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The adenosine di-phosphate (ADP) ribosylation factor (Arf) small guanosine tri-phosphate (GTP)ases function as molecular switches to activate signaling cascades that control membrane organization in eukaryotic cells. In Arf1, the GDP/GTP switch does not occur spontaneously but requires guanine nucleotide exchange factors (GEFs) and membranes. Exchange involves massive conformational changes, including disruption of the core β-sheet. The mechanisms by which this energetically costly switch occurs remain to be elucidated. To probe the switch mechanism, we coupled pressure perturbation with nuclear magnetic resonance (NMR), Fourier Transform infra-red spectroscopy (FTIR), small-angle X-ray scattering (SAXS), fluorescence, and computation. Pressure induced the formation of a classical molten globule (MG) ensemble. Pressure also favored the GDP to GTP transition, providing strong support for the notion that the MG ensemble plays a functional role in the nucleotide switch. We propose that the MG ensemble allows for switching without the requirement for complete unfolding and may be recognized by GEFs. An MG-based switching mechanism could constitute a pervasive feature in Arfs and Arf-like GTPases, and more generally, the evolutionarily related (Ras-like small GTPases) Rags and Gα GTPases., Competing Interests: Competing interests statement:The authors declare no competing interest.

SynDLP, a dynamin-like protein (DLP) encoded in the cyanobacterium Synechocystis sp. PCC 6803, has recently been identified to be structurally highly similar to eukaryotic dynamins. To elucidate structural changes during guanosine triphosphate (GTP) hydrolysis, we solved the cryoelectron microscopy (cryo-EM) structures of oligomeric full-length SynDLP after addition of guanosine diphosphate (GDP) at 4.1 Å and GTP at 3.6-Å resolution as well as a GMPPNP-bound dimer structure of a minimal G-domain construct of SynDLP at 3.8-Å resolution. In comparison with what has been seen in the previously resolved apo structure, we found that the G-domain is tilted upward relative to the stalk upon GTP hydrolysis and that the G-domain dimerizes via an additional extended dimerization domain not present in canonical G-domains. When incubated with lipid vesicles, we observed formation of irregular tubular SynDLP assemblies that interact with negatively charged lipids. Here, we provide the structural framework of a series of different functional SynDLP assembly states during GTP turnover., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Rab3A is a member of the Rab GTPase family involved in synaptic vesicle trafficking. Recent evidence has demonstrated that Rab3A is phosphorylated by leucine-rich repeat kinase 2 (LRRK2) that is implicated in both familial and sporadic forms of Parkinson's disease (PD), and an abnormal increase in Rab3A phosphorylation has been proposed as a cause of PD. Despite the potential importance of Rab3A in PD pathogenesis, its structural information is limited and the effects of bound nucleotides on its biophysical and biochemical properties remain unclear. Here, we show that GDP-bound Rab3A is preferentially phosphorylated by LRRK2 compared with GTP-bound Rab3A. The secondary structure of Rab3A, measured by circular dichroism (CD) spectroscopy, revealed that Rab3A is resistant to heat-induced denaturation at pH 7.4 or 9.0 regardless of the nucleotides bound. In contrast, Rab3A underwent heat-induced denaturation at pH 5.0 at a lower temperature in its GDP-bound form than in its GTP-bound form. The unfolding temperature of Rab3A was studied by differential scanning fluorimetry, which showed a significantly higher unfolding temperature in GTP-bound Rab3A than in GDP-bound Rab3A, with the highest at pH 7.4. These results suggest that Rab3A has unusual thermal stability under physiologically relevant conditions and that bound nucleotides influence both thermal stability and phosphorylation by LRRK2., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Genta Ito reports financial support was provided by Japan Society for the Promotion of Science. Naoko Utsunomiya-Tate reports financial support was provided by Japan Society for the Promotion of Science. Naoko Utsunomiya-Tate reports financial support was provided by Teikyo University Advanced Comprehensive Research Organization. Taisuke Tomita reports financial support was provided by Biogen Inc. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. Gα o K46E has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein βγ subunits. In cells with physiological concentrations of nucleotide, Gα o K46E forms a stable complex with receptors and Gβγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gα o , without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells., (© 2024. The Author(s).)

Dynamin assembles as a helical polymer at the neck of budding endocytic vesicles, constricting the underlying membrane as it progresses through the GTPase cycle to sever vesicles from the plasma membrane. Although atomic models of the dynamin helical polymer bound to guanosine triphosphate (GTP) analogs define earlier stages of membrane constriction, there are no atomic models of the assembled state post-GTP hydrolysis. Here, we used cryo-EM methods to determine atomic structures of the dynamin helical polymer assembled on lipid tubules, akin to necks of budding endocytic vesicles, in a guanosine diphosphate (GDP)-bound, super-constricted state. In this state, dynamin is assembled as a 2-start helix with an inner lumen of 3.4 nm, primed for spontaneous fission. Additionally, by cryo-electron tomography, we trapped dynamin helical assemblies within HeLa cells using the GTPase-defective dynamin K44A mutant and observed diverse dynamin helices, demonstrating that dynamin can accommodate a range of assembled complexes in cells that likely precede membrane fission., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Heterotrimeric GTP-binding proteins (G proteins), consisting of Gα, Gβ and Gγ subunits, transduce signals from a diverse range of extracellular stimuli, resulting in the regulation of numerous cellular and physiological functions in Eukaryotes. According to the classic G protein paradigm established in animal models, the bound guanine nucleotide on a Gα subunit, either guanosine diphosphate (GDP) or guanosine triphosphate (GTP) determines the inactive or active mode, respectively. In plants, there are two types of Gα subunits: canonical Gα subunits structurally similar to their animal counterparts and unconventional extra-large Gα subunits (XLGs) containing a C-terminal domain homologous to the canonical Gα along with an extended N-terminal domain. Both Gα and XLG subunits interact with Gβγ dimers and regulator of G protein signalling (RGS) protein. Plant G proteins are implicated directly or indirectly in developmental processes, stress responses, and innate immunity. It is established that despite the substantial overall similarity between plant and animal Gα subunits, they convey signalling differently including the mechanism by which they are activated. This review emphasizes the unique characteristics of plant Gα subunits and speculates on their unique signalling mechanisms.

