Nucleotide Exchange on RAS Proteins Using Hydrolysable and Non-hydrolysable Nucleotides.

Biochemical and biophysical assays using recombinant RAS require the protein to be in either the active or inactive state. Here we describe methods to exchange the nucleotide present in the purified RAS protein with either GDPßS, GppNHp, or GTP depending on the assay requirement. In addition, we also describe the HPLC method used to validate the exchange process and provide information on the efficiency of the nucleotide exchange.

Measurement of KRAS-GTPase Activity.

Oncogenic mutations in KRAS typically impact the GAP-mediated and intrinsic GTP hydrolysis activity resulting in elevated levels of cellular KRAS-GTP. The development of biochemical assays for GTPase activity provides an opportunity to quantitatively measure the impact of these mutations on GTP hydrolysis. Here we describe a biochemical assay that measures the release of free phosphate upon hydrolysis of the GTP nucleotide and allows the measurement of intrinsic or GAP-stimulated GTP hydrolysis by KRAS. This assay can be used to measure GTPase activity under single turnover conditions.

Biophysical Characterization of RAS-SOS Complexes by Native Mass Spectrometry.

RAS is regulated by specific guanine nucleotide exchange factors, such as Son of Sevenless (SOS), that activates RAS by facilitating the exchange of inactive, GDP-bound RAS with GTP. The catalytic activity of SOS is known to be allosterically modulated by an active, GTP-bound RAS. However, it remains poorly understood how oncogenic RAS mutants interact with SOS and modulate its activity. In this chapter, we describe the application of native mass spectrometry (MS) to monitor the assembly of the catalytic domain of SOS (SOScat) with RAS and cancer-associated mutants. Results from this approach have led to the discovery of different molecular assemblies and distinct conformers of SOScat engaging KRAS. It was also found that KRASG13D exhibits high affinity for SOScat and is a potent allosteric modulator of its SOScat activity. KRASG13D-GTP can allosterically increase the nucleotide exchange rate of KRAS at the active site by more than twofold compared to the wild-type protein. Furthermore, small-molecule RAS•SOS disruptors fail to dissociate KRASG13D•SOScat complexes, underscoring the need for more potent disruptors targeting oncogenic RAS mutants. Taken together, native MS will be instrumental in better understanding the interaction between oncogenic RAS mutants and SOS, which is of crucial importance for development of improved therapeutics.

Real-Time Monitoring of RAS Activity Using In Vitro and In-Cell NMR Spectroscopy.

The activation level of RAS can be determined by GTP hydrolysis rate (khy) and GDP-GTP exchange rates (kex). Either impaired GTP hydrolysis or enhanced GDP-GTP exchange causes the aberrant activation of RAS in oncogenic mutants. Therefore, it is important to quantify the khy and kex for understanding the mechanisms of RAS oncogenesis and drug development. Conventional methods have individually measured the kex and khy of RAS. However, within the intracellular environment, GTP hydrolysis and GDP-GTP exchange reactions occur simultaneously under conditions where GTP concentration is kept constant. In addition, the intracellular activity of RAS is influenced by endogenous regulatory proteins, such as RAS GTPase activating proteins (GAPs) and the guanine-nucleotide exchange factors (GEFs). Here, we describe the in vitro and in-cell NMR methods to estimate the khy and kex simultaneously by measuring the time-dependent changes of the fraction of GTP-bound ratio under the condition of constant GTP concentration.

Molecular dynamics investigation of DNA fragments bound to the anti-HIV protein SAMHD1 reveals alterations in allosteric communications.

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.

Cryo-EM of the Nucleosome Core Particle Bound to Ran-RCC1 Reveals a Dynamic Complex.

Ras-related nuclear protein (Ran) is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) and a regulator of multiple cellular processes. In healthy cells, the GTP-bound form of Ran is concentrated at chromatin, creating a Ran•GTP gradient that provides the driving force for nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation. The Ran•GTP gradient is maintained by the regulator of chromatin condensation 1 (RCC1), a guanine nucleotide exchange factor that accelerates GDP/GTP exchange in Ran. RCC1 interacts with nucleosomes, which are the fundamental repeating units of eukaryotic chromatin. Here, we present a cryo-EM analysis of a trimeric complex composed of the nucleosome core particle (NCP), RCC1, and Ran. While the contacts between RCC1 and Ran in the complex are preserved compared with a previously determined structure of RCC1-Ran, our study reveals that RCC1 and Ran interact dynamically with the NCP and undergo rocking motions on the nucleosome surface. Furthermore, the switch 1 region of Ran, which plays an important role in mediating conformational changes associated with the substitution of GDP and GTP nucleotides in Ras family members, appears to undergo disorder-order transitions and forms transient contacts with the C-terminal helix of histone H2B. Nucleotide exchange assays performed in the presence and absence of NCPs are not consistent with an active role for nucleosomes in nucleotide exchange, at least in vitro. Instead, the nucleosome stabilizes RCC1 and serves as a hub that concentrates RCC1 and Ran to promote efficient Ran•GDP to Ran•GTP conversion.

