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The intrinsically disordered, lipid-modified membrane anchor of small GTPases is emerging as a critical modulator of function through its ability to sort lipids in a conformation-dependent manner. We reviewed recent computational and experimental studies that have begun to shed light on the sequence-ensemble-function relationship in this unique class of lipidated intrinsically disordered regions (LIDRs)., Competing Interests: Declaration of competing interest The authors 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 Elsevier Ltd. All rights reserved.)

Protein structure has been well established to play a key role in determining function; however, intrinsically disordered proteins and regions (IDPs and IDRs) defy this paradigm. IDPs and IDRs exist as an ensemble of structures rather than a stable 3D structure yet play essential roles in many cell-signaling processes. Nearly all Ras superfamily GTPases are tethered to membranes by a lipid tail at the end of a flexible IDR. The sequence of the IDR is a key determinant of membrane localization, and interaction between the IDR and the membrane has been shown to affect signaling in RAS proteins through the modulation of dynamic membrane organization. Here, we utilized atomistic molecular dynamics simulations to study the membrane interaction, conformational dynamics, and lipid sorting of three IDRs from small GTPases Rheb, RhoA, and DiRas3 in model membranes representing their physiological target membranes. We found that complementarity between the lipidated IDR sequence and target membrane lipid composition is a determinant of conformational plasticity. We also show that electrostatic interactions between anionic lipids and basic residues on IDRs are correlated with sampling of semistable conformational substates, and lack of these interactions is associated with greater conformational diversity. Finally, we show that small GTPase IDRs with a polybasic domain alter local lipid composition by segregating anionic lipids and, in some cases, excluding other lipids from their immediate vicinity in favor of anionic lipids.

The basal structure of the bacterial flagellum includes a membrane embedded MS-ring (formed by multiple copies of FliF) and a cytoplasmic C-ring (composed of proteins FliG, FliM and FliN). The SRP-type GTPase FlhF is required for directing the initial flagellar protein FliF to the cell pole, but the mechanisms are unclear. Here, we show that FlhF anchors developing flagellar structures to the polar landmark protein HubP/FimV, thereby restricting their formation to the cell pole. Specifically, the GTPase domain of FlhF interacts with HubP, while a structured domain at the N-terminus of FlhF binds to FliG. FlhF-bound FliG subsequently engages with the MS-ring protein FliF. Thus, the interaction of FlhF with HubP and FliG recruits a FliF-FliG complex to the cell pole. In addition, the modulation of FlhF activity by the MinD-type ATPase FlhG controls the interaction of FliG with FliM-FliN, thereby regulating the progression of flagellar assembly at the pole., (© 2024. The Author(s).)

The sterile alpha motif and histidine-aspartate domain-containing protein 1 (or SAMHD1), a human dNTP-triphosphohydrolase, contributes to HIV-1 restriction in select terminally differentiated cells of the immune system. While the prevailing hypothesis is that the catalytically active form of the protein is an allosterically triggered tetramer, whose HIV-1 restriction properties are attributed to its dNTP - triphosphohydrolase activity, it is also known to bind to ssRNA and ssDNA oligomers. A complete picture of the structure-function relationship of the enzyme is still elusive and the function corresponding to its nucleic acid binding ability is debated. In this in silico study, we investigate the stability, preference and allosteric effects of DNA oligomers bound to SAMHD1. In particular, we compare the binding of DNA and RNA oligomers of the same sequence and also consider the binding of DNA fragments with phosphorothioate bonds in the backbone. The results are compared with the canonical form with the monomers connected by GTP/dATP crossbridges. The simulations indicate that SAMHD1 dimers preferably bind to DNA and RNA oligomers compared to GTP/dATP. However, allosteric communication channels are altered in the nucleic acid acid bound complexes compared to the canonical form. All results are consistent with the hypothesis that the DNA bound form of the protein correspond to an unproductive off-pathway state where the protein is sequestered and not available for dNTP hydrolysis., Competing Interests: Declaration of competing interest The authors 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 Elsevier Inc. All rights reserved.)

Membrane-enclosed organelles are defining features of eukaryotes in distinguishing these organisms from prokaryotes. Specification of distinct membranes is critical to assemble and maintain discrete compartments. Small GTPases and their regulators are the signaling molecules that drive membrane-modifying machineries to the desired location. These signaling molecules include Rab and Rag GTPases, roadblock and longin domain proteins, and TRAPPC3-like proteins. Here, we take a structural approach to assess the relatedness of these eukaryotic-like proteins in Asgard archaea, the closest known prokaryotic relatives to eukaryotes. We find that the Asgard archaea GTPase core domains closely resemble eukaryotic Rabs and Rags. Asgard archaea roadblock, longin and TRAPPC3 domain-containing proteins form dimers similar to those found in the eukaryotic TRAPP and Ragulator complexes. We conclude that the emergence of these protein architectures predated eukaryogenesis, however further adaptations occurred in proto-eukaryotes to allow these proteins to regulate distinct internal membranes., (© 2024. The Author(s).)

The protein kinase Mechanistic Target of Rapamycin (mTOR) in Complex 1 (mTORC1) is regulated in part by the Ras-related GTP-binding proteins (Rag GTPases). Rag GTPases form a heterodimeric complex consisting of either RagA or RagB associated with either RagC or RagD and act to localize mTORC1 to the lysosomal membrane. Until recently, RagA and RagB were thought to be functionally redundant, as were RagC and RagD. However, recent research suggests that the various isoforms differentially activate mTORC1. Here, the mRNA expression and protein abundance of the Rag GTPases was compared across male rat skeletal muscle, heart, liver, kidney, and brain. Whereas mRNA expression of RagA was higher than RagB in nearly all tissues studied, RagB protein abundance was higher than RagA in all tissues besides skeletal muscle. RagC mRNA expression was more abundant or equal to RagD mRNA, and RagD protein was more abundant than RagC protein in all tissues. Moreover, the proportion of RagB in the short isoform was greater than the long in liver, whereas the opposite was true in brain. These results serve to further elucidate Rag GTPase expression and offer potential explanations for the differential responses to amino acids that are observed in different tissues., (© 2024 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Sterile alpha motif histidine-aspartate domain protein 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase that exists in monomeric, dimeric, and tetrameric forms. It is activated by GTP binding to an A1 allosteric site on each monomer subunit, which induces dimerization, a prerequisite for dNTP-induced tetramerization. SAMHD1 is a validated drug target stemming from its inactivation of many anticancer nucleoside drugs leading to drug resistance. The enzyme also possesses a single-strand nucleic acid binding function that promotes RNA and DNA homeostasis by several mechanisms. To discover small molecule inhibitors of SAMHD1, we screened a custom ∼69 000-compound library for dNTPase inhibitors. Surprisingly, this effort yielded no viable hits and indicated that exceptional barriers for discovery of small molecule inhibitors existed. We then took a rational fragment-based inhibitor design approach using a deoxyguanosine (dG) A1 site targeting fragment. A targeted chemical library was synthesized by coupling a 5'-phosphoryl propylamine dG fragment (dGpC 3 NH 2 ) to 376 carboxylic acids (RCOOH). Direct screening of the products (dGpC 3 NHCO-R) yielded nine initial hits, one of which (R = 3-(3'-bromo-[1,1'-biphenyl]), 5a ) was investigated extensively. Amide 5a is a competitive inhibitor against GTP binding to the A1 site and induces inactive dimers that are deficient in tetramerization. Surprisingly, 5a also prevented ssDNA and ssRNA binding, demonstrating that the dNTPase and nucleic acid binding functions of SAMHD1 can be disrupted by a single small molecule. A structure of the SAMHD1- 5a complex indicates that the biphenyl fragment impedes a conformational change in the C-terminal lobe that is required for tetramerization.

