Tumor-derived RHOA mutants interact with effectors in the GDP-bound state.

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 RHOAA161P and RHOAA161V are both fast-cycling mutants with increased guanine nucleotide dissociation/association rates compared with RHOAWT and show reduced GTP-hydrolysis activity. Crystal structures reveal an altered nucleotide association in RHOAA161P and an open nucleotide pocket in RHOAA161V. Both mutations perturb the dynamic properties of RHOA switch regions and shift the conformational landscape important for RHOA activity, as shown by 31P NMR and molecular dynamics simulations. Interestingly, RHOAA161P and RHOAA161V can interact with effectors in the GDP-bound state. 1H-15N HSQC NMR spectra support the existence of an active population in RHOAA161V-GDP. The distinct interaction mechanisms resulting from the mutations likely favor an RHOAWT-like "ON" conformation, endowing GDP-bound state effector binding activity.

A neurodevelopmental disorder mutation locks G proteins in the transitory pre-activated state.

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. GαoK46E 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 ßγ subunits. In cells with physiological concentrations of nucleotide, GαoK46E forms a stable complex with receptors and Gßγ, 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 Gαo, 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.

Capturing RAS oligomerization on a membrane.

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 (SOScat) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOScat. 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.

Exploring the Activation Mechanism of the GPR183 Receptor.

The G protein-coupled receptors (GPCRs) play a pivotal role in numerous biological processes as crucial cell membrane receptors. However, the dynamic mechanisms underlying the activation of GPR183, a specific GPCR, remain largely elusive. To address this, we employed computational simulation techniques to elucidate the activation process and key events associated with GPR183, including conformational changes from inactive to active state, binding interactions with the Gi protein complex, and GDP release. Our findings demonstrate that the association between GPR183 and the Gi protein involves the formation of receptor-specific conformations, the gradual proximity of the Gi protein to the binding pocket, and fine adjustments of the protein conformation, ultimately leading to a stable GPR183-Gi complex characterized by a high energy barrier. The presence of Gi protein partially promotes GPR183 activation, which is consistent with the observation of GPCR constitutive activity test experiments, thus illustrating the reliability of our calculations. Moreover, our study suggests the existence of a stable partially activated state preceding complete activation, providing novel avenues for future investigations. In addition, the relevance of GPR183 for various diseases, such as colitis, the response of eosinophils to Mycobacterium tuberculosis infection, antiviral properties, and pulmonary inflammation, has been emphasized, underscoring its therapeutic potential. Consequently, understanding the activation process of GPR183 through molecular dynamic simulations offers valuable kinetic insights that can aid in the development of targeted therapies.

Effects of bound nucleotides on the secondary structure, thermal stability, and phosphorylation of Rab3A.

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.

Structure of GDP-bound Rab7 Q67L in complex with ORP1L.

Molecular processes are orchestrated by various proteins that promote early endosomes to become late endosomes and eventually fuse with lysosomes, guaranteeing the degradation of the content. Rab7, which is localized to late endosomes, is one of the most well-known GTPases. ORP1L is recruited by Rab7 to facilitate the fusion of late endosomes and lysosomes. Here, we present the structure of GDP-bound Rab7 Q67L with ORP1L. Structural analysis, supported by biochemical and ITC binding experiments, not only provides structural insight into the interactions between the ORP1L ANK domain and Rab7 but also suggests that the GTPase activity of Rab7 does not interfere with its ORP1L-binding capacity.

Stable GDP-tubulin islands rescue dynamic microtubules.

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.

Mitoribosome structure with cofactors and modifications reveals mechanism of ligand binding and interactions with L1 stalk.

The mitoribosome translates mitochondrial mRNAs and regulates energy conversion that is a signature of aerobic life forms. We present a 2.2 Å resolution structure of human mitoribosome together with validated mitoribosomal RNA (rRNA) modifications, including aminoacylated CP-tRNAVal. The structure shows how mitoribosomal proteins stabilise binding of mRNA and tRNA helping to align it in the decoding center, whereas the GDP-bound mS29 stabilizes intersubunit communication. Comparison between different states, with respect to tRNA position, allowed us to characterize a non-canonical L1 stalk, and molecular dynamics simulations revealed how it facilitates tRNA transitions in a way that does not require interactions with rRNA. We also report functionally important polyamines that are depleted when cells are subjected to an antibiotic treatment. The structural, biochemical, and computational data illuminate the principal functional components of the translation mechanism in mitochondria and provide a description of the structure and function of the human mitoribosome.

Dynamics of interdomain rotation facilitates FtsZ filament assembly.

FtsZ, the tubulin homolog essential for bacterial cell division, assembles as the Z-ring at the division site, and directs peptidoglycan synthesis by treadmilling. It is unclear how FtsZ achieves kinetic polarity that drives treadmilling. To obtain insights into fundamental features of FtsZ assembly dynamics independent of peptidoglycan synthesis, we carried out structural and biochemical characterization of FtsZ from the cell wall-less bacteria, Spiroplasma melliferum (SmFtsZ). Interestingly the structures of SmFtsZ, bound to GDP and GMPPNP respectively, were captured as domain swapped dimers. SmFtsZ was found to be a slower GTPase with a higher critical concentration (CC) compared to Escherichia coli FtsZ (EcFtsZ). In FtsZs, a conformational switch from R-state (close) to T-state (open) favors polymerization. We identified that Phe224, located at the interdomain cleft of SmFtsZ, is crucial for R- to T-state transition. SmFtsZF224M exhibited higher GTPase activity and lower CC, whereas the corresponding EcFtsZM225F resulted in cell division defects in E. coli. Our results demonstrate that relative rotation of the domains is a rate-limiting step of polymerization. Our structural analysis suggests that the rotation is plausibly triggered upon addition of a GTP-bound monomer to the filament through interaction of the preformed N-terminal domain (NTD). Hence, addition of monomers to the NTD-exposed end of filament is slower in comparison to the C-terminal domain (CTD) end, thus explaining kinetic polarity. In summary, the study highlights the importance of interdomain interactions and conformational changes in regulating FtsZ assembly dynamics.

