions. We further investigated the interactions between RhebΔCT and the proposed Rheb-binding domain of the regulatory protein FKBP38. The observed weak interactions with the GTP-analogue- (GppNHp-) but not the GDP-bound state, appear to accelerate the GDP to GTP exchange, but only very weakly compared to a genuine GEF. Thus, FKBP38 is most likely not a GEF but a Rheb effector that may function in membrane targeting of Rheb.

In the year 2015, new Zika virus (ZIKV) broke out in Brazil and spread away in more than 80 countries. Scientists directed their efforts toward viral polymerase in attempt to find inhibitors that might interfere with its function. In this study, molecular dynamics simulation (MDS) was performed over 444 ns for a ZIKV polymerase model. Molecular docking (MD) was then performed every 10 ns during the MDS course to ensure the binding of small molecules to the polymerase over the entire time of the simulation. MD revealed the binding ability of four suggested guanosine inhibitors (GIs); (Guanosine substituted with OH and SH (phenyl) oxidanyl in the 2′ carbon of the ribose ring). The GIs were compared to guanosine triphosphate (GTP) and five anti‐hepatitis C virus drugs (either approved or under clinical trials). The mode of binding and the binding performance of GIs to ZIKV polymerase were found to be the same as GTP. Hence, these compounds were capable of competing GTP for the active site. Moreover, GIs bound to ZIKV active site more tightly compared to ribavirin, the wide‐range antiviral drug.

Molecular motors such as kinesin-1 work in small teams to actively shuttle cargos in cells, for example in polarized transport in axons. Here, we examined the potential regulatory role of the nucleotide state of tubulin on the run length of cargos carried by multiple kinesin motors, using an optical trapping-based in vitro assay. Based on a previous report that kinesin binds preferentially to GTP-tubulin-rich microtubules, we anticipated that multiple-kinesin cargos would run substantially greater distances along GMPCPP microtubules than along GDP microtubules. Surprisingly, we did not uncover any significant differences in run length between microtubule types. A combination of single-molecule experiments, comparison with previous theory, and classic microtubule affinity pulldown assays revealed that native kinesin-1 does not bind preferentially to GTP-tubulin-rich microtubules. The apparent discrepancy between our observations and the previous report likely reflects differences in post-translational modifications between the native motors used here and the recombinant motors examined previously. Future investigations will help shed light on the interplay between the motor's post-translational modification and the microtubule's nucleotide-binding state for transport regulation in vivo.

The Rab-GTPase-activating proteins (GAPs) TBC1D1 and TBC1D4 play important roles in the insulin-stimulated translocation of the glucose transporter GLUT4 from intracellular vesicles to the plasma membrane in muscle cells and adipocytes. We identified Rab28 as a substrate for the GAP domains of both TBC1D1 and TBC1D4 in vitro. Rab28 is expressed in adipose cells and skeletal muscle, and its GTP-binding state is acutely regulated by insulin. We found that in intact isolated mouse skeletal muscle, siRNA-mediated knockdown of Rab28 decreases basal glucose uptake. Conversely, in primary rat adipose cells, overexpression of Rab28-Q72L, a constitutively active mutant, increases basal cell surface levels of an epitope-tagged HA-GLUT4. Our results indicate that Rab28 is a novel GTPase involved in the intracellular retention of GLUT4 in insulin target cells.