FlhF and FlhG control the location and number of flagella, respectively, in many polar-flagellated bacteria. The roles of FlhF and FlhG are not well characterized in bacteria that have multiple polar flagella, such as Helicobacter pylori . Deleting flhG in H. pylori shifted the flagellation pattern where most cells had approximately four flagella to a wider and more even distribution in flagellar number. As reported in other bacteria, deleting flhF in H. pylori resulted in reduced motility, hypoflagellation, and the improper localization of flagella to nonpolar sites. Motile variants of H. pylori ∆ flhF mutants that had a higher proportion of flagella localizing correctly to the cell pole were isolated, but we were unable to identify the genetic determinants responsible for the increased localization of flagella to the cell pole. One motile variant though produced more flagella than the Δ flhF parental strain, which apparently resulted from a missense mutation in fliF (encodes the MS ring protein), which changed Asn-255 to aspartate. Recombinant FliF N255D , but not recombinant wild-type FliF, formed ordered ring-like assemblies in vitro that were ~50 nm wide and displayed the MS ring architecture. We infer from these findings that the FliF N225D variant forms the MS ring more effectively in vivo in the absence of FlhF than wild-type FliF. IMPORTANCE Helicobacter pylori colonizes the human stomach where it can cause a variety of diseases, including peptic ulcer disease and gastric cancer. H. pylori uses flagella for motility, which is required for host colonization. FlhG and FlhF control the flagellation patterns in many bacteria. We found that in H. pylori , FlhG ensures that cells have approximately equal number of flagella and FlhF is needed for flagellum assembly and localization. FlhF is proposed to facilitate the assembly of FliF into the MS ring, which is one of the earliest structures formed in flagellum assembly. We identified a FliF variant that assembles the MS ring in the absence of FlhF, which supports the proposed role of FlhF in facilitating MS ring assembly., Competing Interests: The authors declare no conflict of interest.

mTORC1 is a protein kinase complex that controls cellular growth in response to nutrient availability. Amino acid signals are transmitted toward mTORC1 via the Rag/Gtr GTPases and their upstream regulators. An important regulator is LAMTOR, which localizes Rag/Gtr on the lysosomal/vacuole membrane. In human cells, LAMTOR consists of five subunits, but in yeast, only three or four. Currently, it is not known how variation of the subunit stoichiometry may affect its structural organization and biochemical properties. Here, we report a 3.1 Å-resolution structural model of the Gtr-Lam complex in Schizosaccharomyces pombe. We found that SpGtr shares conserved architecture as HsRag, but the intersubunit communication that coordinates nucleotide loading on the two subunits differs. In contrast, SpLam contains distinctive structural features, but its GTP-specific GEF activity toward SpGtr is evolutionarily conserved. Our results revealed unique evolutionary paths of the protein components of the mTORC1 pathway., Competing Interests: Declaration of interests The authors declare no conflict of interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Different protein purification methods exist. Yet, they need to be adapted for specific downstream applications to maintain functional integrity of the recombinant proteins. This study established a purification protocol for lentiviral Vpx (viral protein X) and test its ability to degrade sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) ex vivo in resting CD4 + T cells. For this purpose, we cloned a novel eukaryotic expression plasmid for Vpx including C-terminal 10x His- and HA-tags and confirmed that those tags did not alter the ability to degrade SAMHD1. We optimized purification conditions for Vpx produced in HEK293T cells with CHAPS as detergent and Co-NTA resins yielding the highest solubility and protein amounts. Size exclusion chromatography (SEC) further enhanced the purity of recombinant Vpx proteins. Importantly, nucleofection of resting CD4 + T cells demonstrated that purified recombinant Vpx protein efficiently degraded SAMHD1 in a proteasome-dependent manner. In conclusion, this protocol is suitable for functional downstream applications of recombinant Vpx and might be transferrable to other recombinant proteins with similar functions/properties as lentiviral Vpx., Competing Interests: Declaration of competing interest O.T.K. and H.-M.B. are listed as inventors of patent no. WO2017076880A1. All other authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: SAMHD1 is a deoxynucleotide triphosphohydrolase that restricts replication of HIV-1 in differentiated leucocytes. HIV-1 is not restricted in cycling cells and it has been proposed that this is due to phosphorylation of SAMHD1 at T592 in these cells inactivating the enzymatic activity. To distinguish between theories for how SAMHD1 restricts HIV-1 in differentiated but not cycling cells, we analysed the effects of substitutions at T592 on restriction and dNTP levels in both cycling and differentiated cells as well as tetramer stability and enzymatic activity in vitro., Results: We first showed that HIV-1 restriction was not due to SAMHD1 nuclease activity. We then characterised a panel of SAMHD1 T592 mutants and divided them into three classes. We found that a subset of mutants lost their ability to restrict HIV-1 in differentiated cells which generally corresponded with a decrease in triphosphohydrolase activity and/or tetramer stability in vitro. Interestingly, no T592 mutants were able to restrict WT HIV-1 in cycling cells, despite not being regulated by phosphorylation and retaining their ability to hydrolyse dNTPs. Lowering dNTP levels by addition of hydroxyurea did not give rise to restriction. Compellingly however, HIV-1 RT mutants with reduced affinity for dNTPs were significantly restricted by wild-type and T592 mutant SAMHD1 in both cycling U937 cells and Jurkat T-cells. Restriction correlated with reverse transcription levels., Conclusions: Altogether, we found that the amino acid at residue 592 has a strong effect on tetramer formation and, although this is not a simple "on/off" switch, this does correlate with the ability of SAMHD1 to restrict HIV-1 replication in differentiated cells. However, preventing phosphorylation of SAMHD1 and/or lowering dNTP levels by adding hydroxyurea was not enough to restore restriction in cycling cells. Nonetheless, lowering the affinity of HIV-1 RT for dNTPs, showed that restriction is mediated by dNTP levels and we were able to observe for the first time that SAMHD1 is active and capable of inhibiting HIV-1 replication in cycling cells, if the affinity of RT for dNTPs is reduced. This suggests that the very high affinity of HIV-1 RT for dNTPs prevents HIV-1 restriction by SAMHD1 in cycling cells., (© 2023. The Author(s).)

