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Nucleoside triphosphates are indispensable in numerous biological processes, with enzymes involved in their biogenesis playing pivotal roles in cell proliferation. Pyruvate kinase (PYK), commonly regarded as the terminal glycolytic enzyme that generates ATP in tandem with pyruvate, is also capable of synthesizing a wide range of nucleoside triphosphates from their diphosphate precursors. Despite their substrate promiscuity, some PYKs show preference towards specific nucleotides, suggesting an underlying mechanism for differentiating nucleotide bases. However, the thorough characterization of this mechanism has been hindered by the paucity of nucleotide-bound PYK structures. Here, we present crystal structures of Streptococcus pneumoniae PYK in complex with four different nucleotides. These structures facilitate direct comparison of the protein-nucleotide interactions and offer structural insights into its pronounced selectivity for GTP synthesis. Notably, this selectivity is dependent on a sequence motif in the nucleotide recognition site that is widely present among prokaryotic PYKs, particularly in Firmicutes species. We show that pneumococcal cell growth is significantly impaired when expressing a PYK variant with compromised GTP and UTP synthesis activity, underscoring the importance of PYK in maintaining nucleotide homeostasis. Our findings collectively advance our understanding of PYK biochemistry and prokaryotic metabolism., 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 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Mycobacterial HflX confers resistance against macrolide antibiotics. However, the exact molecular mechanism is poorly understood. To gain further insights, we determined the cryo-EM structures of M. smegmatis (Msm) HflX-50S subunit and 50S subunit-erythromycin (ERY) complexes at a global resolution of approximately 3 Å. A conserved nucleotide A2286 at the gate of nascent peptide exit tunnel (NPET) adopts a swayed conformation in HflX-50S complex and interacts with a loop within the linker helical (LH) domain of MsmHflX that contains an additional 9 residues insertion. Interestingly, the swaying of this nucleotide, which is usually found in the non-swayed conformation, is induced by erythromycin binding. Furthermore, we observed that erythromycin decreases HflX's ribosome-dependent GTP hydrolysis, resulting in its enhanced binding and anti-association activity on the 50S subunit. Our findings reveal how mycobacterial HflX senses the presence of macrolides at the peptide tunnel entrance and confers antibiotic resistance in mycobacteria., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

First-generation KRAS G12C inhibitors, such as sotorasib and adagrasib, are limited by the depth and duration of clinical responses. One potential explanation for their modest clinical activity is the dynamic "cycling" of KRAS between its guanosine diphosphate (GDP)- and guanosine triphosphate (GTP)-bound states, raising controversy about whether targeting the GDP-bound form can fully block this oncogenic driver. We herein report that D3S-001, a next-generation GDP-bound G12C inhibitor with faster target engagement (TE) kinetics, depletes cellular active KRAS G12C at nanomolar concentrations. In the presence of growth factors, such as epithelial growth factor and hepatocyte growth factor, the ability of sotorasib and adagrasib to inhibit KRAS was compromised whereas the TE kinetics of D3S-001 was nearly unaffected, a unique feature differentiating D3S-001 from other GDP-bound G12C inhibitors. Furthermore, the high covalent potency and cellular TE efficiency of D3S-001 contributed to robust antitumor activity preclinically and translated into promising clinical efficacy in an ongoing phase 1 trial (NCT05410145). Significance: The kinetic study presented in this work unveils, for the first time, that a GDP-bound conformation-selective KRAS G12C inhibitor can potentially deplete cellular active KRAS in the presence of growth factors and offers new insights into the critical features that drive preclinical and clinical efficacy for this class of drugs., (©2024 The Authors; Published by the American Association for Cancer Research.)

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.)

By-products like CO₂ and organic acids, produced during Clostridium botulinum growth, appear to inhibit its development and reduce ATP production. A decrease in ATP production creates an imbalance in the ATP/GTP ratio. GTP activates CodY, which regulates BoNT expression. This toxin is released into the extracellular medium. Its light chains act as a specific endopeptidase, targeting SNARE proteins. The specific amino acids released enter the cells and are metabolized by the Stickland reaction, resulting in the synthesis of ATP. This ATP might then be used by histidine kinases to activate Spo0A, the main regulator initiating sporulation, through phosphorylation., Competing Interests: Declaration of competing interest No conflict of interest., (Copyright © 2024 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Metabolic dysregulation is one of the most common causes of pediatric neurodegenerative disorders. However, how the disruption of ubiquitous and essential metabolic pathways predominantly affect neural tissue remains unclear. Here we use mouse models of a childhood neurodegenerative disorder caused by AMPD2 deficiency to study cellular and molecular mechanisms that lead to selective neuronal vulnerability to purine metabolism imbalance. We show that mouse models of AMPD2 deficiency exhibit predominant degeneration of the hippocampal dentate gyrus, despite a general reduction of brain GTP levels. Neurodegeneration-resistant regions accumulate micron-sized filaments of IMPDH2, the rate limiting enzyme in GTP synthesis, while these filaments are barely detectable in the hippocampal dentate gyrus. Furthermore, we show that IMPDH2 filament disassembly reduces GTP levels and impairs growth of neural progenitor cells derived from individuals with human AMPD2 deficiency. Together, our findings suggest that IMPDH2 polymerization prevents detrimental GTP deprivation, opening the possibility of exploring the induction of IMPDH2 assembly as a therapy for neurodegeneration., (© 2024. The Author(s).)