Biomimetic synthesis routes towards the important natural d-mannosyl donor guanosine 5′-diphospho-d-mannose (GDP-Man) rely on kinase-catalyzed nucleotide triphosphate (NTP)-dependent phosphorylations of d-mannose (Man), to give d-mannose 6-phosphate or α-d-mannose 1-phosphate (αMan 1-P) as an intermediate product. A GDP-Man synthesis not requiring the kinase/NTP system would be practical and cost-effective. Here, we have developed a multienzyme cascade towards GDP-Man, characterized in that αMan 1-P was obtained by a diastereoselective phosphatase-catalyzed phosphorylation of Man. α-d-Glucose 1-phosphate (αGlc 1-P), prepared in situ through phosphorylase-catalyzed conversion of sucrose in the presence of inorganic phosphate, was used as an expedient phosphoryl donor. The incipient αMan 1-P and guanosine triphosphate (GTP) were converted into GDP-Man by a highly manno compared to gluco selective nucleotidyltransferase. Pyrophosphatase was additionally required to hydrolyze the pyrophosphate released from the GTP, thus driving the reaction towards GDP-Man. The enzymatic cascade was operated with the αMan 1-P and the GDP-Man formation decoupled from one another (sequential mode) or having all steps run concurrently (simultaneous mode). Detailed time course analysis revealed that kinetic pull due to the constant removal of the intermediate αMan 1-P in simultaneous-mode reactions was important to promote phosphorylation of Man from αGlc 1-P in high efficiency, avoiding loss of sugar 1-phosphates by hydrolysis. Under optimized conditions for the one-pot transformation involving four enzymes, 100 mM (67 g L−1) GDP-Man was prepared from 140 mM sucrose and phosphate, using 400 mM Man as the phosphoryl acceptor. The product was recovered by anion-exchange and size-exclusion chromatography in ≥95% purity in about 50% yield (100 mg). These results demonstrate for the first time the practical use of a phosphorylase-phosphatase combi-catalyst as an alternative to the canonical kinase for the anomeric phosphorylation of the sugar substrate in nucleoside diphospho-sugar synthesis. Phosphorylation from inorganic phosphate via the intermediate αGlc 1-P rather than from NTP, particularly GTP, appears advantageous specifically in cases where the sugar acceptor is a bulk commodity that can be applied in suitable excess to the phosphatase reaction.

Made available in DSpace on 2018-12-11T16:55:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-01 Xanthomonas citri subsp. citri (Xac) is the causative agent of citrus canker, a plant disease that significantly impacts citriculture. In earlier work, we showed that alkylated derivatives of gallic acid have antibacterial action against Xac and target both the cell division protein FtsZ and membrane integrity in Bacillus subtilis. Here, we have purified native XacFtsZ and characterized its GTP hydrolysis and polymerization properties. In a surprising manner, inhibition of XacFtsZ activity by alkyl gallates is not as strong as observed earlier with B. subtilis FtsZ. As the alkyl gallates efficiently permeabilize Xac membranes, we propose that this is the primary mode of antibacterial action of these compounds. Department of Molecular Microbiology Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen Laboratory of Antibiotics and Chemotherapeutics Department of Chemistry and Environmental Sciences Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP) Department of Chemical Engineering Biotechnology and Environmental Technology University of Southern Denmark Laboratory of Antibiotics and Chemotherapeutics Department of Chemistry and Environmental Sciences Institute of Biosciences Humanities and Exact Sciences São Paulo State University (UNESP)

The two second messengers in signalling, cyclic AMP and cyclic GMP, are produced by adenylyl and guanylyl cyclases respectively. Recognition and discrimination of the substrates ATP and GTP by the nucleotidyl cyclases are vital in these reactions. Various apo-, substrate- or inhibitor-bound forms of adenylyl cyclase (AC) structures from transmembrane and soluble ACs have revealed the catalytic mechanism of ATP cyclization reaction. Previously reported structures of guanylyl cyclases represent ligand-free forms and inactive open states of the enzymes and thus do not provide information regarding the exact mode of substrate binding. The structures we present here of the cyclase homology domain of a class III AC from Mycobacterium avium (Ma1120) and its mutant in complex with ATP and GTP in the presence of calcium ion, provide the structural basis for substrate selection by the nucleotidyl cyclases at the atomic level. Precise nature of the enzyme-substrate interactions, novel modes of substrate binding and the ability of the binding pocket to accommodate diverse conformations of the substrates have been revealed by the present crystallographic analysis. This is the first report to provide structures of both the nucleotide substrates bound to a nucleotidyl cyclase. Database Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers: 5D15 (Ma1120(CHD)+ATP.Ca2+), 5D0E (Ma1120(CHD)+GTP.Ca2+), 5D0H (Ma1120(CHD)(KDA -> EGY)+ATP.Ca2+), 5D0G (Ma1120(CHD)(KDA -> EGY)+GTP.Ca2+). Enzymes Adenylyl cyclase (EC number: 4.6.1.1).