By using 31 P NMR, we present evidence that the Rho family GTPase RhoA, similar to Ras GTPases, exists in an equilibrium of conformations when bound to GTP. High-resolution crystal structures of RhoA bound to the GTP analog GMPPNP and to GDP show that they display a similar overall inactive conformation. In contrast to the previously reported crystal structures of GTP analog-bound forms of two RhoA dominantly active mutants (G14V and Q63L), GMPPNP-bound RhoA assumes an open conformation in the Switch I loop with a previously unseen interaction between the γ-phosphate and Pro36, instead of the canonical Thr37. Molecular dynamics simulations found that the oncogenic RhoA G14V mutant displays a reduced flexibility in the Switch regions, consistent with a crystal structure of GDP-bound RhoA G14V . Thus, GDP- and GTP-bound RhoA can present similar inactive conformations, and the molecular dynamics in the Switch regions are likely to have a role in RhoA activation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Human tRNA His guanylyltransferase (HsThg1) catalyzes the 3'-5' addition of guanosine triphosphate (GTP) to the 5'-end (-1 position) of tRNA His , producing mature tRNA His In human cells, cytoplasmic and mitochondrial tRNA His have adenine (A) or cytidine (C), respectively, opposite to G -1 Little attention has been paid to the structural requirements of incoming GTP in 3'-5' nucleotidyl addition by HsThg1. In this study, we evaluated the incorporation efficiencies of various GTP analogs by HsThg1 and compared the reaction mechanism with that of Candida albicans Thg1 (CaThg1). HsThg1 incorporated GTP opposite A or C in the template most efficiently. In contrast to CaThg1, HsThg1 could incorporate UTP opposite A, and guanosine diphosphate (GDP) opposite C. These results suggest that HsThg1 could transfer not only GTP, but also other NTPs, by forming Watson-Crick (WC) hydrogen bonds between the incoming NTP and the template base. On the basis of the molecular mechanism, HsThg1 succeeded in labeling the 5'-end of tRNA His with biotinylated GTP. Structural analysis of HsThg1 was also performed in the presence of the mitochondrial tRNA His Structural comparison of HsThg1 with other Thg1 family enzymes suggested that the structural diversity of the carboxy-terminal domain of the Thg1 enzymes might be involved in the formation of WC base-pairing between the incoming GTP and template base. These findings provide new insights into an unidentified biological function of HsThg1 and also into the applicability of HsThg1 to the 5'-terminal modification of RNAs., (© 2021 Nakamura et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Nucleation of microtubules (MTs) is essential for cellular activities, but its mechanism is unknown because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. Here, combining rapid flush negative stain electron microscopy (EM) and kinetic analysis, we demonstrate that the formation of straight oligomers of critical size is essential for nucleation. Both GDP and GTP tubulin form single-stranded oligomers with a broad range of curvatures, but upon nucleation, the curvature distribution of GTP oligomers is shifted to produce a minor population of straight oligomers. With tubulin having the Y222F mutation in the β subunit, the proportion of straight oligomers increases and nucleation accelerates. Our results support a model in which GTP binding generates a minor population of straight oligomers compatible with lateral association and further growth to MTs. This study suggests that cellular factors involved in nucleation promote it via stabilization of straight oligomers., (© 2021 Ayukawa et al.)

Mycobacterium tuberculosis (M. tuberculosis H 37 Rv) utilizes the signal recognition particle pathway (SRP pathway) system for secretion of various proteins from ribosomes to the extracellular surface which plays an important role in the machinery running inside the bacterium. This system comprises of three major components FtsY, FfH and 4.5S rRNA. This manuscript highlights essential factors responsible for the optimized enzymatic activity of FtsY. Kinetic parameters include V max and K m for the hydrolysis of GTP by ftsY which were 20.25±5.16 μM/min/mg and 39.95±7.7 μM respectively. k cat and catalytic efficiency of the reaction were 0.012±0.003 s -1 and 0.00047±0.0001 μM/s -1 respectively. These values were affected upon changing the standard conditions. Cations (Mg 2+ and Mn 2+ ) play important role in FtsY enzymatic activity as increasing Mg 2+ decrease the activity. Mn 2+ on the other hand is required at higher concentration around 60 mM for carrying optimum GTPase activity. FtsY is hydrolyzing ATP and GDP as well and GDP acts as an inhibitor of the reaction. MD simulation shows effective binding and stabilization of the FtsY complexed structure with GTP, GDP and ATP. Mutational analysis was done at two important residues of GTP binding motif of FtsY, namely, GXXXXGK (K236) and DXXG (D367) and showed that these mutations significantly decrease FtsY GTPase activity. FtsY is comprised of α helices, but this structural pattern was shown to change with increasing concentrations of GTP and ATP which symbolize that these ligands cause significant conformational change by variating the secondary structure to transduce signals required by downstream effectors. This binding favors the functional stabilization of FtsY by destabilization of α-helix integrity. Revealing the hidden aspects of the functioning of FtsY might be an essential part for the understanding of the SRP pathway which is one of the important contributors of M. tuberculosis virulence., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Ribosome biogenesis in eukaryotes requires the participation of several transactivation factors that are involved in the modification, assembly, transport and quality control of the ribosomal subunits. One of these factors is the Large subunit GTPase 1 (Lsg1), a protein that acts as the release factor for the export adaptor named Nonsense-mediated mRNA decay 3 protein (Nmd3) and facilitates the incorporation of the last structural protein uL16 into the 60S subunit. Here, we characterised the recombinant yeast Lsg1 and studied its catalysis and binding properties for guanine nucleotides. We described the interaction of Lsg1 with guanine nucleotides alone and in the presence of the complex Nmd3•60S using fluorescence spectroscopy. Lsg1 has a greater affinity for GTP than for GDP suggesting that in the cell cytoplasm it exists mainly bound to the former. In the presence of 60S subunits loaded with Nmd3, the affinity of Lsg1 for both nucleotides increases but to a larger extent towards GTP. From this observation together with the excess of GTP present in the cytoplasm of exponentially growing cells over that of GDP, we can infer that the pre-ribosomal particle composed by Nmd3•60S acts as a GTP Stabilising Factor for Lsg1. Additionally, Lsg1 undergoes different conformational changes depending on its binding partner or the guanine nucleotides it interacts with. Steady-state kinetic analysis of free Lsg1 indicated slow GTP hydrolysis with values of k cat 1 min -1 and K m of 34 μM., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Small GTPases cycle between active GTP bound and inactive GDP bound forms in live cells. They act as molecular switches and regulate diverse cellular processes at different times and locations in the cell. Spatiotemporal visualization of their activity provides important insights into dynamics of cellular signaling. Conformational sensors for GTPase activity (COSGAs) are based on the conserved GTPase fold and have been used as a versatile approach for imaging small GTPase activity in the cell. Conformational changes upon GDP/GTP binding can be visualized directly in solution, on beads, or in live cells using COSGA by fluorescence lifetime imaging microscopy (FLIM) technique. Herein, we describe the construction of COSGA for imaging K-Ras GTPase activity in live cells.