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.

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.

Ligand efficacy modulates conformational dynamics of the µ-opioid receptor.

The µ-opioid receptor (µOR) is an important target for pain management1 and molecular understanding of drug action on µOR will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance and single-molecule fluorescence resonance energy transfer, how ligand-specific conformational changes of µOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several conformations of the cytoplasmic face of the receptor that interconvert on different timescales, including a pre-activated conformation that is capable of G-protein binding, and a fully activated conformation that markedly reduces GDP affinity within the ternary complex. Interaction of ß-arrestin-1 with the µOR core binding site appears less specific and occurs with much lower affinity than binding of Gi.

Cryo-EM structures of membrane-bound dynamin in a post-hydrolysis state primed for membrane fission.

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.

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.

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.

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.

Adapting recombinant bacterial alkaline phosphatase for nucleotide exchange of small GTPases.

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.

Unlocking translational machinery for antitubercular drug development.

Targeting translational factor proteins (TFPs) presents significant promise for the development of innovative antitubercular drugs. Previous insights from antibiotic binding mechanisms and recently solved 3D crystal structures of Mycobacterium tuberculosis (Mtb) elongation factor thermo unstable-GDP (EF-Tu-GDP), elongation factor thermo stable-EF-Tu (EF-Ts-EF-Tu), and elongation factor G-GDP (EF-G-GDP) have opened up new avenues for the design and development of potent antituberculosis (anti-TB) therapies.

Evolution of optimal growth temperature in Asgard archaea inferred from the temperature dependence of GDP binding to EF-1A.

The archaeal ancestor of eukaryotes apparently belonged to the phylum Asgardarchaeota, but the ecology and evolution of Asgard archaea are poorly understood. The optimal GDP-binding temperature of a translation elongation factor (EF-1A or EF-Tu) has been previously shown to correlate with the optimal growth temperature of diverse prokaryotes. Here, we reconstruct ancestral EF-1A sequences and experimentally measure the optimal GDP-binding temperature of EF-1A from ancient and extant Asgard archaea, to infer the evolution of optimal growth temperatures in Asgardarchaeota. Our results suggest that the Asgard ancestor of eukaryotes was a moderate thermophile, with an optimal growth temperature around 53 °C. The origin of eukaryotes appears to coincide with a transition from thermophilic to mesophilic lifestyle during the evolution of Asgard archaea.

Costoefectividad del régimen combinado de mirabegron/solifenacina en el tratamiento del síndrome de vejiga hiperactiva en Colombia / Cost-effectiveness of the Combined Regimen of Mirabegron/Solifenacin in the Treatment of Overactive Bladder Syndrome in Colombia

Objetivo Evaluar la costoefectividad incremental del régimen combinado de mirabegron/solifenacina en comparación con el uso temprano de toxina botulínica, desde la perspectiva del sistema de salud colombiano, para el tratamiento de adultos con vejiga hiperactiva. Métodos Se empleó un modelo de Markov en que se comparan dos secuencias de tratamiento, una con y otra sin mirabegron/solifenacina, para evaluar la costoefectividad en un horizonte temporal de cinco años. Debido a la perspectiva de análisis, sólo se tuvieron en cuenta los costos médicos directos. La eficacia del tratamiento evaluado y su comparador fue medida en términos de la reducción de episodios diarios de incontinencia y de la frecuencia de micciones. Los costos fueron expresados en pesos colombianos de 2019, y se aplicó una tasa de descuento de 5% tanto para desenlaces como para costos. Resultados Para el caso base, el costo del tratamiento en la secuencia que incluye mirabegron/solifenacina fue mayor, pero generó un mayor número de años de vida ajustados por calidad, y así e obtuvo una razón de costoefectividad incremental de $13.637,184 si se considera el desenlace de reducción de episodios diarios de incontinencia de 50%, y de $29.313,848 si se considera el del 100%. Conclusiones De acuerdo con los resultados de esta evaluación, para un horizonte de análisis de cinco años, la secuencia de tratamiento con mirabegron/solifenacina es una alternativa costoefectiva, si se considera un umbral de disposición a pagar de tres veces el producto interno bruto (PIB) per cápita.

Aim To evaluate the incremental cost-effectiveness of the combined regimen of mirabegron/solifenacin compared with the early use of botulinum toxin, from the perspective of the Colombian health system, for the treatment of adults with overactive bladder. Methods A Markov model comparing two treatment sequences, one with and one without mirabegron/solifenacin, was used to assess cost-effectiveness over a five-year period. Due to the perspective of the analysis, only direct medical costs were considered. The efficacy of the evaluated treatment and its comparator was measured in terms of the reduction in the daily incontinence episodes and the frequency of micturition. The costs were expressed in Colombian pesos of 2019, and a discount rate of 5% was applied for both outcomes and costs. Results For the base case, the cost of the treatment in the sequence that includes mirabegron/solifenacin was higher, but it generated a greater number of quality-adjusted years of life, thus obtaining an incremental cost-effectiveness ratio of $13,637,184 when considering the outcome of 50% of reduction in the daily incontinence episodes, and $29,313,848 when considering 100%. Conclusions According to the results of the present assessment, for a five-year period of analysi, the mirabegron/solifenacin treatment sequence is a cost-effective alternative when considering a threshold of willingness to pay three times the per capita gross domestic product (GDP).