The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of mammalian cell growth that is dysregulated in a number of human diseases, including metabolic syndromes, aging, and cancer. Structural, biochemical, and pharmacological studies that have increased our understanding of how mTORC1 executes growth control often relied upon purified mTORC1 protein. However, current immunoaffinity-based purification methods are expensive, inefficient, and do not necessarily isolate endogenous mTORC1, hampering their overall utility in research. Here we present a simple tool to isolate endogenous mTORC1 from various cellular sources. By recombinantly expressing and isolating mTORC1-binding Rag GTPases from Escherichia coli and using them as affinity probes, we demonstrate that mTORC1 can be isolated from mouse, bovine, and human sources. Our results indicate that mTORC1 isolated by this relatively inexpensive method is catalytically active and amenable to scaling. Collectively, this tool may be utilized to isolate mTORC1 from various cellular sources, organs, and disease contexts, aiding mTORC1-related research., Competing Interests: Conflict of interest Y. H. I. is the founder of Araucaria Laboratories Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Transport of newly synthesized proteins from endoplasmic reticulum (ER) to Golgi is mediated by coat protein complex II (COPII). The assembly and disassembly of COPII vesicles is regulated by the molecular switch Sar1, which is a small GTPase and a component of COPII. Usually a small GTPase binds GDP (inactive form) or GTP (active form). Mammals have two Sar1 isoforms, Sar1a and Sar1b, that have approximately 90% sequence identity. Some experiments demonstrated that these two isoforms had distinct but overlapping functions. Here we found another instance of differing behavior: the alarmone ppGpp could bind to and inhibit the GTPase activity of human Sar1a but could not inhibit the GTPase activity of human Sar1b. The crystal structures of Sar1a⋅ppGpp and Sar1b⋅GDP have been determined. Superposition of the structures shows that ppGpp binds to the nucleotide-binding pocket, its guanosine base, ribose ring and 5'-diphosphate occupying nearly the same positions as for GDP. However, its 3'-diphosphate points away from the active site and, hence, away from the surface of the protein. The overall structure of Sar1a⋅ppGpp is more similar to Sar1b⋅GDP than to Sar1b⋅GTP. We also find that the Asp140-Arg138-water-ligand interaction net is important for the binding of ppGpp to Sar1a. This study provides further evidence showing that there are biochemical differences between the Sar1a and Sar1b isoforms of Sar1., (© 2022 Wiley Periodicals LLC.)

KRas is a small GTPase and membrane-bound signaling protein. Newly synthesized KRas is post-translationally modified with a membrane-anchoring prenyl group. KRas chaperones are therapeutic targets in cancer due to their participation in trafficking oncogenic KRas to membranes. SmgGDS splice variants are chaperones for small GTPases with basic residues in their hypervariable domain (HVR), including KRas. SmgGDS-607 escorts pre-prenylated small GTPases, while SmgGDS-558 escorts prenylated small GTPases. We provide a structural description of farnesylated and fully processed KRas (KRas-FMe) in complex with SmgGDS-558 and define biophysical properties of this interaction. Surface plasmon resonance measurements on biomimetic model membranes quantified the thermodynamics of the interaction of SmgGDS with KRas, and small-angle x-ray scattering was used to characterize complexes of SmgGDS-558 and KRas-FMe structurally. Structural models were refined using Monte Carlo and molecular dynamics simulations. Our results indicate that SmgGDS-558 interacts with the HVR and the farnesylated C-terminus of KRas-FMe, but not its G-domain. Therefore, SmgGDS-558 interacts differently with prenylated KRas than prenylated RhoA, whose G-domain was found in close contact with SmgGDS-558 in a recent crystal structure. Using immunoprecipitation assays, we show that SmgGDS-558 binds the GTP-bound, GDP-bound, and nucleotide-free forms of farnesylated and fully processed KRas in cells, consistent with SmgGDS-558 not engaging the G-domain of KRas. We found that the dissociation constant, K d , for KRas-FMe binding to SmgGDS-558 is comparable with that for the KRas complex with PDEδ, a well-characterized KRas chaperone that also does not interact with the KRas G-domain. These results suggest that KRas interacts in similar ways with the two chaperones SmgGDS-558 and PDEδ. Therapeutic targeting of the SmgGDS-558/KRas complex might prove as useful as targeting the PDEδ/KRas complex in KRas-driven cancers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. All rights reserved.)

The p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. Irgb6 is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive because of lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane-binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs., (© 2021 Saijo-Hamano et al.)

The first stage of the eukaryotic secretory pathway is the packaging of cargo proteins into coat protein complex II (COPII) vesicles exiting the ER. The cytoplasmic COPII vesicle coat machinery is recruited to the ER membrane by the activated, GTP-bound, form of the conserved Sar1 GTPase. Activation of Sar1 on the surface of the ER by Sec12, a membrane-anchored GEF (guanine nucleotide exchange factor), is therefore the initiating step of the secretory pathway. Here we report the structure of the complex between Sar1 and the cytoplasmic GEF domain of Sec12, both from Saccharomyces cerevisiae. This structure, representing a key nucleotide-free activation intermediate, reveals how the potassium ion-binding K loop disrupts the nucleotide-binding site of Sar1. We propose an unexpected orientation of the GEF domain relative to the membrane surface and postulate a mechanism for how Sec12 facilitates membrane insertion of the amphipathic helix exposed by Sar1 upon GTP binding., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The mTOR complex 1 (mTORC1) controls cell growth in response to amino acid levels 1 . Here we report SAR1B as a leucine sensor that regulates mTORC1 signalling in response to intracellular levels of leucine. Under conditions of leucine deficiency, SAR1B inhibits mTORC1 by physically targeting its activator GATOR2. In conditions of leucine sufficiency, SAR1B binds to leucine, undergoes a conformational change and dissociates from GATOR2, which results in mTORC1 activation. SAR1B-GATOR2-mTORC1 signalling is conserved in nematodes and has a role in the regulation of lifespan. Bioinformatic analysis reveals that SAR1B deficiency correlates with the development of lung cancer. The silencing of SAR1B and its paralogue SAR1A promotes mTORC1-dependent growth of lung tumours in mice. Our results reveal that SAR1B is a conserved leucine sensor that has a potential role in the development of lung cancer., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Mitochondrial ribosomes (mitoribosomes) synthesize a critical set of proteins essential for oxidative phosphorylation. Therefore, mitoribosomal function is vital to the cellular energy supply. Mitoribosome biogenesis follows distinct molecular pathways that remain poorly understood. Here, we determine the cryo-EM structures of mitoribosomes isolated from human cell lines with either depleted or overexpressed mitoribosome assembly factor GTPBP5, allowing us to capture consecutive steps during mitoribosomal large subunit (mt-LSU) biogenesis. Our structures provide essential insights into the last steps of 16S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, which require the coordinated action of nine assembly factors. We show that mammalian-specific MTERF4 contributes to the folding of 16S rRNA, allowing 16 S rRNA methylation by MRM2, while GTPBP5 and NSUN4 promote fine-tuning rRNA rearrangements leading to PTC formation. Moreover, our data reveal an unexpected involvement of the elongation factor mtEF-Tu in mt-LSU assembly, where mtEF-Tu interacts with GTPBP5, similar to its interaction with tRNA during translational elongation.