Structural and mechanistic insights into activation of the human RNA ligase RTCB by Archease.

RNA ligases of the RTCB-type play an essential role in tRNA splicing, the unfolded protein response and RNA repair. RTCB is the catalytic subunit of the pentameric human tRNA ligase complex. RNA ligation by the tRNA ligase complex requires GTP-dependent activation of RTCB. This active site guanylylation reaction relies on the activation factor Archease. The mechanistic interplay between both proteins has remained unknown. Here, we report a biochemical and structural analysis of the human RTCB-Archease complex in the pre- and post-activation state. Archease reaches into the active site of RTCB and promotes the formation of a covalent RTCB-GMP intermediate through coordination of GTP and metal ions. During the activation reaction, Archease prevents futile RNA substrate binding to RTCB. Moreover, monomer structures of Archease and RTCB reveal additional states within the RNA ligation mechanism. Taken together, we present structural snapshots along the reaction cycle of the human tRNA ligase.

The RhoGAP RRC-1 is required for the assembly or stability of integrin adhesion complexes and is a member of the PIX pathway in muscle.

GTPases cycle between active GTP bound and inactive GDP bound forms. Exchange of GDP for GTP is catalyzed by guanine nucleotide exchange factors (GEFs). GTPase activating proteins (GAPs) accelerate GTP hydrolysis, to promote the GDP bound form. We reported that the RacGEF, PIX-1, is required for assembly of integrin adhesion complexes (IAC) in striated muscle of Caenorhabditis elegans. In C. elegans, IACs are found at the muscle cell boundaries (MCBs), and bases of sarcomeric M-lines and dense bodies (Z-disks). Screening C. elegans mutants in proteins containing RhoGAP domains revealed that loss of function of rrc-1 results in loss of IAC components at MCBs, disorganization of M-lines and dense bodies, and reduced whole animal locomotion. RRC-1 localizes to MCBs, like PIX-1. The localization of RRC-1 at MCBs requires PIX-1, and the localization of PIX-1 requires RRC-1. Loss of function of CED-10 (Rac) shows lack of PIX-1 and RRC-1 at MCBs. RRC-1 exists in a complex with PIX-1. Transgenic rescue of rrc-1 was achieved with wild type RRC-1 but not RRC-1 with a missense mutation in a highly conserved residue of the RhoGAP domain. Our results are consistent with RRC-1 being a RhoGAP for the PIX pathway in muscle.

Assessing the mechanism of fast-cycling cancer-associated mutations of Rac1 small Rho GTPase.

Rho-GTPases proteins function as molecular switches alternating from an active to an inactive state upon Guanosine triphosphate (GTP) binding and hydrolysis to Guanosine diphosphate (GDP). Among them, Rac subfamily regulates cell dynamics, being overexpressed in distinct cancer types. Notably, these proteins are object of frequent cancer-associated mutations at Pro29 (P29S, P29L, and P29Q). To assess the impact of these mutations on Rac1 structure and function, we performed extensive all-atom molecular dynamics simulations on wild-type (wt) and oncogenic isoforms of this protein in GDP- and GTP-bound states. Our results unprecedentedly elucidate that P29Q/S-induced structural and dynamical perturbations of Rac1 core domain weaken the binding of the catalytic site Mg2+ ion, and reduce the GDP residence time within protein, enhancing the GDP/GTP exchange rate and Rac1 activity. This broadens our knowledge of the role of cancer-associated mutations on small GTPases mechanism supplying valuable information for future drug discovery efforts targeting specific Rac1 isoforms.

Dissecting the mechanism of atlastin-mediated homotypic membrane fusion at the single-molecule level.