The Ras family of proteins is known to play an important role in cellular signal transduction. The oncoprotein Ras is also found to be mutated in ~90% of the pancreatic cancers, of which G12V, G13V, A59G and Q61L are the known hot-spot mutants. These ubiquitous proteins fall in the family of G-proteins, and hence switches between active GTP bound and inactive GDP bound states, which is hindered in most of its oncogenic mutant counterparts. Moreover, Ras being a GTPase has an intrinsic property to hydrolyze GTP to GDP, which is obstructed due to mutations and lends the mutants stuck in constitutively active state leading to oncogenic behavior. In this regard, the present study aims to understand the dynamics involved in the hot-spot mutant A59G-Ras using long 10μs classical MD simulations (5μs for each of the wild-type and mutant systems) and comparing the same with its wild-type counterpart. Advanced analytics using Markov State Model (MSM) based approach has been deployed to comparatively understand the transition path for the wild-type and mutant systems. Roles of crucial residues like Tyr32, Gln61 and Tyr64 have also been established using multivariate PCA analyses. Furthermore, this multivariate PCA analysis also provides crucial features which may be used as reaction coordinates for biased simulations for further studies. The absence of formation of pre-hydrolysis network is also reported for the mutant conformation, using the distance-based analyses (between crucial residues) of the conserved regions. The implications of this study strengthen the hypothesis that the disruption of the pre-hydrolysis network in the mutant A59G ensemble might lead to permanently active oncogenic conformation in the mutant conformers., Competing Interests: The authors have declared that no competing interests exist.

Ribosomes accurately decode mRNA by proofreading each aminoacyl-tRNA that is delivered by the elongation factor EF-Tu 1 . To understand the molecular mechanism of this proofreading step it is necessary to visualize GTP-catalysed elongation, which has remained a challenge 2-4 . Here we use time-resolved cryogenic electron microscopy to reveal 33 ribosomal states after the delivery of aminoacyl-tRNA by EF-Tu•GTP. Instead of locking cognate tRNA upon initial recognition, the ribosomal decoding centre dynamically monitors codon-anticodon interactions before and after GTP hydrolysis. GTP hydrolysis enables the GTPase domain of EF-Tu to extend away, releasing EF-Tu from tRNA. The 30S subunit then locks cognate tRNA in the decoding centre and rotates, enabling the tRNA to bypass 50S protrusions during accommodation into the peptidyl transferase centre. By contrast, the decoding centre fails to lock near-cognate tRNA, enabling the dissociation of near-cognate tRNA both during initial selection (before GTP hydrolysis) and proofreading (after GTP hydrolysis). These findings reveal structural similarity between ribosomes in initial selection states 5,6 and in proofreading states, which together govern the efficient rejection of incorrect tRNA.

Budding yeast septins are essential for cell division and polarity. Septins assemble as palindromic linear octameric complexes. The function and ultra-structural organization of septins are finely governed by their molecular polymorphism. In particular, in budding yeast, the end subunit can stand either as Shs1 or Cdc11. We have dissected, here, for the first time, the behavior of the Shs1 protomer bound to membranes at nanometer resolution, in complex with the other septins. Using electron microscopy, we have shown that on membranes, Shs1 protomers self-assemble into rings, bundles, filaments or two-dimensional gauzes. Using a set of specific mutants we have demonstrated a synergistic role of both nucleotides and lipids for the organization and oligomerization of budding yeast septins. Besides, cryo-electron tomography assays show that vesicles are deformed by the interaction between Shs1 oligomers and lipids. The Shs1-Shs1 interface is stabilized by the presence of phosphoinositides, allowing the visualization of micrometric long filaments formed by Shs1 protomers. In addition, molecular modeling experiments have revealed a potential molecular mechanism regarding the selectivity of septin subunits for phosphoinositide lipids., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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 spectroscopy, 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, favors 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 platform to elucidate the structural basis of RAF activation by RAS and to develop inhibitors that can disrupt the KRAS dimerization. The methodology is applicable to many other farnesylated small GTPases., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bacterial cytokinesis is mediated by the Z-ring, which is formed by the prokaryotic tubulin homolog FtsZ. Recent data indicate that the Z-ring is composed of small patches of FtsZ protofilaments that travel around the bacterial cell by treadmilling. Treadmilling involves a switch from a relaxed (R) state, favored for monomers, to a tense (T) conformation, which is favored upon association into filaments. The R conformation has been observed in numerous monomeric FtsZ crystal structures and the T conformation in Staphylococcus aureus FtsZ crystallized as assembled filaments. However, while Escherichia coli has served as a main model system for the study of the Z-ring and the associated divisome, a structure has not yet been reported for E. coli FtsZ. To address this gap, structures were determined of the E. coli FtsZ mutant FtsZ(L178E) with GDP and GTP bound to 1.35 and 1.40 Å resolution, respectively. The E. coli FtsZ(L178E) structures both crystallized as straight filaments with subunits in the R conformation. These high-resolution structures can be employed to facilitate experimental cell-division studies and their interpretation in E. coli., (open access.)

Membrane dynamic processes require Arf GTPase activation by guanine nucleotide exchange factors (GEFs) with a Sec7 domain. Cytohesin family Arf GEFs function in signaling and cell migration through Arf GTPase activation on the plasma membrane and endosomes. In this study, the structural organization of two cytohesins (Grp1 and ARNO) was investigated in solution by size exclusion-small angle X-ray scattering and negative stain-electron microscopy and on membranes by dynamic light scattering, hydrogen-deuterium exchange-mass spectrometry and guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange assays. The results suggest that cytohesins form elongated dimers with a central coiled coil and membrane-binding pleckstrin-homology (PH) domains at opposite ends. The dimers display significant conformational heterogeneity, with a preference for compact to intermediate conformations. Phosphoinositide-dependent membrane recruitment is mediated by one PH domain at a time and alters the conformational dynamics to prime allosteric activation by Arf-GTP. A structural model for membrane targeting and allosteric activation of full-length cytohesin dimers is discussed., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Translation elongation factor EF1A delivers aminoacyl-tRNA to the ribosome in a GTP-bound form, and is released from the ribosome in a GDP-bound form. This association/dissociation cycle proceeds efficiently via a marked conformational change in EF1A. EF1A function is dependent on the ribosomal "stalk" protein of the ribosomal large subunit, although the precise mechanism of action of the stalk on EF1A remains unclear. Here, we clarify the binding mode of archaeal stalk aP1 to GTP-bound aEF1A associated with aPelota. Intriguingly, the C-terminal domain (CTD) of aP1 binds to aEF1A•GTP with a similar affinity to aEF1A•GDP. We have also determined the crystal structure of the aP1-CTD•aEF1A•GTP•aPelota complex at 3.0 Å resolution. The structure shows that aP1-CTD binds to a space between domains 1 and 3 of aEF1A. Biochemical analyses show that this binding is crucial for protein synthesis. Comparison of the structures of aP1-CTD•aEF1A•GTP and aP1-CTD•aEF1A•GDP demonstrates that the binding mode of aP1 changes markedly upon a conformational switch between the GTP- and GDP-bound forms of aEF1A. Taking into account biochemical data, we infer that aP1 employs its structural flexibility to bind to aEF1A before and after GTP hydrolysis for efficient protein synthesis.