Selective KRAS G12C inhibitors have been developed to covalently lock the oncogene in the inactive GDP-bound state. Two of these molecules, sotorasib and adagrasib, are approved for the treatment of adult patients with KRAS G12C -mutated previously treated advanced non-small cell lung cancer. Drug treatment imposes selective pressures leading to the outgrowth of drug-resistant variants. Mass sequencing from patients' biopsies identified a number of acquired KRAS mutations -both in cis and in trans- in resistant tumors. We demonstrate here that disease progression in vivo can also occur due to adaptive mechanisms and increased KRAS-GTP loading. Using the preclinical tool tri-complex KRAS G12C -selective covalent inhibitor, RMC-4998 (also known as RM-029), that targets the active GTP-bound (ON) state of the oncogene, we provide a proof-of-concept that the clinical stage KRAS G12C (ON) inhibitor RMC-6291 alone or in combination with KRAS G12C (OFF) drugs can be an alternative potential therapeutic strategy to circumvent resistance due to increased KRAS-GTP loading., (© 2024. The Author(s).)

RHOA mutations are found at diverse residues in various cancer types, implying mutation- and cell-specific mechanisms of tumorigenesis. Here, we focus on the underlying mechanisms of two gain-of-function RHOA mutations, A161P and A161V, identified in adult T-cell leukemia/lymphoma. We find that RHOA A161P and RHOA A161V are both fast-cycling mutants with increased guanine nucleotide dissociation/association rates compared with RHOA WT and show reduced GTP-hydrolysis activity. Crystal structures reveal an altered nucleotide association in RHOA A161P and an open nucleotide pocket in RHOA A161V . Both mutations perturb the dynamic properties of RHOA switch regions and shift the conformational landscape important for RHOA activity, as shown by 31 P NMR and molecular dynamics simulations. Interestingly, RHOA A161P and RHOA A161V can interact with effectors in the GDP-bound state. 1 H- 15 N HSQC NMR spectra support the existence of an active population in RHOA A161V -GDP. The distinct interaction mechanisms resulting from the mutations likely favor an RHOA WT -like "ON" conformation, endowing GDP-bound state effector binding activity., (© 2024. The Author(s).)

RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOS cat ) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOS cat . These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation., Competing Interests: Competing interests statement:The authors declare no competing interest.

The purine nucleotides ATP and GTP are made from the common precursor inosine monophosphate (IMP). Maintaining the correct balance of these nucleotides for optimal cell growth is controlled in part by the enzyme IMP dehydrogenase (IMPDH), which catalyzes the first dedicated step of GTP biosynthesis. The regulation of IMPDH mRNA and protein levels in the yeast S. cerevisiae grown in liquid culture has been studied in some detail, but regulation of IMPDH protein under conditions of cellular crowding on a solid substrate has not been examined. Here, we report real-time, live-cell analysis of the accumulation of the Imd2 isoform of IMPDH in yeast cells forming a monolayer colony in a microfluidic device over a 50-hour time course. We observe two distinct phases of increased Imd2 accumulation: a guanine-insensitive phase early in outgrowth and a guanine-sensitive phase later, when cells become crowded. We show that the IMPDH inhibitor mycophenolic acid enhances both phases of increase. Deletion of a transcription attenuator upstream of the mRNA start site that decreases Imd2 mRNA synthesis in the presence of high GTP increases the baseline level of Imd2 protein 10-fold and abolishes guanine-sensitive but not guanine-insensitive induction. Our results suggest that at least two mechanisms of yeast Imd2 regulation exist, the known GTP-dependent attenuation of RNA polymerase II elongation and a GTP concentration-independent pathway that may be controlled by cell growth state. Live-cell analysis of IMPDH protein levels in a growing yeast colony confirms a known mechanism of regulation and provides evidence for an additional mode of regulation., Importance: This study used live-cell microscopy to track changes in the level of a key enzyme in GTP nucleotide biosynthesis, inosine monophosphate dehydrogenase (IMPDH), during growth of a brewers yeast colony over 2 days in a microfluidic device. The results show that feedback regulation via transcription attenuation allows cells to adapt to nutrient limitation in the crowded environs of a yeast colony. They also identify a novel mode of regulation of IMPDH level that is not driven by guanine nucleotide availability., Competing Interests: The authors declare no conflict of interest.

RAS proteins are conserved guanosine triphosphate (GTP) hydrolases (GTPases) that act as molecular binary switches and play vital roles in numerous cellular processes. Upon GTP binding, RAS GTPases adopt an active conformation and interact with specific proteins termed RAS effectors that contain a conserved ubiquitin-like domain, thereby facilitating downstream signaling. Over 50 effector proteins have been identified in the human proteome, and many have been studied as potential mediators of RAS-dependent signaling pathways. Biochemical and structural analyses have provided mechanistic insights into these effectors, and studies using model organisms have complemented our understanding of their role in physiology and disease. Yet, many critical aspects regarding the dynamics and biological function of RAS-effector complexes remain to be elucidated. In this review, we discuss the mechanisms and functions of known RAS effector proteins, provide structural perspectives on RAS-effector interactions, evaluate their significance in RAS-mediated signaling, and explore their potential as therapeutic targets., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Allosteric regulation of inosine 5'-monophosphate dehydrogenase (IMPDH), an essential enzyme of purine metabolism, contributes to the homeostasis of adenine and guanine nucleotides. However, the precise molecular mechanism of IMPDH regulation in bacteria remains unclear. Using biochemical and cryo-EM approaches, we reveal the intricate molecular mechanism of the IMPDH allosteric regulation in mycobacteria. The enzyme is inhibited by both GTP and (p)ppGpp, which bind to the regulatory CBS domains and, via interactions with basic residues in hinge regions, lock the catalytic core domains in a compressed conformation. This results in occlusion of inosine monophosphate (IMP) substrate binding to the active site and, ultimately, inhibition of the enzyme. The GTP and (p)ppGpp allosteric effectors bind to their dedicated sites but stabilize the compressed octamer by a common mechanism. Inhibition is relieved by the competitive displacement of GTP or (p)ppGpp by ATP allowing IMP-induced enzyme expansion. The structural knowledge and mechanistic understanding presented here open up new possibilities for the development of allosteric inhibitors with antibacterial potential., (© 2024. The Author(s).)