Homotypic membrane fusion of the endoplasmic reticulum (ER) is mediated by dynamin-like GTPase atlastin (ATL). This fundamental process relies on GTP-dependent domain rearrangements in the N-terminal region of ATL (ATLcyto), including the GTPase domain and three-helix bundle (3HB). However, its conformational dynamics during the GTPase cycle remain elusive. Here, we combine single-molecule FRET imaging and molecular dynamics simulations to address this conundrum. Different from the prevailing model, ATLcyto can form a loose crossover dimer upon GTP binding, which is tightened by GTP hydrolysis for membrane fusion. Furthermore, the α-helical motif between the 3HB and transmembrane domain, which is embedded in the surface of the lipid bilayer and self-associates in the crossover dimer, is required for ATL function. To recycle the proteins, Pi release, which disassembles the dimer, activates frequent relative movements between the GTPase domain and 3HB, and subsequent GDP dissociation alters the conformational preference of the ATLcyto monomer for entering the next reaction cycle. Finally, we found that two disease-causing mutations affect human ATL1 activity by destabilizing GTP binding-induced loose crossover dimer formation and the membrane-embedded helix, respectively. These results provide insights into ATL-mediated homotypic membrane fusion and the pathological mechanisms of related disease.

Biofilm formation of Hafnia paralvei induced by c-di-GMP through facilitating bcsB gene expression promotes spoilage of Yellow River carp (Cyprinus carpio).

Hafnia paralvei, a Gram-negative foodborne pathogen, is found ubiquitously in various aquatic animals and seafoods, which can form biofilm as a dominant virulence factor that contributes to its pathogenesis. However, the biofilm formation mechanism of H. paralvei and its effect on food spoilage has not been fully characterized. Here we show that biofilm formation, is regulated by c-di-GMP which mediated by bcsB, can increase the spoilage ability of H. paralvei. We found that GTP was added exogenously to enhance the synthesis of c-di-GMP, which further promoted biofilm formation. The gene dgcC, one of 11 genes encoding GGDEF domain-containing proteins in H. paralvei, was significantly upregulated with GTP as substrate. The upregulation of dgcC contributes to a significant increase of c-di-GMP and the formation of biofilm. In addition, the overexpression of dgcC induced upregulation of bcsB, a reported effector protein encoding gene, which was further demonstrated that overexpression of bcsB can encourage the synthesis of bacterial cellulose and biofilm formation. The effect of biofilm formation induced by c-di-GMP on spoilage of Yellow River carp (Cyprinus carpio) was evaluated by sensory evaluation, the total viable count, and the total volatile basic nitrogen, which showed that biofilm formation can significantly increase the spoilage ability of H. paralvei on C. carpio. Our findings provide the regulation of c-di-GMP on expression of bcsB, that can contribute to biofilm formation and spoilage ability of H. paralvei, which is favor to understanding the pathogenesis of Hafnia paralvei and its role in food spoilage.

Coupling of zinc and GTP binding drives G-domain folding in Acinetobacter baumannii ZigA.

COG0523 proteins, also known as nucleotide-dependent metallochaperones, are a poorly understood class of small P-loop G3E GTPases. Multiple family members play critical roles in bacterial pathogen survival during an infection as part of the adaptive response to host-mediated "nutritional immunity." Our understanding of the structure, dynamics, and molecular-level function of COG0523 proteins, apart from the eukaryotic homolog, Zng1, remains in its infancy. Here, we use X-ray absorption spectroscopy to establish that Acinetobacter baumannii (Ab) ZigA coordinates ZnII using all three cysteines derived from the invariant CXCC motif to form an S3(N/O) coordination complex, a feature inconsistent with the ZnII-bound crystal structure of a distantly related COG0523 protein of unknown function from Escherichia coli, EcYjiA. The binding of ZnII and guanine nucleotides is thermodynamically linked in AbZigA, and this linkage is more favorable for the substrate GTP relative to the product GDP. Part of this coupling originates with nucleotide-induced stabilization of the G-domain tertiary structure as revealed by global thermodynamics measurements and hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS also reveals that the HDX behavior of the G2 (switch 1) loop is highly sensitive to nucleotide status and becomes more exchange labile in the GDP (product)-bound state. Significant long-range perturbation of local stability in both the G-domain and the C-terminal domain define a candidate binding pocket for a client protein that appears sensitive to nucleotide status (GDP versus GTP). We place these new insights into the structure, dynamics, and energetics of intermolecular metal transfer into the context of a model for AbZigA metallochaperone function.

Green tea polyphenols inhibit TBBPA-induced lung injury via enhancing antioxidant capacity and modulating the NF-κB pathway in mice.