Mitochondrial ribosomes are specialized for the synthesis of membrane proteins responsible for oxidative phosphorylation. Mammalian mitoribosomes have diverged considerably from the ancestral bacterial ribosomes and feature dramatically reduced ribosomal RNAs. The structural basis of the mammalian mitochondrial ribosome assembly is currently not well understood. Here we present eight distinct assembly intermediates of the human large mitoribosomal subunit involving seven assembly factors. We discover that the NSUN4-MTERF4 dimer plays a critical role in the process by stabilizing the 16S rRNA in a conformation that exposes the functionally important regions of rRNA for modification by the MRM2 methyltransferase and quality control interactions with the conserved mitochondrial GTPase MTG2 that contacts the sarcin-ricin loop and the immature active site. The successive action of these factors leads to the formation of the peptidyl transferase active site of the mitoribosome and the folding of the surrounding rRNA regions responsible for interactions with tRNAs and the small ribosomal subunit.

Ribosome biogenesis requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. Particularly, maturation of the peptidyl transferase center (PTC) is mediated by conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial large ribosomal subunit (mtLSU) using endogenous complex purification, in vitro reconstitution and cryo-EM. Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch and progression to a near-mature PTC state. Additionally, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results provide a framework for understanding step-wise PTC folding as a critical conserved quality control checkpoint.

SAMHD1 is a fundamental regulator of cellular dNTPs that catalyzes their hydrolysis into 2'-deoxynucleoside and triphosphate, restricting the replication of viruses, including HIV-1, in CD4 + myeloid lineage and resting T-cells. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome (AGS) and certain cancers. More recently, SAMHD1 has been linked to anticancer drug resistance and the suppression of the interferon response to cytosolic nucleic acids after DNA damage. Here, we probe dNTP hydrolysis and inhibition of SAMHD1 using the R p and S p diastereomers of dNTPαS nucleotides. Our biochemical and enzymological data show that the α-phosphorothioate substitution in S p -dNTPαS but not R p -dNTPαS diastereomers prevents Mg 2+ ion coordination at both the allosteric and catalytic sites, rendering SAMHD1 unable to form stable, catalytically active homotetramers or hydrolyze substrate dNTPs at the catalytic site. Furthermore, we find that S p -dNTPαS diastereomers competitively inhibit dNTP hydrolysis, while R p -dNTPαS nucleotides stabilize tetramerization and are hydrolyzed with similar kinetic parameters to cognate dNTPs. For the first time, we present a cocrystal structure of SAMHD1 with a substrate, R p -dGTPαS, in which an Fe-Mg-bridging water species is poised for nucleophilic attack on the P α . We conclude that it is the incompatibility of Mg 2+ , a hard Lewis acid, and the α-phosphorothioate thiol, a soft Lewis base, that prevents the S p -dNTPαS nucleotides coordinating in a catalytically productive conformation. On the basis of these data, we present a model for SAMHD1 stereospecific hydrolysis of R p -dNTPαS nucleotides and for a mode of competitive inhibition by S p -dNTPαS nucleotides that competes with formation of the enzyme-substrate complex.

mTORC1 is a central player in cell growth, a process that is tightly regulated by the availability of nutrients and that controls various aspects of metabolism in the normal cell and in severe diseases such as cancers. mTORC1 is a large multiprotein complex, composed of the kinase subunit mTOR, of Ragulator, which attaches mTOR to the lysosome membrane, of the atypical Rag GTPases and the small GTPase RheB, whose nucleotide states directly dictate its localization to the lysosome and its kinase activity, and of RAPTOR, an adaptor that assembles the complex. The activity of the Rag GTPases is further controlled by the GATOR1 and folliculin complexes, which regulate their GTP/GDP conversion. Here, we review recent structures of important components of the mTORC1 machinery, determined by cryo-electron microscopy for the most part, which allow to reconstitute the architecture of active mTORC1 at near atomic resolution. Notably, we discuss how these structures shed new light on the roles of Rag GTPases and their regulators in mTORC1 regulation, and the perspectives that they open towards understanding the inner workings of mTORC1 on the lysosomal membrane., (© 2021 médecine/sciences – Inserm.)

OptoPB is an optogenetic tool engineered by fusion of the phosphoinositide (PI)-binding polybasic domain of Rit1 (Rit-PB) to a photoreactive light-oxygen-voltage (LOV) domain. OptoPB selectively and reversibly binds the plasma membrane (PM) under blue light excitation, and in the dark, it releases back to the cytoplasm. However, the molecular mechanism of optical regulation and lipid recognition is still unclear. Here using nuclear magnetic resonance (NMR) spectroscopy, liposome pulldown assay, and surface plasmon resonance (SPR), we find that OptoPB binds to membrane mimetics containing di- or triphosphorylated phosphatidylinositols, particularly phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), an acidic phospholipid predominantly located in the eukaryotic PM. In the dark, steric hindrance prevented this protein-membrane interaction, while 470 nm blue light illumination activated it. NMR titration and site-directed mutagenesis revealed that both cationic and hydrophobic Rit-PB residues are essential to the membrane interaction, indicating that OptoPB binds the membrane via a specific PI(4,5)P 2 -dependent mechanism.

