Serine 39 in the GTP-binding domain of Drp1 is involved in shaping mitochondrial morphology.

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.

The IMPDH cytoophidium couples metabolism and fetal development in mice.

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.

Molecular mechanism for recognition of the cargo adapter Rab6GTP by the dynein adapter BicD2.

Rab6 is a key modulator of protein secretion. The dynein adapter Bicaudal D2 (BicD2) recruits the motors cytoplasmic dynein and kinesin-1 to Rab6GTP-positive vesicles for transport; however, it is unknown how BicD2 recognizes Rab6. Here, we establish a structural model for recognition of Rab6GTP 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 Rab6GTP that abolish binding to BicD2 also result in reduced co-migration of Rab6GTP/BicD2 in cells, validating our model. These mutations also severely diminished the motility of Rab6-positive vesicles in cells, highlighting the importance of the Rab6GTP/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.

Molecular Mechanism of Phosphorylation-Mediated Impacts on the Conformation Dynamics of GTP-Bound KRAS Probed by GaMD Trajectory-Based Deep Learning.

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 Mg2+ 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.

Tumour-selective activity of RAS-GTP inhibition in pancreatic cancer.

Broad-spectrum RAS inhibition has the potential to benefit roughly a quarter of human patients with cancer whose tumours are driven by RAS mutations1,2. 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 variants3. More than 90% of cases of human pancreatic ductal adenocarcinoma (PDAC) are driven by activating mutations in KRAS4. 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.

Crystal structure of NRAS Q61K with a ligand-induced pocket near switch II.

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.

Insights into the ATP / GTP selectivity of a GTPase, adenylosuccinate synthetase from Leishmania donovani.

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 (593SAXD596) 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.

Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers.

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.

Concurrent inhibition of oncogenic and wild-type RAS-GTP for cancer therapy.

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 611. 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 cancer2,3. Nevertheless, KRASG12C mutations account for only around 15% of KRAS-mutated cancers4,5, 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 (KRASG12X). 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 KRASG12C 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).

Conformational Modulation of Tissue Transglutaminase via Active Site Thiol Alkylating Agents: Size Does Not Matter.

TG2 is a unique member of the transglutaminase family as it undergoes a dramatic conformational change, allowing its mutually exclusive function as either a cross-linking enzyme or a G-protein. The enzyme's dysregulated activity has been implicated in a variety of pathologies (e.g., celiac disease, fibrosis, cancer), leading to the development of a wide range of inhibitors. Our group has primarily focused on the development of peptidomimetic targeted covalent inhibitors, the nature and size of which were thought to be important features to abolish TG2's conformational dynamism and ultimately inhibit both its activities. However, we recently demonstrated that the enzyme was unable to bind guanosine triphosphate (GTP) when catalytically inactivated by small molecule inhibitors. In this study, we designed a library of models targeting covalent inhibitors of progressively smaller sizes (15 to 4 atoms in length). We evaluated their ability to inactivate TG2 by measuring their respective kinetic parameters kinact and KI. Their impact on the enzyme's ability to bind GTP was then evaluated and subsequently correlated to the conformational state of the enzyme, as determined via native PAGE and capillary electrophoresis. All irreversible inhibitors evaluated herein locked TG2 in its open conformation and precluded GTP binding. Therefore, we conclude that steric bulk and structural complexity are not necessary factors to consider when designing TG2 inhibitors to abolish G-protein activity.

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.

Enhancement of intrinsic guanine fluorescence by protonation in DNA of various structures.

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.

RPGR is a guanine nucleotide exchange factor for the small GTPase RAB37 required for retinal function via autophagy regulation.

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.

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.

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.

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.

GTP-Bound N-Ras Conformational States and Substates Are Modulated by Membrane and Point Mutation.

Oncogenic Ras proteins are known to present multiple conformational states, as reported by the great variety of crystallographic structures. The GTP-bound states are grouped into two main states: the "inactive" state 1 and the "active" state 2. Recent reports on H-Ras have shown that state 2 exhibits two substates, directly related to the orientation of Tyr32: toward the GTP-bound pocket and outwards. In this paper, we show that N-Ras exhibits another substate of state 2, related to a third orientation of Tyr32, toward Ala18 and parallel to the GTP-bound pocket. We also show that this substate is highly sampled in the G12V mutation of N-Ras and barely present in its wild-type form, and that the G12V mutation prohibits the sampling of the GTPase-activating protein (GAP) binding substate, rendering this mutation oncogenic. Furthermore, using molecular dynamics simulations, we explore the importance of the membrane on N-Ras' conformational state dynamics and its strong influence on Ras protein stability. Moreover, the membrane has a significant influence on the conformational (sub)states sampling of Ras. This, in turn, is of crucial importance in the activation/deactivation cycle of Ras, due to the binding of guanine nucleotide exchange factor proteins (GEFs)/GTPase-activating proteins (GAPs).