Microtubules are dynamic polymers that interconvert between phases of growth and shrinkage, yet they provide structural stability to cells. Growth involves hydrolysis of GTP-tubulin to GDP-tubulin, which releases energy that is stored within the microtubule lattice and destabilizes it; a GTP cap at microtubule ends is thought to prevent GDP subunits from rapidly dissociating and causing catastrophe. Here, using in vitro reconstitution assays, we show that GDP-tubulin, usually considered inactive, can itself assemble into microtubules, preferentially at the minus end, and promote persistent growth. GDP-tubulin-assembled microtubules are highly stable, displaying no detectable spontaneous shrinkage. Strikingly, islands of GDP-tubulin within dynamic microtubules stop shrinkage events and promote rescues. Microtubules thus possess an intrinsic capacity for stability, independent of accessory proteins. This finding provides novel mechanisms to explain microtubule dynamics., (© 2024 Bagdadi et al.)

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).)

Small GTPases switch between GDP- and GTP-bound states during cell signaling. The ADP-ribosylation factor (ARF) family of small GTPases is involved in vesicle trafficking. Although evolutionarily well conserved, little is known about ARF and ARF-like GTPases in plants. We characterized biochemical properties and cellular localization of the essential small ARF-like GTPase TITAN 5 (TTN5; also known as HALLIMASCH, ARL2 and ARLC1) from Arabidopsis thaliana, and two TTN5 proteins with point mutants in conserved residues, TTN5T30N and TTN5Q70L, that were expected to be unable to perform nucleotide exchange and GTP hydrolysis, respectively. TTN5 exhibited very rapid intrinsic nucleotide exchange and remarkably low GTP hydrolysis activity, functioning as a non-classical small GTPase being likely present in a GTP-loaded active form. We analyzed signals from YFP-TTN5 and HA3-TTN5 by in situ immunolocalization in Arabidopsis seedlings and through use of a transient expression system. Colocalization with endomembrane markers and pharmacological treatments suggests that TTN5 can be present at the plasma membrane and that it dynamically associates with membranes of vesicles, Golgi stacks and multivesicular bodies. Although TTN5Q70L mirrored wild-type TTN5 behavior, the TTN5T30N mutant differed in some aspects. Hence, the unusual rapid nucleotide exchange activity of TTN5 is linked with its membrane dynamics, and TTN5 likely has a role in vesicle transport within the endomembrane system., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

A host of metabolic enzymes reversibly self-assemble to form membrane-less, intracellular filaments under normal physiological conditions and in response to stress. Often, these enzymes reside at metabolic control points, suggesting that filament formation affords an additional regulatory mechanism. Examples include cytidine-5'-triphosphate (CTP) synthase (CTPS), which catalyzes the rate-limiting step for the de novo biosynthesis of CTP; inosine-5'-monophosphate dehydrogenase (IMPDH), which controls biosynthetic access to guanosine-5'-triphosphate (GTP); and ∆ 1 -pyrroline-5-carboxylate (P5C) synthase (P5CS) that catalyzes the formation of P5C, which links the Krebs cycle, urea cycle, and proline metabolism. Intriguingly, CTPS can exist in co-assemblies with IMPDH or P5CS. Since GTP is an allosteric activator of CTPS, the association of CTPS and IMPDH filaments accords with the need to coordinate pyrimidine and purine biosynthesis. Herein, a hypothesis is presented furnishing a biochemical connection underlying co-assembly of CTPS and P5CS filaments - potent inhibition of CTPS by glutamate γ-semialdehyde, the open-chain form of P5C., (© 2024 The Author(s). BioEssays published by Wiley Periodicals LLC.)

Mitofusins (Mfn1 and Mfn2) are the mitochondrial outer-membrane fusion proteins in mammals and belong to the dynamin superfamily of multidomain GTPases. Recent structural studies of truncated variants lacking alpha helical transmembrane domains suggested that Mfns dimerize to promote the approximation and the fusion of the mitochondrial outer membranes upon the hydrolysis of guanine 5'-triphosphate disodium salt (GTP). However, next to the presence of GTP, the fusion activity seems to require multiple regulatory factors that control the dynamics and kinetics of mitochondrial fusion through the formation of Mfn1-Mfn2 heterodimers. Here, we purified and reconstituted the full-length murine Mfn2 protein into giant unilamellar vesicles (GUVs) with different lipid compositions. The incubation with GTP resulted in the fusion of Mfn2-GUVs. High-speed video-microscopy showed that the Mfn2-dependent membrane fusion pathway progressed through a zipper mechanism where the formation and growth of an adhesion patch eventually led to the formation of a membrane opening at the rim of the septum. The presence of physiological concentration (up to 30 mol%) of dioleoyl-phosphatidylethanolamine (DOPE) was shown to be a requisite to observe GTP-induced Mfn2-dependent fusion. Our observations show that Mfn2 alone can promote the fusion of micron-sized DOPE-enriched vesicles without the requirement of regulatory cofactors, such as membrane curvature, or the assistance of other proteins., Competing Interests: Competing interests statement:The authors declare no competing interest.

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.)

The wildtype H-Ras protein functions as a molecular switch in a variety of cell signaling pathways, and mutations to key residues result in a constitutively active oncoprotein. However, there is some debate regarding the mechanism of the intrinsic GTPase activity of H-Ras. It has been hypothesized that ordered water molecules are coordinated at the active site by Q61, a highly transforming amino acid site, and Y32, a position that has not previously been investigated. Here, we examine the electrostatic contribution of the Y32 position to GTP hydrolysis by comparing the rate of GTP hydrolysis of Y32X mutants to the vibrational energy shift of each mutation measured by a nearby thiocyanate vibrational probe to estimate changes in the electrostatic environment caused by changes at the Y32 position. We further compared vibrational energy shifts for each mutation to the hydration potential of the respective side chain and demonstrated that Y32 is less critical for recruiting water molecules into the active site to promote hydrolysis than Q61. Our results show a clear interplay between a steric contribution from Y32 and an electrostatic contribution from Q61 that are both critical for intrinsic GTP hydrolysis.