Cell signaling by small G proteins uses an ON to OFF signal based on conformational changes following the hydrolysis of GTP to GDP and release of dihydrogen phosphate (P i ). The catalytic mechanism of GTP hydrolysis by RhoA is strongly accelerated by a GAP protein and is now well defined, but timing of inorganic phosphate release and signal change remains unresolved. We have generated a quaternary complex for RhoA-GAP-GDP-P i . Its 1.75 Å crystal structure shows geometry for ionic and hydrogen bond coordination of GDP and P i in an intermediate state. It enables the selection of a QM core for DFT exploration of a 20 H-bonded network. This identifies serial locations of the two mobile protons from the original nucleophilic water molecule, showing how they move in three rational steps to form a stable quaternary complex. It also suggests how two additional proton transfer steps can facilitate P i release., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ras superfamily GTPases are molecular switches that cycle between GDP-bound inactive state and GTP-bound active state to control many signaling pathways. Emerging evidence suggests that several Ras superfamily GTPases, including RhoF, do not follow the classical GDP/GTP exchange cycle; they act as constitutively active GTP-bound proteins due to their fast activities of GDP/GTP exchange (termed as 'fast-cycling' GTPases). To understand the molecular basis of the fast-cycling GTPases, we generated a GTPase active recombinant RhoF and examined its function and structure. Two point mutations in the switch I/II regions (Q77L and P45S, corresponding to Q61L and P29S of Rac1) significantly reduced the GTPase activity of RhoF, suggesting a conserved mechanism of GTP hydrolysis between RhoF and other RAS superfamily GTPases. However, in contrary to the previous evidence, RhoF represented a slow GDP/GTP exchange activity that dissociates GDP very slowly on a day-to-week time scale, in our experiment using fluorescently labeled GDP. The slow GDP dissociation was accelerated by Mg 2+ chelation and canonical fast-cycling mutations, F44L (corresponding to F28L of Rac1) and P45S. NMR and dynamic light scattering data revealed a multimeric structure of RhoF that can switch between different conformations depending on the GTP/GDP-bound state. Overall, our study suggests that (1) RhoF shares a conserved mechanism of GTP hydrolysis with other RAS superfamily GTPases, but (2) RhoF adopts a unique multimeric structure. Our study also argues that (3) the emerging concept of the fast-cycling GTPases for RhoF should be validated using an alternative assay that does not rely on fluorescently labeled GDP (251 words)., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Intraflagellar transport (IFT) relies on motor proteins and the IFT complex to construct cilia and flagella. The IFT complex subunit IFT22/RabL5 has sequence similarity with small GTPases although the nucleotide specificity is unclear because of non-conserved G4/G5 motifs. We show that IFT22 specifically associates with G-nucleotides and present crystal structures of IFT22 in complex with GDP, GTP, and with IFT74/81. Our structural analysis unravels an unusual GTP/GDP-binding mode of IFT22 bypassing the classical G4 motif. The GTPase switch regions of IFT22 become ordered upon complex formation with IFT74/81 and mediate most of the IFT22-74/81 interactions. Structure-based mutagenesis reveals that association of IFT22 with the IFT complex is essential for flagellum construction in Trypanosoma brucei although IFT22 GTP-loading is not strictly required., (© 2019 The Authors.)

Ran is an evolutionarily conserved GTPase crucial in regulating various cell divisions, including mitosis and meiosis. A previous study showed that the knockdown of RAN1 inhibited macronuclear amitosis with the abnormal organization of intramacronuclear microtubules in Tetrahymena thermophila. This study aimed to further investigate the effects of the inducible expression of wild-type Ran1 (Ran1WT), GTP-bound Ran1-mimetic (Ran1Q70L), and GDP-bound Ran1-mimetic (Ran1T25N) on cytoplasmic microtubule assembly during amitosis of T. thermophila, based on previous studies about their effects on intramacronuclear microtubule. The mutant strains of T. thermophila for inducible expression of Ran1WT/T25N/Q70L by Cd^(2+) were constructed. The inducibly expressed HA-Ran1Q70L/T25N distributed asymmetrically across the macronuclear envelope during amitosis. At the lower level of inducible expression, only Ran1T25N showed a significant decreasing effect on T. thermophila reproduction, macronuclear amitosis and cytokinesis. At the higher level of inducible expression, Ran1WT/Q70L/T25N inhibited T. thermophila reproduction, macronuclear amitosis and cytokinesis, and the inhibitive effect of Ran1T25N was the most significant. The inducible expression of Ran1WT/Q70L/T25N led to defects in amitosis and cytokinesis with abnormal cytoplasmic microtubule assembly. These results further confirmed the regulatory function of Ran1 on amitosis and suggested a novel role of Ran1 in cytokinesis and the alignment of cytoplasmic microtubules in T. thermophila.

Ras-related C3 botulinum toxin substrate 1 (Rac1) plays critical roles in the maintenance of cell morphology by cycling between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states. Rac1 P29S mutant is known to strongly promote oncogenesis by facilitating its intrinsic GDP dissociation and thereby increasing the level of the GTP-bound state. Here, we used solution nuclear magnetic resonance spectroscopy to investigate the activation mechanism of the oncogenic P29S mutant. We demonstrate that the conformational landscape is markedly altered in the mutant, and the preexisting equilibrium is shifted toward the conformation with reduced affinity for Mg 2+ , a cofactor that is critical for maintaining stable GDP binding. Our results suggest that the alternation of the preexisting conformational equilibrium of proteins is one of the fundamental mechanisms underlying their oncogenic activities.

Single and double tubulin rings were studied under a range of conditions and during microtubule (MT) assembly and disassembly. Here, tubulin was purified from porcine brain and used without any further modifications or additives that promote ring assembly. The structure of single GDP-rich tubulin rings was determined by cryo-transmission electron microscopy and synchrotron solution X-ray scattering. The scattering curves were fitted to atomic models, using our state-of-the-art analysis software, D+ . We found that there is a critical concentration for ring formation, which increased with GTP concentration with temperature. MT assembly or disassembly, induced by changes in temperature, was analyzed by time-resolved small-angle X-ray scattering. During MT assembly, the fraction of rings and unassembled dimers simultaneously decreased. During MT disassembly, the mass fraction of dimers increased. The increase in the concentration of rings was delayed until the fraction of dimers was sufficiently high. We verified that pure dimers, eluted via size-exclusion chromatography, could also form rings. Interestingly, X-ray radiation triggered tubulin ring disassembly. The concentration of disassembled rings versus exposure time followed a first-order kinetics. The disassembly rate constant and initial concentration were determined. X-ray radiation-triggered disassembly was used to determine the concentration of rings. We confirmed that following a temperature jump, the mass fraction of rings decreased and then stabilized at a constant value during the first stage of the MT assembly kinetics. This study sheds light on the most basic assembly and disassembly conditions for in vitro single GDP-rich tubulin rings and their relation to MT kinetics.