Tetrabromobisphenol A (TBBPA) is a global pollutant. When TBBPA is absorbed by the body through various routes, it can have a wide range of harmful effects on the body. Green tea polyphenols (GTPs) can act as antioxidants, resisting the toxic effects of TBBPA on animals. The effects and mechanisms of GTP and TBBPA on oxidative stress, inflammation and apoptosis in the mouse lung are unknown. Therefore, we established in vivo and in vitro models of TBBPA exposure and GTP antagonism using C57 mice and A549 cells and examined the expression of factors related to oxidative stress, autophagy, inflammation and apoptosis. The results of the study showed that the increase in reactive oxygen species (ROS) levels after TBBPA exposure decreased the expression of autophagy-related factors Beclin1, LC3-II, ATG3, ATG5, ATG7 and ATG12 and increased the expression of p62; oxidative stress inhibits autophagy levels. The increased expression of the pro-inflammatory factors IL-1ß, IL-6 and TNF-α decreased the expression of the anti-inflammatory factor IL-10 and activation of the NF-κB p65/TNF-α pathway. The increased expression of Bax, caspase-3, caspase-7 and caspase-9 and the decreased expression of Bcl-2 activate apoptosis-related pathways. The addition of GTP attenuated oxidative stress levels, restored autophagy inhibition and reduced the inflammation and apoptosis levels. Our results suggest that GTP can attenuate the toxic effects of TBBPA by modulating ROS, reducing oxidative stress levels, increasing autophagy and attenuating inflammation and apoptosis in mouse lung and A549 cells. These results provide fundamental information for exploring the antioxidant mechanism of GTP and further for studying the toxic effects of TBBPA.

Unraveling the Significance of Mg2+ Dependency and Nucleotide Binding Specificity of H-RAS.

RAS is a small GTPase and acts as a binary molecular switch; the transition from its active to inactive state plays a crucial role in various cell signaling processes. Molecular dynamics simulations at the atomistic level suggest that the absence of cofactor Mg2+ ion generally leads to pronounced structural changes in the Switch-I than Switch-II regions and assists GTP binding. The presence of the Mg2+ ion also restricts the rotation of ϒ phosphate and enhances the hydrolysis rate of GTP. Further, the simulations reveal that the stability of the protein is almost uncompromised when Mg2+ is replaced with Zn2+ and not the Ca2+ ion. The specificity of H-RAS to GTP was evaluated by substituting with ATP and CTP, which indicates that the binding pocket tolerates purine bases over pyrimidine bases. However, the D119 residue specifically interacts with the guanine base and serves as one of the primary interactions that leads to the selectivity of GTP over ATP. The ring displacement of 32Y serves as gate dynamics in H-RAS which are important for its interaction with GAP for the nucleotide exchange and is restricted in the presence of ATP. Finally, the point mutations 61, 16, and 32 influence the structural changes, specifically in the Switch-II region, which are expected to impact the GTP hydrolysis and thus are termed oncogenic mutations.

Unveiling the State Transition Mechanisms of Ras Proteins through Enhanced Sampling and QM/MM Simulations.

In cells, wild-type RasGTP complexes exist in two distinct states: active State 2 and inactive State 1. These complexes regulate their functions by transitioning between the two states. However, the mechanisms underlying this state transition have not been clearly elucidated. To address this, we conducted a detailed simulation study to characterize the energetics of the stable states involved in the state transitions of the HRasGTP complex, specifically from State 2 to State 1. This was achieved by employing multiscale quantum mechanics/molecular mechanics and enhanced sampling molecular dynamics methods. Based on the simulation results, we constructed the two-dimensional free energy landscapes that provide crucial information about the conformational changes of the HRasGTP complex from State 2 to State 1. Furthermore, we also explored the conformational changes from the intermediate state to the product state during guanosine triphosphate hydrolysis. This study on the conformational changes involved in the HRas state transitions serves as a valuable reference for understanding the corresponding events of both KRas and NRas as well.

Performance of large language models on advocating the management of meningitis: a comparative qualitative study.

OBJECTIVES: We aimed to examine the adherence of large language models (LLMs) to bacterial meningitis guidelines using a hypothetical medical case, highlighting their utility and limitations in healthcare. METHODS: A simulated clinical scenario of a patient with bacterial meningitis secondary to mastoiditis was presented in three independent sessions to seven publicly accessible LLMs (Bard, Bing, Claude-2, GTP-3.5, GTP-4, Llama, PaLM). Responses were evaluated for adherence to good clinical practice and two international meningitis guidelines. RESULTS: A central nervous system infection was identified in 90% of LLM sessions. All recommended imaging, while 81% suggested lumbar puncture. Blood cultures and specific mastoiditis work-up were proposed in only 62% and 38% sessions, respectively. Only 38% of sessions provided the correct empirical antibiotic treatment, while antiviral treatment and dexamethasone were advised in 33% and 24%, respectively. Misleading statements were generated in 52%. No significant correlation was found between LLMs' text length and performance (r=0.29, p=0.20). Among all LLMs, GTP-4 demonstrated the best performance. DISCUSSION: Latest LLMs provide valuable advice on differential diagnosis and diagnostic procedures but significantly vary in treatment-specific information for bacterial meningitis when introduced to a realistic clinical scenario. Misleading statements were common, with performance differences attributed to each LLM's unique algorithm rather than output length. CONCLUSIONS: Users must be aware of such limitations and performance variability when considering LLMs as a support tool for medical decision-making. Further research is needed to refine these models' comprehension of complex medical scenarios and their ability to provide reliable information.