FtsZ is highly conserved among bacteria and plays an essential role in bacterial cell division. The tense conformation of FtsZ bound to GTP assembles into a straight filament via head-to-tail associations, and then the upper subunit of FtsZ hydrolyzes GTP bound to the lower FtsZ subunit. The subunit with GDP bound disassembles accompanied by a conformational change in the subunit from the tense to relaxed conformation. Although crystal structures of FtsZ derived from several bacterial species have been determined, the conformational change from the relaxed to tense conformation has only been observed in Staphylococcus aureus FtsZ (SaFtsZ). Recent cryo-electron microscopy analyses revealed the three-dimensional reconstruction of the protofilament, in which tense molecules assemble via head-to-tail associations. However, the lower resolution of the protofilament suggested that the flexibility of the FtsZ protomers between the relaxed and tense conformations caused them to form in less-strict alignments. Furthermore, this flexibility may also prevent FtsZs other than SaFtsZ from crystalizing in the tense conformation, suggesting that the flexibility of bacterial FtsZs differs. In this study, molecular dynamics simulations were performed using SaFtsZ and Bacillus subtilis FtsZ in several situations, which suggested that different features of the FtsZs affect their conformational stability., (© 2024. The Author(s).)

In many animal species, the oocyte meiotic spindle, which is required for chromosome segregation, forms without centrosomes. In some systems, Ran-GEF on chromatin initiates spindle assembly. We found that in Caenorhabditis elegans oocytes, endogenously-tagged Ran-GEF dissociates from chromatin during spindle assembly but re-associates during meiotic anaphase. Meiotic spindle assembly occurred after auxin-induced degradation of Ran-GEF, but anaphase I was faster than controls and extrusion of the first polar body frequently failed. In search of a possible alternative pathway for spindle assembly, we found that soluble tubulin concentrates in the nuclear volume during germinal vesicle breakdown. We found that the concentration of soluble tubulin in the metaphase spindle region is enclosed by ER sheets which exclude cytoplasmic organelles including mitochondria and yolk granules. Measurement of the volume occupied by yolk granules and mitochondria indicated that volume exclusion would be sufficient to explain the concentration of tubulin in the spindle volume. We suggest that this concentration of soluble tubulin may be a redundant mechanism promoting spindle assembly near chromosomes., (© 2024 Gong et al.)

κB-Ras (NF-κB inhibitor-interacting Ras-like protein) GTPases are small Ras-like GTPases but harbor interesting differences in important sequence motifs. They act in a tumor-suppressive manner as negative regulators of Ral (Ras-like) GTPase and NF-κB signaling, but little is known about their mode of function. Here, we demonstrate that, in contrast to predictions based on primary structure, κB-Ras GTPases possess hydrolytic activity. Combined with low nucleotide affinity, this renders them fast-cycling GTPases that are predominantly GTP-bound in cells. We characterize the impact of κB-Ras mutations occurring in tumors and demonstrate that nucleotide binding affects κB-Ras stability but is not strictly required for RalGAP (Ral GTPase-activating protein) binding. This demonstrates that κB-Ras control of RalGAP/Ral signaling occurs in a nucleotide-binding- and switch-independent fashion., (© 2024 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Eukaryotic and archaeal translation initiation factor 2 in complex with GTP delivers the initiator methionyl-tRNA to the small ribosomal subunit. Over the past 20 years, thanks to the efforts of various research groups, including ours, this factor from the archaeon Sulfolobus solfataricus and its individual subunits have been crystallized in ten different space groups. Analysis of the molecular packing in these crystals makes it possible to better understand the roles of functionally significant switches and other elements of the nucleotide-binding pocket during the function of the factor as well as the influence of external effects on its transition between active and inactive states.

Understanding the diversity of DNA structure and functions in biology requires tools to study this biomolecule selectively and thoroughly. Fluorescence methods are powerful technique for non-invasive research. Due to the low quantum yield, the intrinsic fluorescence of nucleotides has not been considered for use in the detection and differentiation of nucleic acid bases. Here, we have studied the influence of protonation of nucleotides on their fluorescence properties. We show that protonation of ATP and GTP leads to enhanced intrinsic fluorescence. Fluorescence enhancement at acidic pH has been observed for double-stranded DNA and single-stranded oligonucleotides. The formation of G4 secondary structures apparently protected certain nucleotides from protonation, resulting in less pronounced fluorescence enhancement. Furthermore, acid-induced depurination under protonation was less noticeable in G4 structures than in double-stranded and single-stranded DNA. We show that changes in the intrinsic fluorescence of guanine can be used as a sensitive sensor for changes in the structure of the DNA and for the protonation of specific nucleotides., (Copyright © 2024 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