Ribosome biogenesis is a fundamental multi-step cellular process that culminates in the formation of ribosomal subunits, whose production and modification are regulated by numerous biogenesis factors. In this study, we analyze physiologic prokaryotic ribosome biogenesis by isolating bona fide pre-50S subunits from an Escherichia coli strain with the biogenesis factor ObgE, affinity tagged at its native gene locus. Our integrative structural approach reveals a network of interacting biogenesis factors consisting of YjgA, RluD, RsfS, and ObgE on the immature pre-50S subunit. In addition, our study provides mechanistic insight into how the GTPase ObgE, in concert with other biogenesis factors, facilitates the maturation of the 50S functional core and reveals both conserved and divergent evolutionary features of ribosome biogenesis between prokaryotes and eukaryotes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmatis gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA-MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Secretion-associated and ras-related protein 1 (Sar1) is a small GTPase that plays an important role in the transport of protein coated with coat protein complex II vesicles. However, its alternative roles in the biological processes of Procambarus clarkii remain unclear. Here, a sar1 gene (named as Pc-sar1) with an open reading frame of 582 bp from P. clarkii was identified. Pc-sar1 was expressed in all examined tissues with highest expression levels in muscle, which was determined by real-time PCR and western blotting. After the induction of lipopolysaccharide (LPS) and polycytidylic acid (Poly I: C), the transcriptional levels of Pc-sar1 differed in hepatopancreas, gill, muscle and intestine. In contrast, the expression of Pc-sar1 was upregulated by 20-hydroxyecdysone in these four tissues. In addition, the RNA interference of Pc-sar1 significantly affected the expression levels of immune and hormone-related genes. These results indicate that Pc-sar1 is involved in the innate immune response and ecdysteroid signaling pathway., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The Rag GTPases (Rags) recruit mTORC1 to the lysosomal membrane in response to nutrients, where it is then activated in response to energy and growth factor availability. The lysosomal folliculin (FLCN) complex (LFC) consists of the inactive Rag dimer, the pentameric scaffold Ragulator, and the FLCN:FNIP2 (FLCN-interacting protein 2) GTPase activating protein (GAP) complex, and prevents Rag dimer activation during amino acid starvation. How the LFC is disassembled upon amino acid refeeding is an outstanding question. Here we show that the cytoplasmic tail of the human lysosomal solute carrier family 38 member 9 (SLC38A9) destabilizes the LFC and thereby triggers GAP activity of FLCN:FNIP2 toward RagC. We present the cryo-EM structures of Rags in complex with their lysosomal anchor complex Ragulator and the cytoplasmic tail of SLC38A9 in the pre- and post-GTP hydrolysis state of RagC, which explain how SLC38A9 destabilizes the LFC and so promotes Rag dimer activation.

The Rho family of GTPases consists of 20 members including RhoE. Here, we discover the existence of a short isoform of RhoE designated as RhoEα, the first Rho GTPase isoform generated from alternative translation. Translation of this new isoform is initiated from an alternative start site downstream of and in-frame with the coding region of the canonical RhoE. RhoEα exhibits a similar subcellular distribution while its protein stability is higher than RhoE. RhoEα contains binding capability to RhoE effectors ROCK1, p190RhoGAP and Syx. The distinct transcriptomes of cells with the expression of RhoE and RhoEα, respectively, are demonstrated. The data propose distinctive and overlapping biological functions of RhoEα compared to RhoE. In conclusion, this study reveals a new Rho GTPase isoform generated from alternative translation. The discovery provides a new scope of understanding the versatile functions of small GTPases and underlines the complexity and diverse roles of small GTPases.

The assembly of a septin filament requires that homologous monomers must distinguish between one another in establishing appropriate interfaces with their neighbors. To understand this phenomenon at the molecular level, we present the first four crystal structures of heterodimeric septin complexes. We describe in detail the two distinct types of G-interface present within the octameric particles, which must polymerize to form filaments. These are formed between SEPT2 and SEPT6 and between SEPT7 and SEPT3, and their description permits an understanding of the structural basis for the selectivity necessary for correct filament assembly. By replacing SEPT6 by SEPT8 or SEPT11, it is possible to rationalize Kinoshita's postulate, which predicts the exchangeability of septins from within a subgroup. Switches I and II, which in classical small GTPases provide a mechanism for nucleotide-dependent conformational change, have been repurposed in septins to play a fundamental role in molecular recognition. Specifically, it is switch I which holds the key to discriminating between the two different G-interfaces. Moreover, residues which are characteristic for a given subgroup play subtle, but pivotal, roles in guaranteeing that the correct interfaces are formed., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Cell polarity is fundamental to the development of both eukaryotes and prokaryotes, yet the mechanisms behind its formation are not well understood. Here we found that, phytohormone auxin-induced, sterol-dependent nanoclustering of cell surface transmembrane receptor kinase 1 (TMK1) is critical for the formation of polarized domains at the plasma membrane (PM) during the morphogenesis of cotyledon pavement cells (PC) in Arabidopsis. Auxin-induced TMK1 nanoclustering stabilizes flotillin1-associated ordered nanodomains, which in turn promote the nanoclustering of ROP6 GTPase that acts downstream of TMK1 to regulate cortical microtubule organization. In turn, cortical microtubules further stabilize TMK1- and flotillin1-containing nanoclusters at the PM. Hence, we propose a new paradigm for polarity formation: A diffusive signal triggers cell polarization by promoting cell surface receptor-mediated nanoclustering of signaling components and cytoskeleton-mediated positive feedback that reinforces these nanodomains into polarized domains.

Vesicles that are coated by coat protein complex II (COPII) are the primary mediators of vesicular traffic from the endoplasmic reticulum to the Golgi apparatus. Secretion-associated Ras-related GTPase 1 (SAR1) is a small GTPase that is part of COPII and, upon GTP binding, recruits the other COPII proteins to the endoplasmic reticulum membrane. Mammals have two SAR1 paralogs that genetic data suggest may have distinct physiological roles, e.g. in lipoprotein secretion in the case of SAR1B. Here we identified two amino acid clusters that have conserved SAR1 paralog-specific sequences. We observed that one cluster is adjacent to the SAR1 GTP-binding pocket and alters the kinetics of GTP exchange. The other cluster is adjacent to the binding site for two COPII components, SEC31 homolog A COPII coat complex component (SEC31) and SEC23. We found that the latter cluster confers to SAR1B a binding preference for SEC23A that is stronger than that of SAR1A for SEC23A. Unlike SAR1B, SAR1A was prone to oligomerize on a membrane surface. SAR1B knockdown caused loss of lipoprotein secretion, overexpression of SAR1B but not of SAR1A could restore secretion, and a divergent cluster adjacent to the SEC31/SEC23-binding site was critical for this SAR1B function. These results highlight that small primary sequence differences between the two mammalian SAR1 paralogs lead to pronounced biochemical differences that significantly affect COPII assembly and identify a specific function for SAR1B in lipoprotein secretion, providing insights into the mechanisms of large cargo secretion that may be relevant for COPII-related diseases., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Melville et al.)