The Ras family of small guanine nucleotide-binding proteins behave as molecular switches: they are switched off and inactive when bound to GDP but can be activated by GTP binding in response to signal transduction pathways. Early structural analysis showed that two regions of the protein, which change conformation depending on the nucleotide present, mediate this switch. A large number of X-ray, NMR and simulation studies have shown that this is an over-simplification. The switch regions themselves are highly dynamic and can exist in distinct sub-states in the GTP-bound form that have different affinities for other proteins. Furthermore, regions outside the switches have been found to be sensitive to the nucleotide state of the protein, indicating that allosteric change is more widespread than previously thought. Taken together, the accrued knowledge about small G protein structures, allostery and dynamics will be essential for the design and testing of the next generation of inhibitors, both orthosteric and allosteric, as well as for understanding their mode of action., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Constitutively active G protein α subunits cause cancer, cholera, Sturge-Weber syndrome, and other disorders. Therapeutic intervention by targeted inhibition of constitutively active Gα subunits in these disorders has yet to be achieved. We found that constitutively active Gα q in uveal melanoma (UM) cells was inhibited by the cyclic depsipeptide FR900359 (FR). FR allosterically inhibited guanosine diphosphate-for-guanosine triphosphate (GDP/GTP) exchange to trap constitutively active Gα q in inactive, GDP-bound Gαβγ heterotrimers. Allosteric inhibition of other Gα subunits was achieved by the introduction of an FR-binding site. In UM cells driven by constitutively active Gα q , FR inhibited second messenger signaling, arrested cell proliferation, reinstated melanocytic differentiation, and stimulated apoptosis. In contrast, FR had no effect on BRAF -driven UM cells. FR promoted UM cell differentiation by reactivating polycomb repressive complex 2 (PRC2)-mediated gene silencing, a heretofore unrecognized effector system of constitutively active Gα q in UM. Constitutively active Gα q and PRC2 therefore provide therapeutic targets for UM. The development of FR analogs specific for other Gα subunit subtypes may provide novel therapeutic approaches for diseases driven by constitutively active Gα subunits or multiple G protein-coupled receptors (GPCRs) where targeting a single receptor is ineffective., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Rotavirus infection has emerged as an important cause of complications in organ transplantation recipients and might play a role in the pathogenesis of inflammatory bowel disease (IBD). 6-Thioguanine (6-TG) has been widely used as an immunosuppressive drug for organ recipients and treatment of IBD in the clinic. This study aims to investigate the effects and mode-of-action of 6-TG on rotavirus replication. Human intestinal Caco2 cell line, 3D model of human primary intestinal organoids, laboratory rotavirus strain (SA11) and patient-derived rotavirus isolates were used. We have demonstrated that 6-TG significantly inhibits rotavirus replication in these intestinal epithelium models. Importantly, gene knockdown or knockout of Rac1, the cellular target of 6-TG, significantly inhibited rotavirus replication, indicating the supportive role of Rac1 for rotavirus infection. We have further demonstrated that 6-TG can effectively inhibit the active form of Rac1 (GTP-Rac1), which essentially mediates the anti-rotavirus effect of 6-TG. Consistently, ectopic over-expression of GTP-Rac1 facilitates but an inactive Rac1 (N17) or a specific Rac1 inhibitor (NSC23766) inhibits rotavirus replication. In conclusion, we have identified 6-TG as an effective inhibitor of rotavirus replication via the inhibition of Rac1 activation. Thus, for transplantation patients or IBD patients infected with rotavirus or at risk of rotavirus infection, the choice of 6-TG as a treatment appears rational., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

The atypical Rho GTPase RhoD has previously been shown to have a major impact on the organization and function of the actin filament system. However, when first discovered, RhoD was found to regulate endosome trafficking and dynamics and we therefore sought to investigate this regulation in more detail. We found that exogenously expressed RhoD in human fibroblasts localized to vesicles and the plasma membrane and that the active GTP-bound conformation was required for the plasma membrane localization but not for vesicle localization. In contrast to the GTPase deficient atypical Rho GTPases, which have a stalled GTPase activity, RhoD has an elevated intrinsic GDP/GTP exchange activity, rendering the protein constitutively active. Importantly, RhoD can still hydrolyze GTP and we found that an intact GTPase activity was required for efficient fusion of RhoD-positive vesicles. RhoD has a unique N-terminal extension of 14 amino acid residues, which is not present in the classical Rho GTPases RhoA, Cdc42 and Rac1. Deletion of this N-terminal motif often lead to clustering of RhoD positive vesicles, which were found accumulated at the peripheral membrane border. In addition, the number of vesicles per cell was increased manifold, suggesting that the N-terminal motif has an important regulatory role in vesicle dynamics., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Protein prenylation involves the attachment of a hydrophobic isoprenoid moiety to the C-terminus of proteins. Several small GTPases, including members of the Ras and Rho subfamilies, require prenylation for their normal and pathological functions. Recent work has suggested that SmgGDS proteins regulate the prenylation of small GTPases in vivo. Using RhoA as a representative small GTPase, we directly test this hypothesis using biochemical assays and present a mechanism describing the mode of prenylation regulation. SmgGDS-607 completely inhibits RhoA prenylation catalyzed by protein geranylgeranyltransferase I (GGTase-I) in an in vitro radiolabel incorporation assay. SmgGDS-607 inhibits prenylation by binding to and blocking access to the C-terminal tail of the small GTPase (substrate sequestration mechanism) rather than via inhibition of the prenyltransferase activity. The reactivity of GGTase-I with RhoA is unaffected by addition of nucleotides. In contrast, the affinity of SmgGDS-607 for RhoA varies with the nucleotide bound to RhoA; SmgGDS-607 has a higher affinity for RhoA-GDP compared to RhoA-GTP. Consequently, the prenylation blocking function of SmgGDS-607 is regulated by the bound nucleotide. This work provides mechanistic insight into a novel pathway for the regulation of small GTPase protein prenylation by SmgGDS-607 and demonstrates that peptides are a good mimic for full-length proteins when measuring GGTase-I activity.