Nuclear export of circular RNA.

Circular RNAs (circRNAs), which are increasingly being implicated in a variety of functions in normal and cancerous cells1-5, are formed by back-splicing of precursor mRNAs in the nucleus6-10. circRNAs are predominantly localized in the cytoplasm, indicating that they must be exported from the nucleus. Here we identify a pathway that is specific for the nuclear export of circular RNA. This pathway requires Ran-GTP, exportin-2 and IGF2BP1. Enhancing the nuclear Ran-GTP gradient by depletion or chemical inhibition of the major protein exporter CRM1 selectively increases the nuclear export of circRNAs, while reducing the nuclear Ran-GTP gradient selectively blocks circRNA export. Depletion or knockout of exportin-2 specifically inhibits nuclear export of circRNA. Analysis of nuclear circRNA-binding proteins reveals that interaction between IGF2BP1 and circRNA is enhanced by Ran-GTP. The formation of circRNA export complexes in the nucleus is promoted by Ran-GTP through its interactions with exportin-2, circRNA and IGF2BP1. Our findings demonstrate that adaptors such as IGF2BP1 that bind directly to circular RNAs recruit Ran-GTP and exportin-2 to export circRNAs in a mechanism that is analogous to protein export, rather than mRNA export.

Ran-GTP assembles a specialized spindle structure for accurate chromosome segregation in medaka early embryos.

Despite drastic cellular changes during cleavage, a mitotic spindle assembles in each blastomere to accurately segregate duplicated chromosomes. Mechanisms of mitotic spindle assembly have been extensively studied using small somatic cells. However, mechanisms of spindle assembly in large vertebrate embryos remain little understood. Here, we establish functional assay systems in medaka (Oryzias latipes) embryos by combining CRISPR knock-in with auxin-inducible degron technology. Live imaging reveals several unexpected features of microtubule organization and centrosome positioning that achieve rapid, accurate cleavage. Importantly, Ran-GTP assembles a dense microtubule network at the metaphase spindle center that is essential for chromosome segregation in early embryos. This unique spindle structure is remodeled into a typical short, somatic-like spindle after blastula stages, when Ran-GTP becomes dispensable for chromosome segregation. We propose that despite the presence of centrosomes, the chromosome-derived Ran-GTP pathway has essential roles in functional spindle assembly in large, rapidly dividing vertebrate early embryos, similar to acentrosomal spindle assembly in oocytes.

Structure of the GDP-bound state of the SRP GTPase FlhF.

The GTPase FlhF, a signal recognition particle (SRP)-type enzyme, is pivotal for spatial-numerical control and bacterial flagella assembly across diverse species, including pathogens. This study presents the X-ray structure of FlhF in its GDP-bound state at a resolution of 2.28 Å. The structure exhibits the classical N- and G-domain fold, consistent with related SRP GTPases such as Ffh and FtsY. Comparative analysis with GTP-loaded FlhF elucidates the conformational changes associated with GTP hydrolysis. These topological reconfigurations are similarly evident in Ffh and FtsY, and play a pivotal role in regulating the functions of these hydrolases.

Native architecture of a human GBP1 defense complex for cell-autonomous immunity to infection.

All living organisms deploy cell-autonomous defenses to combat infection. In plants and animals, large supramolecular complexes often activate immune proteins for protection. In this work, we resolved the native structure of a massive host-defense complex that polymerizes 30,000 guanylate-binding proteins (GBPs) over the surface of gram-negative bacteria inside human cells. Construction of this giant nanomachine took several minutes and remained stable for hours, required guanosine triphosphate hydrolysis, and recruited four GBPs plus caspase-4 and Gasdermin D as a cytokine and cell death immune signaling platform. Cryo-electron tomography suggests that GBP1 can adopt an extended conformation for bacterial membrane insertion to establish this platform, triggering lipopolysaccharide release that activated coassembled caspase-4. Our "open conformer" model provides a dynamic view into how the human GBP1 defense complex mobilizes innate immunity to infection.