Rac proteins are classified as a subfamily of the Rho family of small G proteins. They are important molecular switches which act as key signal transducers regulating a wide variety of processes in the cell. DjRac1, a novel Rac gene from planarian Dugesia japonica was cloned by RACE method and characterized. This cDNA contains 851 bp with a putative open reading frame of 190 amino acids. It has a predicted molecular mass of 21.12 kDa and an isoelectric point of 8.42. Whole-mount in situ hybridization and relative quantitative real-time PCR were used to study the spatial and temporal expression pattern of DjRac1 from 1 to 7 days in the regenerating planarians. Results showed that the expression of DjRac1 was concentrated in the blastema and the transcription level of DjRac1 was significantly upregulated after amputation within three days, suggesting DjRac1 might participate in the process of regeneration in planarian., Competing Interests: Declaration of competing interest We have no conflicts of interest with other people or organizations., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The Ral proteins (RalA and RalB) are small G proteins of the Ras family that have been implicated in exocytosis, endocytosis, transcriptional regulation and mitochondrial fission, as well as having a role in tumourigenesis. RalA and RalB are activated downstream of the master regulator, Ras, which causes the nucleotide exchange of GDP for GTP. Here we report the 1 H, 15  N and 13 C resonance assignments of RalA in its active form bound to the GTP analogue GMPPNP. We also report the backbone assignments of RalA in its inactive, GDP-bound form. The assignments give insight into the switch regions, which change conformation upon nucleotide exchange. These switch regions are invisible in the spectra of the active, GMPPNP bound form but the residues proximal to the switches can be monitored. RalA is also an important drug target due to its over activation in some cancers and these assignments will be extremely useful for NMR-based screening approaches.

Even though the Obg protein is essential for bacterial viability, the cellular functions of this universally conserved GTPase remain enigmatic. Moreover, the influence of GTP and GDP binding on the activity of this protein is largely unknown. Previously, we identified a mutant isoform of ObgE (the Obg protein of Escherichia coli ) that triggers cell death. In this research we explore the biochemical requirements for the toxic effect of this mutant ObgE* isoform, using cell death as a readily accessible read-out for protein activity. Both the absence of the N-terminal domain and a decreased GTP binding affinity neutralize ObgE*-mediated toxicity. Moreover, a deletion in the region that connects the N-terminal domain to the G domain likewise abolishes toxicity. Taken together, these data indicate that GTP binding by ObgE* triggers a conformational change that is transmitted to the N-terminal domain to confer toxicity. We therefore conclude that ObgE*-GTP, but not ObgE*-GDP, is the active form of ObgE* that is detrimental to cell viability. Based on these data, we speculate that also for wild-type ObgE, GTP binding triggers conformational changes that affect the N-terminal domain and thereby control ObgE function., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

mTORC1 controls anabolic and catabolic processes in response to nutrients through the Rag GTPase heterodimer, which is regulated by multiple upstream protein complexes. One such regulator, FLCN-FNIP2, is a GTPase activating protein (GAP) for RagC/D, but despite its important role, how it activates the Rag GTPase heterodimer remains unknown. We used cryo-EM to determine the structure of FLCN-FNIP2 in a complex with the Rag GTPases and Ragulator. FLCN-FNIP2 adopts an extended conformation with two pairs of heterodimerized domains. The Longin domains heterodimerize and contact both nucleotide binding domains of the Rag heterodimer, while the DENN domains interact at the distal end of the structure. Biochemical analyses reveal a conserved arginine on FLCN as the catalytic arginine finger and lead us to interpret our structure as an on-pathway intermediate. These data reveal features of a GAP-GTPase interaction and the structure of a critical component of the nutrient-sensing mTORC1 pathway., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The tumor suppressor folliculin (FLCN) enables nutrient-dependent activation of the mechanistic target of rapamycin complex 1 (mTORC1) protein kinase via its guanosine triphosphatase (GTPase) activating protein (GAP) activity toward the GTPase RagC. Concomitant with mTORC1 inactivation by starvation, FLCN relocalizes from the cytosol to lysosomes. To determine the lysosomal function of FLCN, we reconstituted the human lysosomal FLCN complex (LFC) containing FLCN, its partner FLCN-interacting protein 2 (FNIP2), and the RagA GDP :RagC GTP GTPases as they exist in the starved state with their lysosomal anchor Ragulator complex and determined its cryo-electron microscopy structure to 3.6 angstroms. The RagC-GAP activity of FLCN was inhibited within the LFC, owing to displacement of a catalytically required arginine in FLCN from the RagC nucleotide. Disassembly of the LFC and release of the RagC-GAP activity of FLCN enabled mTORC1-dependent regulation of the master regulator of lysosomal biogenesis, transcription factor E3, implicating the LFC as a checkpoint in mTORC1 signaling., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase regulates growth in response to nutrients and growth factors. Nutrients promote its translocation to the lysosomal surface, where its Raptor subunit interacts with the Rag guanosine triphosphatase (GTPase)-Ragulator complex. Nutrients switch the heterodimeric Rag GTPases among four different nucleotide-binding states, only one of which (RagA/B•GTP-RagC/D•GDP) permits mTORC1 association. We used cryo-electron microscopy to determine the structure of the supercomplex of Raptor with Rag-Ragulator at a resolution of 3.2 angstroms. Our findings indicate that the Raptor α-solenoid directly detects the nucleotide state of RagA while the Raptor "claw" threads between the GTPase domains to detect that of RagC. Mutations that disrupted Rag-Raptor binding inhibited mTORC1 lysosomal localization and signaling. By comparison with a structure of mTORC1 bound to its activator Rheb, we developed a model of active mTORC1 docked on the lysosome., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo-electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagA GTP /RagC GDP nucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Small GTPases are key regulators of cellular events, and their dysfunction causes many types of cancer. They serve as molecular switches by cycling between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states. GTPases are deactivated by GTPase-activating proteins (GAPs) and are activated by guanine-nucleotide exchange factors (GEFs). The intrinsic GTP hydrolysis activity of small GTPases is generally low and is accelerated by GAPs. GEFs promote GDP dissociation from small GTPases to allow for GTP binding, which results in a conformational change of two highly flexible segments, called switch I and switch II, that enables binding of the gamma phosphate and allows small GTPases to interact with downstream effectors. For several decades, crystal structures of many GEFs and GAPs have been reported and have shown tremendous structural diversity. In this review, we focus on the latest structural studies of GEFs. Detailed pictures of the variety of GEF mechanisms at atomic resolution can provide insights into new approaches for drug discovery., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Ras family small GTPases undergo prenylation (such as farnesylation) for proper localization to the plasma membrane, where they can initiate oncogenic signaling pathways. Small GTP-binding protein GDP-dissociation stimulator (SmgGDS) proteins are chaperones that bind and traffic small GTPases, although their exact cellular function is unknown. Initially, SmgGDS proteins were classified as guanine nucleotide exchange factors, but recent findings suggest that SmgGDS proteins also regulate prenylation of small GTPases in vivo in a substrate-selective manner. SmgGDS-607 recognizes the polybasic region and the CAA X box of several small GTPases and inhibits prenylation by impeding their entry into the geranylgeranylation pathway. Here, using recombinant and purified enzymes for prenylation and protein-binding assays, we demonstrate that SmgGDS-607 differentially regulates farnesylation of several small GTPases. SmgGDS-607 inhibited farnesylation of some proteins, such as DiRas1, by sequestering the protein and limiting modification catalyzed by protein farnesyltransferase (FTase). We found that the competitive binding affinities of the small GTPase for SmgGDS-607 and FTase dictate the extent of this inhibition. Additionally, we discovered that SmgGDS-607 increases the rate of farnesylation of HRas by enhancing product release from FTase. Our work indicates that SmgGDS-607 binds to a broad range of small GTPases and does not require a PBR for recognition. Together, these results provide mechanistic insight into SmgGDS-607-mediated regulation of farnesylation of small GTPases and suggest that SmgGDS-607 has multiple modes of substrate recognition., (© 2019 García-Torres and Fierke.)