At the molecular level, the dynamic instability (random growth and shrinkage) of the microtubule (MT) is driven by the nucleotide state (GTP vs GDP) in the β subunit of the tubulin dimers at the MT cap. Here, we use large-scale molecular dynamics (MD) simulations and normal-mode analysis (NMA) to characterize the effect of a single GTP cap layer on tubulin octamers composed of two neighboring protofilaments (PFs). We utilize recently reported high-resolution structures of dynamic MTs to simulate a GDP octamer both with and without a single GTP cap layer. We perform multiple replicas of long-time atomistic MD simulations (3 replicas, 0.3 μs for each replica, 0.9 μs for each octamer system, and 1.8 μs total) of both octamers. We observe that a single GTP cap layer induces structural differences in neighboring PFs, finding that one PF possesses a gradual curvature, compared to the second PF which possesses a kinked conformation. This results in either curling or splaying between these PFs. We suggest that this is due to asymmetric strengths of longitudinal contacts between the two PFs. Furthermore, using NMA, we calculate mechanical properties of these octamer systems and find that octamer system with a single GTP cap layer possesses a lower flexural rigidity.

The single most frequent cancer-causing mutation across all heterotrimeric G proteins is R201C in Gαs. The current model explaining the gain-of-function activity of the R201 mutations is through the loss of GTPase activity and resulting inability to switch off to the GDP state. Here, we find that the R201C mutation can bypass the need for GTP binding by directly activating GDP-bound Gαs through stabilization of an intramolecular hydrogen bond network. Having found that a gain-of-function mutation can convert GDP into an activator, we postulated that a reciprocal mutation might disrupt the normal role of GTP. Indeed, we found R228C, a loss-of-function mutation in Gαs that causes pseudohypoparathyroidism type 1a (PHP-Ia), compromised the adenylyl cyclase-activating activity of Gαs bound to a non-hydrolyzable GTP analog. These findings show that disease-causing mutations in Gαs can subvert the canonical roles of GDP and GTP, providing new insights into the regulation mechanism of G proteins., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Microtubules (MTs) are key components of the cytoskeleton and play a central role in cell division and development. MT assembly is known to be associated with a structural change in [Formula: see text]-tubulin dimers from kinked to straight conformations. How GTP binding renders individual dimers polymerization-competent, however, is still unclear. Here, we have characterized the conformational dynamics and energetics of unassembled tubulin using atomistic molecular dynamics and free energy calculations. Contrary to existing allosteric and lattice models, we find that GTP-tubulin favors a broad range of almost isoenergetic curvatures, whereas GDP-tubulin has a much lower bending flexibility. Moreover, irrespective of the bound nucleotide and curvature, two conformational states exist differing in location of the anchor point connecting the monomers that affects tubulin bending, with one state being strongly favored in solution. Our findings suggest a new combined model in which MTs incorporate and stabilize flexible GTP-dimers with a specific anchor point state., Competing Interests: MI, HG No competing interests declared, (© 2018, Igaev et al.)

RAS mutations are found in 30% of all human cancers, with KRAS the most frequently mutated among the three RAS isoforms (KRAS, NRAS, and HRAS). However, directly targeting oncogenic KRAS with small molecules in the nucleotide-binding site has been difficult because of the high affinity of KRAS for GDP and GTP. We designed an engineered allele of KRAS and a covalent inhibitor that competes for GTP and GDP. This ligand-receptor combination demonstrates that the high affinity of GTP and GDP for RAS proteins can be overcome with a covalent inhibitor and a suitably engineered binding site. The covalent inhibitor irreversibly modifies the protein at the engineered nucleotide-binding site and is able to compete with GDP and GTP. This provides a new tool for studying KRAS function and suggests strategies for targeting the nucleotide-binding site of oncogenic RAS proteins.

Covalent inhibitors of K-Ras(G12C) have been reported that exclusively recognize the GDP state. Here, we utilize disulfide tethering of a non-natural cysteine (K-Ras(M72C)) to identify a new switch-II pocket (S-IIP) binding ligand (2C07) that engages the active GTP state. Co-crystal structures of 2C07 bound to H-Ras(M72C) reveal binding in a cryptic groove we term S-IIG. In the GppNHp state, 2C07 binding to a modified S-IIP pushes switch I away from the nucleotide, breaking the network of polar contacts essential for adopting the canonical GTP state. Biochemical studies show that 2C07 alters nucleotide preference and inhibits SOS binding and catalyzed nucleotide exchange. 2C07 was converted to irreversible covalent analogs, which target both nucleotide states, inhibit PI3K activation in vitro, and function as occupancy probes to detect reversible engagement in competition assays. Targeting both nucleotide states opens the possibility of inhibiting oncogenic mutants of Ras, which exist predominantly in the GTP state in cells., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

K-Ras, a molecular switch that regulates cell growth, apoptosis and metabolism, is activated when it undergoes a conformation change upon binding GTP and is deactivated following the hydrolysis of GTP to GDP. Hydrolysis of GTP in water is accelerated by coordination to K-Ras, where GTP adopts a high-energy conformation approaching the transition state. The G12A mutation reduces intrinsic K-Ras GTP hydrolysis by an unexplained mechanism. Here, crystal structures of G12A K-Ras in complex with GDP, GTP, GTPγS and GppNHp, and of Q61A K-Ras in complex with GDP, are reported. In the G12A K-Ras-GTP complex, the switch I region undergoes a significant reorganization such that the Tyr32 side chain points towards the GTP-binding pocket and forms a hydrogen bond to the GTP γ-phosphate, effectively stabilizing GTP in its precatalytic state, increasing the activation energy required to reach the transition state and contributing to the reduced intrinsic GTPase activity of G12A K-Ras mutants.

ZipA protein from Escherichia coli is one of the essential components of the division proto-ring that provides membrane tethering to the septation FtsZ protein. A sedimentation assay was used to measure the equilibrium binding of FtsZ-GDP and FtsZ-GTP to ZipA immobilized at controlled densities on the surface of microbeads coated with a phospholipid mixture resembling the composition of E. coli membrane. We found that for both nucleotide-bound species, the amount of bound FtsZ exceeds the monolayer capacity of the ZipA immobilized beads at high concentrations of free FtsZ. In the case of FtsZ-GDP, equilibrium binding does not appear to be saturable, whereas in the case of FtsZ-GTP equilibrium binding appears to be saturable. The difference between the two modes of binding is attributed to the difference between the composition of oligomers of free FtsZ-GDP and free FtsZ-GTP formed in solution.