MutT proteins belong to the Nudix hydrolase superfamily that includes a diverse group of Mg 2+ requiring enzymes. These proteins use a generalized substrate, nucleoside diphosphate linked to a chemical group X (NDP-X), to produce nucleoside monophosphate (NMP) and the moiety X linked with phosphate (XP). E. coli MutT (EcoMutT) and mycobacterial MutT1 (MsmMutT1) belong to the Nudix hydrolase superfamily that utilize 8-oxo-(d)GTP (referring to both 8-oxo-GTP or 8-oxo-dGTP). However, predominant products of their activities are different. While EcoMutT produces 8-oxo-(d)GMP, MsmMutT1 gives rise to 8-oxo-(d)GDP. Here, we show that the altered cleavage specificities of the two proteins are largely a consequence of the variation at the equivalent of Gly37 (G37) in EcoMutT to Lys (K65) in the MsmMutT1. Remarkably, mutations of G37K (EcoMutT) and K65G (MsmMutT1) switch their cleavage specificities to produce 8-oxo-(d)GDP, and 8-oxo-(d)GMP, respectively. Further, a time course analysis using 8-oxo-GTP suggests that MsmMutT1(K65G) hydrolyses 8-oxo-(d)GTP to 8-oxo-(d)GMP in a two-step reaction via 8-oxo-(d)GDP intermediate. Expectedly, unlike EcoMutT (G37K) and MsmMutT1, EcoMutT and MsmMutT1 (K65G) rescue an E. coli ΔmutT strain, better by decreasing A to C mutations., Competing Interests: Declaration of Competing Interest None to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Bacterial cell division is crucial for replication and requires careful coordination via proteins collectively called the divisome. The tubulin-like GTPase FtsZ is the master regulator of this process and serves to recruit downstream divisome proteins and regulate their activities. Upon assembling at mid-cell, FtsZ exhibits treadmilling motion driven by GTP binding and hydrolysis. Treadmilling is proposed to play roles in Z-ring condensation and in distribution and regulation of peptidoglycan (PG) cell wall enzymes. FtsZ polymer superstructure and dynamics are central to its function, yet their regulation is incompletely understood. We addressed these gaps in knowledge by evaluating the contribution of GTPase activity to FtsZ's function in vitro and in Caulobacter crescentus cells. We observed that a lethal mutation that abrogates FtsZ GTP hydrolysis impacts FtsZ dynamics and Z-ring positioning, but not constriction. Aberrant Z-ring positioning was due to insensitivity to the FtsZ regulator MipZ when GTPase activity is reduced. Z-ring mislocalization resulted in DNA damage, likely due to constriction over the nucleoid. Collectively, our results indicate that GTP hydrolysis serves primarily to position the Z-ring at mid-cell in Caulobacter . Proper Z-ring localization is required for effective coordination with chromosome segregation to prevent DNA damage and ensure successful cell division.

How nucleocytoplasmic transport (NCT) rates change due to cellular physiology-mediated fluctuations in GTP availability remains unclear. In this issue, Scott et al. (https://doi.org/10.1083/jcb.202308152) demonstrate that cell migration, spreading, and nucleocytoskeletal coupling impact GTP levels, thereby regulating NCT, RNA export, and protein synthesis., (© 2024 Luxton.)

Nucleocytoplasmic transport (NCT), the facilitated diffusion of cargo molecules between the nucleus and cytoplasm through nuclear pore complexes (NPCs), enables numerous fundamental eukaryotic cellular processes. Ran GTPase uses cellular energy in the direct form of GTP to create a gradient across the nuclear envelope (NE) that drives the majority of NCT. We report here that changes in GTP availability resulting from altered cellular physiology modulate the rate of NCT, as monitored using synthetic and natural cargo, and the dynamics of Ran itself. Cell migration, cell spreading, and/or modulation of the cytoskeleton or its connection to the nucleus alter GTP availability and thus rates of NCT, regulating RNA export and protein synthesis. These findings support a model in which changes in cellular physiology that alter GTP availability can regulate the rate of NCT, impacting fundamental cellular processes that extensively utilize NCT., (© 2024 Scott et al.)

Gliomas are the most common primary tumors of the central nervous system, with approximately half of patients presenting with the most aggressive form of glioblastoma. Although several molecular markers for glioma have been identified, they are not sufficient to predict the prognosis due to the extensive genetic heterogeneity within glioma. Our study reveals that the ratio of IMPDH1 to IMPDH2 expression levels serves as a molecular indicator for glioma treatment prognosis. Patients with a higher IMPDH1/IMPDH2 ratio exhibit a worse prognosis, while those with a lower ratio display a more favorable prognosis. We further demonstrate that IMPDH1 plays a crucial role in maintaining cellular GTP/GDP levels following DNA damage compared to IMPDH2. In the absence of IMPDH1, cells experience an imbalance in the GTP/GDP ratio, impairing DNA damage repair capabilities and rendering them more sensitive to TMZ. This study not only introduces a novel prognostic indicator for glioma clinical diagnosis but also offers innovative insights for precise and stratified glioma treatment., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Continuous fusion and fission are critical for mitochondrial health. In this study, we further characterize the role played by dynamin-related protein 1 (Drp1) in mitochondrial fission. We show that a single amino acid change in Drp1 at position 39 from serine to alanine (S39A) within the GTP-binding (GTPase) domain results in a fused mitochondrial network in human SH-SY5Y neuroblastoma cells. Interestingly, the phosphorylation of Ser-616 and Ser-637 of Drp1 remains unaffected by the S39A mutation, and mitochondrial bioenergetic profile and cell viability in the S39A mutant were comparable to those observed in the control. This leads us to propose that the serine 39 residue of Drp1 plays a crucial role in mitochondrial distribution through its involvement in the GTPase activity. Furthermore, this amino acid mutation leads to structural anomalies in the mitochondrial network. Taken together, our results contribute to a better understanding of the function of the Drp1 protein., (© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