Purpose Trimethoprim is a folate biosynthesis inhibitor. Tetrahydrofolates are essential for the transfer of C1 units in several biochemical pathways including purine, thymine, methionine, and glycine biosynthesis. This study addressed the effects of folate biosynthesis inhibition on bacterial physiology. Experimental design Two complementary proteomic approaches were employed to analyze the response of Bacillus subtilis to trimethoprim. Acute changes in protein synthesis rates were monitored by radioactive pulse labeling of newly synthesized proteins and subsequent 2DE analysis. Changes in protein levels were detected using gel-free quantitative MS. Results Proteins involved in purine and histidine biosynthesis, the σB -dependent general stress response, and sporulation were upregulated. Most prominently, the PurR-regulon required for de novo purine biosynthesis was derepressed indicating purine depletion. The general stress response was activated energy dependently and in a subpopulation of treated cultures an early onset of sporulation was observed, most likely triggered by low guanosine triphosphate levels. Supplementation of adenosine triphosphate, adenosine, and guanosine to the medium substantially decreased antibacterial activity, showing that purine depletion becomes the bottleneck in trimethoprim-treated B. subtilis. Conclusions and clinical relevance The frequently prescribed antibiotic trimethoprim causes purine depletion in B. subtilis, which can be complemented by supplementing purines to the medium.

Translational GTPases (trGTPases) play key roles in facilitating protein synthesis on the ribosome. Despite the high degree of evolutionary conservation in the sequences of their GTP‐binding domains, the rates of GTP hydrolysis and nucleotide exchange vary broadly between different trGTPases. EF‐Tu, one of the best‐characterized model G proteins, evolved an exceptionally rapid and tightly regulated GTPase activity, which ensures rapid and accurate incorporation of amino acids into the nascent chain. Other trGTPases instead use the energy of GTP hydrolysis to promote movement or to ensure the forward commitment of translation reactions. Recent data suggest the GTPase mechanism of EF‐Tu and provide an insight in the catalysis of GTP hydrolysis by its unusual activator, the ribosome. Here we summarize these advances in understanding the functional cycle and the regulation of trGTPases, stimulated by the elucidation of their structures on the ribosome and the progress in dissecting the reaction mechanism of GTPases. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 463–475, 2016.

Interpretation of the experiments showing that the Ras-GAP protein complex maintains activity in guanosine triphosphate (GTP) hydrolysis upon replacement of Glu61 in Ras with its unnatural nitro analog, NGln, is an important issue for understanding details of chemical transformations at the enzyme active site. By using molecular modeling we demonstrate that both glutamine and its nitro analog in the aci-nitro form participate in the reaction of GTP hydrolysis at the stages of proton transfer and formation of inorganic phosphate. The computed structures and the energy profiles for the complete pathway from the enzyme-substrate to enzyme-product complexes for the wild-type and mutated Ras suggest that the reaction mechanism is not affected by this mutation.

Summary Zoospore chemotaxis to soybean isoflavones is essential in the early stages of infection by the oomycete pathogen Phytophthora sojae. Previously, we have identified a G-protein α subunit encoded by PsGPA1 which regulates the chemotaxis and pathogenicity of P. sojae. In the present study, we used affinity purification to identify PsGPA1-interacting proteins, including PsHint1, a histidine triad (HIT) domain-containing protein orthologous to human HIT nucleotide-binding protein 1 (HINT1). PsHint1 interacted with both the guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms of PsGPA1. An analysis of the gene-silenced transformants revealed that PsHint1 was involved in the chemotropic response of zoospores to the isoflavone daidzein. During interaction with a susceptible soybean cultivar, PsHint1-silenced transformants displayed significantly reduced infectious hyphal extension and caused a strong cell death in plants. In addition, the transformants displayed defective cyst germination, forming abnormal germ tubes that were highly branched and exhibited apical swelling. These results suggest that PsHint1 not only regulates chemotaxis by interacting with PsGPA1, but also participates in a Gα-independent pathway involved in the pathogenicity of P. sojae.

The control of rRNA synthesis and, thereby, translation is vital for adapting to changing environmental conditions. The decrease of rRNA is a common feature of the stringent response, which is elicited by the rapid synthesis of (p)ppGpp. Here we analysed the properties and regulation of one representative rRNA operon of Staphylococcus aureus under stringent conditions and during growth. The promoters, P1 and P2, are severely downregulated at low intracellular guanosine triphosphate (GTP) concentrations either imposed by stringent conditions or in a guanine auxotroph guaBA mutant. In a (p)ppGpp(0) strain, the GTP level increased under stringent conditions, and rRNA transcription was upregulated. The correlation of the intracellular GTP levels and rRNA promoter activity could be linked to GTP nucleotides in the initiation region of both promoters at positions between +1 and +4. This indicates that not only transcriptional initiation, but also the first steps of elongation, requires high concentrations of free nucleotides. However, the severe downregulation of rRNA in post-exponential growth phase is independent of (p)ppGpp, the composition of the initiation region and the intracellular nucleotide pool. In summary, rRNA transcription in S. aureus is only partially and presumably indirectly controlled by (p)ppGpp.