Amyotrophic Lateral Sclerosis and Frontotemporal Dementia (ALS-FTD) are devastating neurodegenerative disease affecting motoneurons from the spinal chord and neurons from the frontal and temporal cortex, respectively. The most common genetic cause for ALS-FTD is an expansion of GGGGCC repeats within the first intron of the C9ORF72 gene. However, little is known on the function of C9ORF72. Recently, other and we found that C9ORF72 forms a stable complex with the SMCR8 and WDR41 proteins. This complex acts as a GDP/GTP exchange factor for the small RAB GTPases Rab8a and Rab39b. Since Rab8 and Rab39 are involved in macroautophagy, we tested the role of C9ORF72 in this mechanism. Decrease expression of C9ORF72 in neuronal cultures leads to autophagy dysfunction characterized by accumulation of aggregates of p62/SQSTM1. However, loss of C9ORF72 expression does not cause major neuronal cell death, suggesting that a second stress may be required to promote cell toxicity. Intermediate size of polyglutamine repeats within Ataxin-2 (ATXN2) is an important genetic modifier of ALS-FTD. We found that decrease expression of C9ORF72 synergizes the toxicity and aggregation of ATXN2 with intermediate size of polyglutamine (30Q). Overall, our data suggest that reduce expression of C9ORF72 causes suboptimal autophagy that sensitizes neurons to a second stress. These data suggest that reduce expression of C9ORF72 may partly contribute to ALS-FTD pathogenesis.

Simple reversible competitive inhibition of nucleotide binding of GTP to Ras family GTPases has long been recognized as an unlikely approach to manipulating the activity of such proteins for experimental or therapeutic purposes. This is due to the high affinity of GTP to GTPases coupled with high cellular GTP concentrations, but also to problems of specificity for the highly conserved binding sites in GTPases. A recent approach suggested that these problems might be overcome by using GDP derivatives that can undergo a covalent reaction with disease specific mutants, in particular addressing inhibition of KRas G12C using GDP equipped with an electrophilic group at the β-phosphate. We show here that a major drawback to this approach is a loss of reversible affinity of such β-modified derivatives for Ras of at least 10 4 compared to GTP and GDP. With the help of a thorough kinetic characterization, we show that this leads to covalent reaction times that are too slow to make the compounds attractive for intracellular use, but that generation of a hypothetical reactive GDP derivative that retains the high reversible affinity of GDP/GTP to Ras might be a viable alternative.

Gluten-induced aggregation of K562 cells represents an in vitro model reproducing the early steps occurring in the small bowel of celiac patients exposed to gliadin. Despite the clear involvement of TG2 in the activation of the antigen-presenting cells, it is not yet clear in which compartment it occurs. Herein we study the calcium-dependent aggregation of these cells, using either cell-permeable or cell-impermeable TG2 inhibitors. Gluten induces efficient aggregation when calcium is absent in the extracellular environment, while TG2 inhibitors do not restore the full aggregating potential of gluten in the presence of calcium. These findings suggest that TG2 activity is not essential in the cellular aggregation mechanism. We demonstrate that gluten contacts the cells and provokes their aggregation through a mechanism involving the A-gliadin peptide 31-43. This peptide also activates the cell surface associated extracellular TG2 in the absence of calcium. Using a bioinformatics approach, we identify the possible docking sites of this peptide on the open and closed TG2 structures. Peptide docks with the closed TG2 structure near to the GTP/GDP site, by establishing molecular interactions with the same amino acids involved in stabilization of GTP binding. We suggest that it may occur through the displacement of GTP, switching the TG2 structure from the closed to the active open conformation. Furthermore, docking analysis shows peptide binding with the β-sandwich domain of the closed TG2 structure, suggesting that this region could be responsible for the different aggregating effects of gluten shown in the presence or absence of calcium. We deduce from these data a possible mechanism of action by which gluten makes contact with the cell surface, which could have possible implications in the celiac disease onset.

The Fe-type nitrile hydratase activator protein from Rhodococcus equi TG328-2 (ReNHase TG328-2) was successfully expressed and purified. Sequence analysis and homology modeling suggest that it is a G3E P-loop guanosine triphosphatase (GTPase) within the COG0523 subfamily. Kinetic studies revealed that the Fe-type activator protein is capable of hydrolyzing GTP to GDP with a k cat value of 1.2 × 10 -3  s -1 and a K m value of 40 μM in the presence of 5 mM MgCl 2 in 50 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid at a pH of 8.0. The addition of divalent metal ions, such as Co(II), which binds to the ReNHase TG328-2 activator protein with a K d of 2.9 μM, accelerated the rate of GTP hydrolysis, suggesting that GTP hydrolysis is potentially connected to the proposed metal chaperone function of the ReNHase TG328-2 activator protein. Circular dichroism data reveal a significant conformational change upon the addition of GTP, which may be linked to the interconnectivity of the cofactor binding sites, resulting in an activator protein that can be recognized and can bind to the NHase α-subunit. A combination of these data establishes, for the first time, that the ReNHase TG328-2 activator protein falls into the COG0523 subfamily of G3E P-loop GTPases, many of which play a role in metal homeostasis processes., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

G proteins are part of the G-protein-coupled receptor (GPCR) signal transduction cascade in which they transfer a signal from the membrane-embedded GPCR to other proteins in the cell. In the case of the inhibitory G-protein heterotrimer, permanent N-terminal myristoylation can transiently localize the Gα i subunit at the membrane as well as crucially influence Gα i 's function in the GTP-bound conformation. The attachment of lipids to proteins is known to be essential for membrane trafficking; however, our results suggest that lipidation is also important for protein-protein interactions during signal transduction. Here we investigate the effect of myristoylation on the structure and dynamics of soluble Gα i1 and its possible implication for signal transduction. A 2 μs classical molecular dynamics simulation of a myristoylated Gα i1 -GTP complex suggests that the myristoyl-induced conformational changes of the switch II and alpha helical domains create new possibilities for protein-protein interactions and emphasize the importance of permanent lipid attachment for the conformation and functional tunability of signaling proteins.