As the important hub of many cellular signaling networks, KRAS (Kirsten rat sarcoma viral oncogene homologue) has been identified as a tumor biomarker. It is the frequently mutated oncogene in human cancers, and KRAS protein activation caused by mutations, such as G12D, has been found in many human tumors tissues. Although, there are two specific allosteric sites (AS1 and AS2) on the KRAS protein that can be used as the targets for inhibitor development, the difference of regulatory mechanisms between two individual allosteric sites still not be reported. Here, using molecular dynamics simulations combined with molecular mechanics generalized born surface area (MM/GBSA) analysis, we found that both of the inhibitors, located at AS1 and AS2, were able to reduce the binding free energy between wild type, mutant KRAS (G12/D/V/S/C) and GTP remarkably, however the effect of inhibitors on the binding free energy between wild type, mutant KRAS and GDP was limited. In addition, the degree of decrease of binding free energy between KRAS and GTP caused by inhibitors at AS2 was significantly greater than that caused by inhibitors at AS1. Further analysis revealed that both inhibitors at AS1 and AS2 were able to regulate the fluctuation of Switch Ⅰ and Switch Ⅱ to expand the pocket of the orthosteric site (GTP binding site), thereby reducing the binding of KRAS to GTP. Noteworthy there was significant differences in the regulatory preferences on Switch Ⅰ and Switch Ⅱ between two type inhibitor. The inhibitor at AS2 mainly regulated Switch Ⅱ to affect the pocket of the orthosteric site, while the inhibitor at AS1 mainly expand the pocket of the orthosteric site by regulating the fluctuation of Switch Ⅰ. Our study compared the differences between two type inhibitors in regulating the KRAS protein activity and revealed the advantages of the AS2 as the small molecule drug target, aiming to provide theoretical guidance for the research of novel KRAS protein inhibitors., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Many GTPases have been shown to utilize ATP too as the phosphoryl donor. Both GTP and ATP are important molecules in the cellular environments and play multiple and discrete functional role within the cells. In our present study, we showed that one of the purine metabolic enzymes Adenylosuccinate synthetase from Leishmania donovani (LdAdSS) which belongs to the BioD-superfamily of GTPases can also carry out the catalysis by hydrolysing ATP instead of its cognate substrate GTP albeit with less efficiency. Biochemical and biophysical studies indicated its ability to bind to ATP too but at a higher concentration of ATP compared to that of GTP. Sequence analysis and molecular dynamic simulations suggested that residues of the switch loop and the G4-G5 ( 593 SAXD 596 ) connected motif of LdAdSS plays a role in determining the nucleotide specificity. Though the crucial interaction between Asp596 and the nucleotide is broken when ATP is bound, interactions between the Ala594 and the adenine ring of ATP could still hold ATP in the GTP binding site. The results of the present study suggested that though LdAdSS is GTP specific, it still shows ATP hydrolysing activity., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr Anju Pappachan reports was provided by Central University of Gujarat. Mochi Jigneshkumar Amritlal reports was provided by Central University of Gujarat. Jani Jaykumar reports was provided by Central University of Gujarat. Tak Kiran reports was provided by Central University of Gujarat. 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 Elsevier Inc. All rights reserved.)

RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985)., Significance: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897., (©2024 The Authors; Published by the American Association for Cancer Research.)

The RAS isoforms (KRAS, HRAS and NRAS) have distinct cancer type-specific profiles. NRAS mutations are the second most prevalent RAS mutations in skin and hematological malignancies. Although RAS proteins were considered undruggable for decades, isoform and mutation-specific investigations have produced successful RAS inhibitors that are either specific to certain mutants, isoforms (pan-KRAS) or target all RAS proteins (pan-RAS). While extensive structural and biochemical investigations have focused mainly on K- and H-RAS mutations, NRAS mutations have received less attention, and the most prevalent NRAS mutations in human cancers, Q61K and Q61R, are rare in K- and H-RAS. This manuscript presents a crystal structure of the NRAS Q61K mutant in the GTP-bound form. Our structure reveals a previously unseen pocket near switch II induced by the binding of a ligand to the active form of the protein. This observation reveals a binding site that can potentially be exploited for development of inhibitors against mutant NRAS. Furthermore, the well-resolved catalytic site of this GTPase bound to native GTP provides insight into the stalled GTP hydrolysis observed for NRAS-Q61K., 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 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Membrane protrusions are fundamental to cellular functions like migration, adhesion, and communication and depend upon dynamic reorganization of the cytoskeleton. GAP-dependent GTP hydrolysis of Arf proteins regulates actin-dependent membrane remodeling. Here, we show that dAsap regulates membrane protrusions in S2R+ cells by a mechanism that critically relies on its ArfGAP domain and relocalization of actin regulators, SCAR, and Ena. While our data reinforce the preference of dAsap for Arf1 GTP hydrolysis in vitro, we demonstrate that induction of membrane protrusions in S2R+ cells depends on Arf6 inactivation. This study furthers our understanding of how dAsap-dependent GTP hydrolysis maintains a balance between active and inactive states of Arf6 to regulate cell shape., (© 2024 Federation of European Biochemical Societies.)

The biosynthesis pathways of coenzyme A (CoA) in most archaea involve several unique enzymes including dephospho-CoA kinase (DPCK) that converts dephospho-CoA to CoA in the final step of CoA biosynthesis in all domains of life. The archaeal DPCK is unrelated to the analogous bacterial and eukaryotic enzymes and shows no significant sequence similarity to any proteins with known structures. Unusually, the archaeal DPCK utilizes GTP as the phosphate donor although the analogous bacterial and eukaryotic enzymes are ATP-dependent kinases. Here, we report the crystal structure of DPCK and its complex with GTP and a magnesium ion from the archaeal hyperthermophile Thermococcus kodakarensis. The crystal structure demonstrates why GTP is the preferred substrate of this kinase. We also report the activity analyses of site-directed mutants of crucial residues determined based on sequence conservation and the crystal structure. From these results, the key residues involved in the reaction of phosphoryl transfer and the possible dephospho-CoA binding site are inferred., (© 2024 Wiley Periodicals LLC.)

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.)