Small GTPases alternatively bind GDP/GTP guanine nucleotides to gate signaling pathways that direct most cellular processes. Numerous GTPases are implicated in oncogenesis, particularly the three RAS isoforms HRAS, KRAS, and NRAS and the RHO family GTPase RAC1. Signaling networks comprising small GTPases are highly connected, and there is some evidence of direct biochemical cross-talk between their functional G-domains. The activation potential of a given GTPase is contingent on a codependent interaction with the nucleotide and a Mg 2+ ion, which bind to individual variants with distinct affinities coordinated by residues in the GTPase nucleotide-binding pocket. Here, we utilized a selective-labeling strategy coupled with real-time NMR spectroscopy to monitor nucleotide exchange, GTP hydrolysis, and effector interactions of multiple small GTPases in a single complex system. We provide insight into nucleotide preference and the role of Mg 2+ in activating both WT and oncogenic mutant enzymes. Multiplexing revealed guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP), and effector-binding specificities in mixtures of GTPases and resolved that the three related RAS isoforms are biochemically equivalent. This work establishes that direct quantitation of the nucleotide-bound conformation is required to accurately determine an activation potential for any given GTPase, as small GTPases such as RAS-like proto-oncogene A (RALA) or the G12C mutant of KRAS display fast exchange kinetics but have a high affinity for GDP. Furthermore, we propose that the G-domains of small GTPases behave autonomously in solution and that nucleotide cycling proceeds independently of protein concentration but is highly impacted by Mg 2+ abundance., (© 2019 Killoran and Smith.)

Giant viruses have extraordinarily large dsDNA genomes, and exceptionally, they encode various components of the translation apparatus, including tRNAs, aminoacyl-tRNA synthetases and translation factors. Here, we focused on the elongation factor 1 (EF1) family of viral translational GTPases (trGTPases), using computational and functional approaches to shed light on their functions. Multiple sequence alignment indicated that these trGTPases clustered into two groups epitomized by members of Mimiviridae and Marseilleviridae, respectively. trGTPases in the first group were more closely related to GTP-binding protein 1 (GTPBP1), whereas trGTPases in the second group were closer to eEF1A, eRF3 and Hbs1. Functional characterization of representative GTPBP1-like trGTPases (encoded by Hirudovirus, Catovirus and Moumouvirus) using in vitro reconstitution revealed that they possess eEF1A-like activity and can deliver cognate aa-tRNAs to the ribosomal A site during translation elongation. By contrast, representative eEF1A/eRF3/Hbs1-like viral trGTPases, encoded by Marseillevirus and Lausannevirus, have eRF3-like termination activity and stimulate peptide release by eRF1. Our analysis identified specific aspects of the functioning of these viral trGTPases with eRF1 of human, amoebal and Marseillevirus origin., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Noonan syndrome (NS) is characterized by distinctive craniofacial appearance, short stature, and congenital heart disease. Approximately 80% of individuals with NS harbor mutations in genes whose products are involved in the RAS/mitogen-activating protein kinase (MAPK) pathway. However, the underlying genetic causes in nearly 20% of individuals with NS phenotype remain unexplained. Here, we report four de novo RRAS2 variants in three individuals with NS. RRAS2 is a member of the RAS subfamily and is ubiquitously expressed. Three variants, c.70_78dup (p.Gly24_Gly26dup), c.216A>T (p.Gln72His), and c.215A>T (p.Gln72Leu), have been found in cancers; our functional analyses showed that these three changes induced elevated association of RAF1 and that they activated ERK1/2 and ELK1. Notably, prominent activation of ERK1/2 and ELK1 by p.Gln72Leu associates with the severe phenotype of the individual harboring this change. To examine variant pathogenicity in vivo, we generated zebrafish models. Larvae overexpressing c.70_78dup (p.Gly24_Gly26dup) or c.216A>T (p.Gln72His) variants, but not wild-type RRAS2 RNAs, showed craniofacial defects and macrocephaly. The same dose injection of mRNA encoding c.215A>T (p.Gln72Leu) caused severe developmental impairments and low dose overexpression of this variant induced craniofacial defects. In contrast, the RRAS2 c.224T>G (p.Phe75Cys) change, located on the same allele with p.Gln72His in an individual with NS, resulted in no aberrant in vitro or in vivo phenotypes by itself. Together, our findings suggest that activating RRAS2 mutations can cause NS and expand the involvement of RRAS2 proto-oncogene to rare germline disorders., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Aberrant signaling through pathways controlling cell response to extracellular stimuli constitutes a central theme in disorders affecting development. Signaling through RAS and the MAPK cascade controls a variety of cell decisions in response to cytokines, hormones, and growth factors, and its upregulation causes Noonan syndrome (NS), a developmental disorder whose major features include a distinctive facies, a wide spectrum of cardiac defects, short stature, variable cognitive impairment, and predisposition to malignancies. NS is genetically heterogeneous, and mutations in more than ten genes have been reported to underlie this disorder. Despite the large number of genes implicated, about 10%-20% of affected individuals with a clinical diagnosis of NS do not have mutations in known RASopathy-associated genes, indicating that additional unidentified genes contribute to the disease, when mutated. By using a mixed strategy of functional candidacy and exome sequencing, we identify RRAS2 as a gene implicated in NS in six unrelated subjects/families. We show that the NS-causing RRAS2 variants affect highly conserved residues localized around the nucleotide binding pocket of the GTPase and are predicted to variably affect diverse aspects of RRAS2 biochemical behavior, including nucleotide binding, GTP hydrolysis, and interaction with effectors. Additionally, all pathogenic variants increase activation of the MAPK cascade and variably impact cell morphology and cytoskeletal rearrangement. Finally, we provide a characterization of the clinical phenotype associated with RRAS2 mutations., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