Mutations in the human leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of hereditary Parkinson's disease (PD). LRRK2 belongs to the Roco family of proteins, which are characterized by the presence of a Ras of complex proteins domain (Roc), a C-terminal of Roc domain (COR) and a kinase domain. Despite intensive research, much remains unknown about activity and the effect of PD-associated mutations. Recent biochemical and structural studies suggest that LRRK2 and Roco proteins are noncanonical G-proteins that do not depend on guanine nucleotide exchange factors or GTPase-activating proteins for activation. In this review, we will discuss the unusual G-protein cycle of LRRK2 in the context of the complex intramolecular LRRK2 activation mechanism., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Septins are GTP-binding proteins that are highly conserved among eukaryotes and which are usually membrane-associated. They have been linked to several critical cellular functions such as exocytosis and ciliogenesis, but little mechanistic detail is known. Their assembly into filaments and membrane binding properties are incompletely understood and that is specially so for non-human septins where such information would offer therapeutic potential. In this study we use Schistosoma mansoni, exhibiting just four septin genes, as a simpler model for characterizing the septin structure and organization. We show that the biochemical and biophysical proprieties of its SmSEPT5 and SmSEPT10 septins are consistent with their human counterparts of subgroups SEPT2 and SEPT6, respectively. By succeeding to isolate stable constructs comprising distinct domains of SmSEPT5 and SmSEPT10 we were able to infer the influence of terminal interfaces in the oligomerization and membrane binding properties. For example, both proteins tended to form oligomers interacting by the N- and C-terminal interfaces in a nucleotide independent fashion but form heterodimers via the G interface, which are nucleotide dependent. Furthermore, we report for the first time that it is the C-terminus of SmSETP10, rather than the N-terminal polybasic region found in other septins, that mediates its binding to liposomes. Upon binding we observe formation of discrete lipo-protein clusters and higher order septin structures, making our system an exciting model to study interactions of septins with biological membranes., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the α5 helix of Gαi1, especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in Gαi1 by inserting 4 alanine amino acids into the α5 helix between residues Gln(333) and Phe(334) (Ins4A). This extends the length of the α5 helix without disturbing the β6-α5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His(57), Phe(189), Phe(191), and Phe(336); it also disturbs the rotation of the α5 helix and the π-π interaction between His(57) and Phe(189) In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between α5, α1, and β2-β3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the α5 helix for G protein release from the receptor after GTP binding., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein-coupled receptor (GPCR) activation. Agonist-receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape.

Is nucleotide exchange sufficient to activate K-Ras4B? To signal, oncogenic rat sarcoma (Ras) anchors in the membrane and recruits effectors by exposing its effector lobe. With the use of NMR and molecular dynamics (MD) simulations, we observed that in solution, farnesylated guanosine 5'-diphosphate (GDP)-bound K-Ras4B is predominantly autoinhibited by its hypervariable region (HVR), whereas the GTP-bound state favors an activated, HVR-released state. On the anionic membrane, the catalytic domain adopts multiple orientations, including parallel (∼180°) and perpendicular (∼90°) alignments of the allosteric helices, with respect to the membrane surface direction. In the autoinhibited state, the HVR is sandwiched between the effector lobe and the membrane; in the active state, with membrane-anchored farnesyl and unrestrained HVR, the catalytic domain fluctuates reinlessly, exposing its effector-binding site. Dimerization and clustering can reduce the fluctuations. This achieves preorganized, productive conformations. Notably, we also observe HVR-autoinhibited K-Ras4B-GTP states, with GDP-bound-like orientations of the helices. Thus, we propose that the GDP/GTP exchange may not be sufficient for activation; instead, our results suggest that the GDP/GTP exchange, HVR sequestration, farnesyl insertion, and orientation/localization of the catalytic domain at the membrane conjointly determine the active or inactive state of K-Ras4B. Importantly, K-Ras4B-GTP can exist in active and inactive states; on its own, GTP binding may not compel K-Ras4B activation.-Jang, H., Banerjee, A., Chavan, T. S, Lu, S., Zhang, J., Gaponenko, V., Nussinov, R. The higher level of complexity of K-Ras4B activation at the membrane., (© FASEB.)

The small GTPases from the rat sarcoma (Ras) superfamily are a heterogeneous group of proteins of about 21 kDa that act as molecular switches, modulating cell signaling pathways and controlling diverse cellular processes. They are active when bound to guanosine triphosphate (GTP) and inactive when bound to guanosine diphosphate (GDP). Ras homolog enriched in brain (Rheb) is a member of the Ras GTPase superfamily and a key activator of the mammalian/mechanistic target of rapamycin complex 1 (mTORC1). We recently determined that microspherule protein 1 (MCRS1) maintains Rheb at lysosomal surfaces in an amino acid-dependent manner. MCRS1 depletion promotes the formation of the GDP-bound form of Rheb, which is then delocalized from the lysosomal platform and transported to endocytic recycling vesicles, leading to mTORC1 inactivation. During this delocalization process, Rheb-GDP remains farnesylated and associated with cellular endomembranes. These findings provide new insights into the regulation of small GTPases, whose activity depends on both their GTP/GDP switch state and their capacity to move between different cellular membrane-bound compartments. Dynamic spatial transport between compartments makes it possible to alter the proximity of small GTPases to their activatory sites depending on the prevailing physiological and cellular conditions.

Introduction: Nucleoside diphosphate kinase (NDK), conserved across bacteria to humans, synthesises NTP from NDP and ATP. The eukaryotic homologue, the NDPK, uses ATP to phosphorylate the tubulin-bound GDP to GTP for tubulin polymerisation. The bacterial cytokinetic protein FtsZ, which is the tubulin homologue, also uses GTP for polymerisation. Therefore, we examined whether NDK can interact with FtsZ to convert FtsZ-bound GDP and/or free GDP to GTP to trigger FtsZ polymerisation., Methods: Recombinant and native NDK and FtsZ proteins of Mycobacterium smegmatis and Mycobacterium tuberculosis were used as the experimental samples. FtsZ polymersation was monitored using 90° light scattering and FtsZ polymer pelleting assays. The γ32P-GTP synthesised by NDK from GDP and γ32P-ATP was detected using thin layer chromatography and quantitated using phosphorimager. The FtsZ bound 32P-GTP was quantitated using phosphorimager, after UV-crosslinking, followed by SDS-PAGE. The NDK-FtsZ interaction was determined using Ni2+-NTA-pulldown assay and co-immunoprecipitation of the recombinant and native proteins in vitro and ex vivo, respectively., Results: NDK triggered instantaneous polymerisation of GDP-precharged recombinant FtsZ in the presence of ATP, similar to the polymerisation of recombinant FtsZ (not GDP-precharged) upon the direct addition of GTP. Similarly, NDK triggered polymerisation of recombinant FtsZ (not GDP-precharged) in the presence of free GDP and ATP as well. Mutant NDK, partially deficient in GTP synthesis from ATP and GDP, triggered low level of polymerisation of MsFtsZ, but not of MtFtsZ. As characteristic of NDK's NTP substrate non-specificity, it used CTP, TTP, and UTP also to convert GDP to GTP, to trigger FtsZ polymerisation. The NDK of one mycobacterial species could trigger the polymerisation of the FtsZ of another mycobacterial species. Both the recombinant and the native NDK and FtsZ showed interaction with each other in vitro and ex vivo, alluding to the possibility of direct phosphorylation of FtsZ-bound GDP by NDK., Conclusion: Irrespective of the bacterial species, NDK interacts with FtsZ in vitro and ex vivo and, through the synthesis of GTP from FtsZ-bound GDP and/or free GDP, and ATP (CTP/TTP/UTP), triggers FtsZ polymerisation. The possible biological context of this novel activity of NDK is presented.