Human K-Ras protein, which is a member of the GTPase Ras family, hydrolyzes GTP to GDP and concomitantly converts from its active to its inactive state. It is a key oncoprotein, because several mutations, particularly those at residue position 12, occur with a high frequency in a wide range of human cancers. The K-Ras protein is therefore an important target for developing therapeutic anti-cancer agents. In this work we report the almost complete sequence-specific resonance assignments of wild-type and the oncogenic G12C and G12D mutants in the GTP-complexed active forms, including the functionally important Switch I and Switch II regions. These assignments serve as the basis for a comprehensive functional dynamics study of wild-type K-Ras and its G12 mutants., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20-30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment., Competing Interests: Declaration of Competing interest The authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cell motility universally relies on spatial regulation of focal adhesion complexes (FAs) connecting the substrate to cellular motors. In bacterial FAs, the Adventurous gliding motility machinery (Agl-Glt) assembles at the leading cell pole following a Mutual gliding-motility protein (MglA)-guanosine 5'-triphosphate (GTP) gradient along the cell axis. Here, we show that GltJ, a machinery membrane protein, contains cytosolic motifs binding MglA-GTP and AglZ and recruiting the MreB cytoskeleton to initiate movement toward the lagging cell pole. In addition, MglA-GTP binding triggers a conformational shift in an adjacent GltJ zinc-finger domain, facilitating MglB recruitment near the lagging pole. This prompts GTP hydrolysis by MglA, leading to complex disassembly. The GltJ switch thus serves as a sensor for the MglA-GTP gradient, controlling FA activity spatially.

The phosphorylation of different sites produces a significant effect on the conformational dynamics of KRAS. Gaussian accelerated molecular dynamics (GaMD) simulations were combined with deep learning (DL) to explore the molecular mechanism of the phosphorylation-mediated effect on conformational dynamics of the GTP-bound KRAS. The DL finds that the switch domains are involved in obvious differences in conformation contacts and suggests that the switch domains play a key role in the function of KRAS. The analyses of free energy landscapes (FELs) reveal that the phosphorylation of pY32, pY64, and pY137 leads to more disordered states of the switch domains than the wild-type (WT) KRAS and induces conformational transformations between the closed and open states. The results from principal component analysis (PCA) indicate that principal motions PC1 and PC2 are responsible for the closed and open states of the phosphorylated KRAS. Interaction networks were analyzed and the results verify that the phosphorylation alters interactions of GTP and magnesium ion Mg 2+ with the switch domains. It is concluded that the phosphorylation pY32, pY64, and pY137 tune the activity of KRAS through changing conformational dynamics and interactions of the switch domains. We anticipated that this work could provide theoretical aids for deeply understanding the function of KRAS.

The cytoophidium is an evolutionarily conserved subcellular structure formed by filamentous polymers of metabolic enzymes. In vertebrates, inosine monophosphate dehydrogenase (IMPDH), which catalyses the rate-limiting step in guanosine triphosphate (GTP) biosynthesis, is one of the best-known cytoophidium-forming enzymes. Formation of the cytoophidium has been proposed to alleviate the inhibition of IMPDH, thereby facilitating GTP production to support the rapid proliferation of certain cell types such as lymphocytes, cancer cells and pluripotent stem cells (PSCs). However, past studies lacked appropriate models to elucidate the significance of IMPDH cytoophidium under normal physiological conditions. In this study, we demonstrate that the presence of IMPDH cytoophidium in mouse PSCs correlates with their metabolic status rather than pluripotency. By introducing IMPDH2 Y12C point mutation through genome editing, we established mouse embryonic stem cell (ESC) lines incapable of forming IMPDH polymers and the cytoophidium. Our data indicate an important role of IMPDH cytoophidium in sustaining a positive feedback loop that couples nucleotide biosynthesis with upstream metabolic pathways. Additionally, we find that IMPDH2 Y12C mutation leads to decreased cell proliferation and increased DNA damage in teratomas, as well as impaired embryo development following blastocoel injection. Further analysis shows that IMPDH cytoophidium assembly in mouse embryonic development begins after implantation and gradually increases throughout fetal development. These findings provide insights into the regulation of IMPDH polymerisation in embryogenesis and its significance in coordinating cell metabolism and development., (© 2024. The Author(s).)

Rab6 is a key modulator of protein secretion. The dynein adapter Bicaudal D2 (BicD2) recruits the motors cytoplasmic dynein and kinesin-1 to Rab6 GTP -positive vesicles for transport; however, it is unknown how BicD2 recognizes Rab6. Here, we establish a structural model for recognition of Rab6 GTP by BicD2, using structure prediction and mutagenesis. The binding site of BicD2 spans two regions of Rab6 that undergo structural changes upon the transition from the GDP- to GTP-bound state, and several hydrophobic interface residues are rearranged, explaining the increased affinity of the active GTP-bound state. Mutations of Rab6 GTP that abolish binding to BicD2 also result in reduced co-migration of Rab6 GTP /BicD2 in cells, validating our model. These mutations also severely diminished the motility of Rab6-positive vesicles in cells, highlighting the importance of the Rab6 GTP /BicD2 interaction for overall motility of the multi-motor complex that contains both kinesin-1 and dynein. Our results provide insights into trafficking of secretory and Golgi-derived vesicles and will help devise therapies for diseases caused by BicD2 mutations, which selectively affect the affinity to Rab6 and other cargoes., (© 2024 Zhao et al.)