RAS subfamily proteins regulates cell growth promoting signaling processes by cycling between active (GTP-bound) and inactive (GDP-bound) states. Different RAS isoforms, though structurally similar, exhibit functional specificity and are associated with different types of cancers and developmental disorders. Understanding the dynamical differences between the isoforms is crucial for the design of inhibitors that can selectively target a particular malfunctioning isoform. In this study, we provide a comprehensive comparison of the dynamics of all the three RAS isoforms (HRAS, KRAS, and NRAS) using extensive molecular dynamics simulations in both the GDP- (total of 3.06 μs) and GTP-bound (total of 2.4 μs) states. We observed significant differences in the dynamics of the isoforms, which rather interestingly, varied depending on the type of the nucleotide bound and the simulation temperature. Both SwitchI (Residues 25-40) and SwitchII (Residues 59-75) differ significantly in their flexibility in the three isoforms. Furthermore, Principal Component Analysis showed that there are differences in the conformational space sampled by the GTP-bound RAS isoforms. We also identified a previously unreported pocket, which opens transiently during MD simulations, and can be targeted to regulate nucleotide exchange reaction or possibly interfere with membrane localization. Further, we present the first simulation study showing GDP destabilization in the wild-type RAS protein. The destabilization of GDP/GTP occurred only in 1/50 simulations, emphasizing the need of guanine nucleotide exchange factors (GEFs) to accelerate such an extremely unfavorable process. This observation along with the other results presented in this article further support our previously hypothesized mechanism of GEF-assisted nucleotide exchange.

The free energy profiles for the chemical reaction of the guanosine triphosphate hydrolysis GTP + H2O → GDP + Pi by Ras-GAP for the wild-type and G13V mutated Ras were computed by using molecular dynamics protocols with the QM(ab initio)/MM potentials. The results are consistent with the recent measurements of reaction kinetics in Ras-GAP showing about two-order reduction of the rate constant upon G13V mutation in Ras: the computed activation barrier on the free energy profile is increased by 3 kcal/mol upon the G13V replacement. The major reason for a higher energy barrier is a shift of the "arginine finger" (R789 from GAP) from the favorable position in the active site. The results of simulations provide support for the mechanism of the reference reaction according to which the Q61 side chain directly participates in chemical transformations at the proton transfer stage.

Dynamin is a 96 kDa protein that has multiple oligomerization states that influence its GTPase activity. A number of different dynamin effectors, including lipids, actin filaments, and SH3-domain containing proteins, have been implicated in the regulation of dynamin oligomerization, though their roles in influencing dynamin oligomerization have been studied predominantly in vitro using recombinant proteins. Here, we identify higher order dynamin oligomers such as rings and helices in vitro and in live cells using fluorescence lifetime imaging microscopy (FLIM). FLIM detected GTP- and actin-dependent dynamin oligomerization at distinct cellular sites, including the cell membrane and transition zones where cortical actin transitions into stress fibers. Our study identifies a major role for direct dynamin-actin interactions and dynamin’s GTPase activity in the regulation of dynamin oligomerization in cells.

In the present research, we have studied the inoculation effects of two root-associated plant growth-promoting rhizobacteria (PGPR) in rice and provide the pieces of evidence that the inoculation of the PGPR could potentially result in inducing the expression of the salt stress-related RAB18 plant gene under varying degrees of salinity stress. The sequenced putative gene of RAB18 of Oryza sativa in this study is 740 bp long, has a content of 44.4%, and a molecular weight of 492 102.00 Da. BLAST homology patterns revealed sequence similarity with the previously sequenced RAB in model plant species. We demonstrate the mode of action of this stress-related protein by performing comparative modeling of Q10RT8 (Os03g0146000 protein, homolog of the sequenced RAB18; O. sativa subsp. japonica) using energy minimization, molecular dynamic simulations, and molecular docking of a guanosine triphosphate (GTP) ligand with the protein. The docking results indicated that Ser21, Ala22, Lys25, Asp68, Ala70, Glu73, and Arg74 are important determinant residues for functional interaction with the GTP ligand. The present research contributes to the understanding of the PGPR inoculation in salinity stress. Additionally, it provides the layout of the understanding of the molecular interactions between RAB and GTP ligand. Copyright © 2014 John Wiley & Sons, Ltd.