CgtA is an essential bacterial GTPase protein involved in multiple cellular activities. In the presence of 50S ribosome, its GTPase activity increases significantly. Through sequential deletions of CgtA protein of Vibrio cholerae (CgtA vc ) we found that its N terminal Obg domain is essential for ribosome binding and augmenting the ribosome mediated GTPase activity. Strategic deletions of the three glycine rich loops of Obg domain revealed that loop 1 of Obg domain is involved in anchoring the protein into the 50S, whereas, loop 2 & loop 3 are involved in conveying the effect of interaction of the Obg domain with the 50S to the GTPase domain through an interdomain linker, followed by GTP hydrolysis. On the other hand, the non-conserved C-terminal domain (CTD) is not directly involved in ribosome binding but shows negative impact on GTPase activity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ras-related C3 botulinum toxin substrate 1 (Rac1) participates in many biological processes. In this study, a Rac1 gene was identified in the crayfish Procambarus clarkii with an open reading frame of 579 bp that encoded 192 amino acids. This predicted 21.4 kDa protein was highly homologous to those in other invertebrates. Real-time PCR analysis revealed that Pc-Rac1 was expressed in all examined tissues with the highest expression level in hemocytes. The transcriptional expression level of Pc-Rac1 was significantly upregulated in hemocytes and hepatopancreas after lipopolysaccharide (LPS) or polyinosinic: polycytidylic acid (poly I: C) induction. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis suggested that a recombinant Pc-Rac1 protein was successfully expressed in E. coli. Far-western blot analysis demonstrated that Rac1 can interact with the PBD domain of p21-activated kinase 1 (PAK1). RNA interference of Pc-Rac1 affected the mRNA expression levels of immune-related genes lectin, Toll, crustin, TNF, ALF and cactus. These results suggest that Pc-Rac1 is involved in the innate immune responses in P. clarkii., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Many biological processes have to occur at specific locations on the cell membrane. These locations are often specified by the localised activity of small GTPase proteins. Some processes require the formation of a single cluster of active GTPase, also called unipolar polarisation (here "polarisation"), whereas others need multiple coexisting clusters. Moreover, sometimes the pattern of GTPase clusters is dynamically regulated after its formation. This raises the question how the same interacting protein components can produce such a rich variety of naturally occurring patterns. Most currently used models for GTPase-based patterning inherently yield polarisation. Such models may at best yield transient coexistence of at most a few clusters, and hence fail to explain several important biological phenomena. These existing models are all based on mass conservation of total GTPase and some form of direct or indirect positive feedback. Here, we show that either of two biologically plausible modifications can yield stable coexistence: including explicit GTPase turnover, i.e., breaking mass conservation, or negative feedback by activation of an inhibitor like a GAP. Since we start from two different polarising models our findings seem independent of the precise self-activation mechanism. By studying the net GTPase flows among clusters, we provide insight into how these mechanisms operate. Our coexistence models also allow for dynamical regulation of the final pattern, which we illustrate with examples of pollen tube growth and the branching of fungal hyphae. Together, these results provide a better understanding of how cells can tune a single system to generate a wide variety of biologically relevant patterns., Competing Interests: The authors have declared that no competing interests exist.

In September 2018, conference organizers Nava Segev (University of Illinois, Chicago) and Marino Zerial (MPI, Dresden) hosted the 5th FASEB Meeting in Small GTPases in Membrane Processes: Trafficking, Autophagy and Disease at the National Conference Center in Leesburg, Virginia. With over 100 attendees from across the globe sharing their varied expertise and interests, we came together with the common goal of gaining a better understanding of how small GTPases and their regulators act in both canonical and non-canonical pathways to conduct a diversity of essential cellular functions. A broad range of disciplines was covered in this meeting, including the study of biophysical and structural properties of these proteins, functional studies to get at the roles of these proteins in various cellular contexts (eg, ciliary function, mitophagy, cell motility, cell cycle, and development), and translational approaches to understand the greater implications of small GTPases and their regulators in multicellular systems and disease pathology. This meeting provided attendees with the opportunity to discuss pressing questions that are driving the study of small GTPases and to explore directions for the future. Of particular note, both formal talks and informal discussions very clearly highlighted the clinical importance of these proteins and pathways, the ways in which cutting edge imaging technologies are expanding our understanding of them, and the need to work better in groups to tackle the larger questions of how GTPases contribute to cellular homeostasis or dysfunction. In this meeting report, we focus upon these three themes, as they have the potential to help shape our future studies of both the biology of small GTPases and their roles in a wide array of fundamental cellular functions., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Small GTPases regulate many aspects of cell logistics by alternating between an inactive, GDP-bound form and an active, GTP-bound form. This nucleotide switch is coupled to a cytosol/membrane cycle, such that GTP-bound small GTPases carry out their functions at the periphery of endomembranes. A global understanding of the molecular determinants of the interaction of small GTPases with membranes and of the resulting supramolecular organization is beginning to emerge from studies of model systems. Recent studies highlighted that small GTPases establish multiple interactions with membranes involving their lipid anchor, their lipididated hypervariable region and elements in their GTPase domain, which combine to determine the strength, specificity and orientation of their association with lipids. Thereby, membrane association potentiates small GTPase interactions with GEFs, GAPs and effectors through colocalization and positional matching. Furthermore, it leads to small GTPase nanoclustering and to lipid demixing, which drives the assembly of molecular platforms in which proteins and lipids co-operate in producing high-fidelity signals through feedback and feedforward loops. Although still fragmentary, these observations point to an integrated model of signaling by membrane-attached small GTPases that involves a diversity of direct and indirect interactions, which can inspire new therapeutic strategies to block their activities in diseases., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)