RAS oncogenes (collectively NRAS, HRAS and especially KRAS) are among the most frequently mutated genes in cancer, with common driver mutations occurring at codons 12, 13 and 61 1 . Small molecule inhibitors of the KRAS(G12C) oncoprotein have demonstrated clinical efficacy in patients with multiple cancer types and have led to regulatory approvals for the treatment of non-small cell lung cancer 2,3 . Nevertheless, KRAS G12C mutations account for only around 15% of KRAS-mutated cancers 4,5 , and there are no approved KRAS inhibitors for the majority of patients with tumours containing other common KRAS mutations. Here we describe RMC-7977, a reversible, tri-complex RAS inhibitor with broad-spectrum activity for the active state of both mutant and wild-type KRAS, NRAS and HRAS variants (a RAS(ON) multi-selective inhibitor). Preclinically, RMC-7977 demonstrated potent activity against RAS-addicted tumours carrying various RAS genotypes, particularly against cancer models with KRAS codon 12 mutations (KRAS G12X ). Treatment with RMC-7977 led to tumour regression and was well tolerated in diverse RAS-addicted preclinical cancer models. Additionally, RMC-7977 inhibited the growth of KRAS G12C cancer models that are resistant to KRAS(G12C) inhibitors owing to restoration of RAS pathway signalling. Thus, RAS(ON) multi-selective inhibitors can target multiple oncogenic and wild-type RAS isoforms and have the potential to treat a wide range of RAS-addicted cancers with high unmet clinical need. A related RAS(ON) multi-selective inhibitor, RMC-6236, is currently under clinical evaluation in patients with KRAS-mutant solid tumours (ClinicalTrials.gov identifier: NCT05379985)., (© 2024. The Author(s).)

G-protein-coupled receptors (GPCRs) activate heterotrimeric G proteins by stimulating guanine nucleotide exchange in the Gα subunit 1 . To visualize this mechanism, we developed a time-resolved cryo-EM approach that examines the progression of ensembles of pre-steady-state intermediates of a GPCR-G-protein complex. By monitoring the transitions of the stimulatory G s protein in complex with the β 2 -adrenergic receptor at short sequential time points after GTP addition, we identified the conformational trajectory underlying G-protein activation and functional dissociation from the receptor. Twenty structures generated from sequential overlapping particle subsets along this trajectory, compared to control structures, provide a high-resolution description of the order of main events driving G-protein activation in response to GTP binding. Structural changes propagate from the nucleotide-binding pocket and extend through the GTPase domain, enacting alterations to Gα switch regions and the α5 helix that weaken the G-protein-receptor interface. Molecular dynamics simulations with late structures in the cryo-EM trajectory support that enhanced ordering of GTP on closure of the α-helical domain against the nucleotide-bound Ras-homology domain correlates with α5 helix destabilization and eventual dissociation of the G protein from the GPCR. These findings also highlight the potential of time-resolved cryo-EM as a tool for mechanistic dissection of GPCR signalling events., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Broad-spectrum RAS inhibition has the potential to benefit roughly a quarter of human patients with cancer whose tumours are driven by RAS mutations 1,2 . RMC-7977 is a highly selective inhibitor of the active GTP-bound forms of KRAS, HRAS and NRAS, with affinity for both mutant and wild-type variants 3 . More than 90% of cases of human pancreatic ductal adenocarcinoma (PDAC) are driven by activating mutations in KRAS 4 . Here we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models. We observed broad and pronounced anti-tumour activity across models following direct RAS inhibition at exposures that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumour versus normal tissues. Treated tumours exhibited waves of apoptosis along with sustained proliferative arrest, whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. In the autochthonous KPC mouse model, RMC-7977 treatment resulted in a profound extension of survival followed by on-treatment relapse. Analysis of relapsed tumours identified Myc copy number gain as a prevalent candidate resistance mechanism, which could be overcome by combinatorial TEAD inhibition in vitro. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS-GTP inhibition in the setting of PDAC and identify a promising candidate combination therapeutic regimen to overcome monotherapy resistance., (© 2024. The Author(s).)

Roco proteins entered the limelight after mutations in human LRRK2 were identified as a major cause of familial Parkinson's disease. LRRK2 is a large and complex protein combining a GTPase and protein kinase activity, and disease mutations increase the kinase activity, while presumably decreasing the GTPase activity. Although a cross-communication between both catalytic activities has been suggested, the underlying mechanisms and the regulatory role of the GTPase domain remain unknown. Several structures of LRRK2 have been reported, but structures of Roco proteins in their activated GTP-bound state are lacking. Here, we use single-particle cryo-electron microscopy to solve the structure of a bacterial Roco protein (CtRoco) in its GTP-bound state, aided by two conformation-specific nanobodies: Nb Roco1 and Nb Roco2 . This structure presents CtRoco in an active monomeric state, featuring a very large GTP-induced conformational change using the LRR-Roc linker as a hinge. Furthermore, this structure shows how Nb Roco1 and Nb Roco2 collaborate to activate CtRoco in an allosteric way. Altogether, our data provide important new insights into the activation mechanism of Roco proteins, with relevance to LRRK2 regulation, and suggest new routes for the allosteric modulation of their GTPase activity., Competing Interests: CG, GG, MF, CG, WV No competing interests declared, (© 2024, Galicia et al.)

Although the small GTPase RAB37 acts as an organizer of autophagosome biogenesis, the upstream regulatory mechanism of autophagy via guanosine diphosphate (GDP)-guanosine triphosphate (GTP) exchange in maintaining retinal function has not been determined. We found that retinitis pigmentosa GTPase regulator (RPGR) is a guanine nucleotide exchange factor that activates RAB37 by accelerating GDP-to-GTP exchange. RPGR directly interacts with RAB37 via the RPGR-RCC1-like domain to promote autophagy through stimulating exchange. Rpgr knockout (KO) in mice leads to photoreceptor degeneration owing to autophagy impairment in the retina. Notably, the retinopathy phenotypes of Rpgr KO retinas are rescued by the adeno-associated virus-mediated transfer of pre-trans-splicing molecules, which produce normal Rpgr mRNAs via trans-splicing in the Rpgr KO retinas. This rescue upregulates autophagy through the re-expression of RPGR in KO retinas to accelerate GDP-to-GTP exchange; thus, retinal homeostasis reverts to normal. Taken together, these findings provide an important missing link for coordinating RAB37 GDP-GTP exchange via the RPGR and retinal homeostasis by autophagy regulation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)