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.

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.

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.

Unveiling the catalytic mechanism of GTP hydrolysis in microtubules.

Microtubules (MTs) are large cytoskeletal polymers, composed of αß-tubulin heterodimers, capable of stochastically converting from polymerizing to depolymerizing states and vice versa. Depolymerization is coupled with hydrolysis of guanosine triphosphate (GTP) within ß-tubulin. Hydrolysis is favored in the MT lattice compared to a free heterodimer with an experimentally observed rate increase of 500- to 700-fold, corresponding to an energetic barrier lowering of 3.8 to 4.0 kcal/mol. Mutagenesis studies have implicated α-tubulin residues, α:E254 and α:D251, as catalytic residues completing the ß-tubulin active site of the lower heterodimer in the MT lattice. The mechanism for GTP hydrolysis in the free heterodimer, however, is not understood. Additionally, there has been debate concerning whether the GTP-state lattice is expanded or compacted relative to the GDP state and whether a "compacted" GDP-state lattice is required for hydrolysis. In this work, extensive quantum mechanics/molecular mechanics simulations with transition-tempered metadynamics free-energy sampling of compacted and expanded interdimer complexes, as well as a free heterodimer, have been carried out to provide clear insight into the GTP hydrolysis mechanism. α:E254 was found to be the catalytic residue in a compacted lattice, while in the expanded lattice, disruption of a key salt bridge interaction renders α:E254 less effective. The simulations reveal a barrier decrease of 3.8 ± 0.5 kcal/mol for the compacted lattice compared to a free heterodimer, in good agreement with experimental kinetic measurements. Additionally, the expanded lattice barrier was found to be 6.3 ± 0.5 kcal/mol higher than compacted, demonstrating that GTP hydrolysis is variable with lattice state and slower at the MT tip.

Binding modes of GDP, GTP and GNP to NRAS deciphered by using Gaussian accelerated molecular dynamics simulations.

Probing binding modes of GDP, GTP and GNP to NRAS are of significance for understanding the regulation mechanism on the activity of RAS proteins. Four separate Gaussian accelerated molecular dynamics (GaMD) simulations were performed on the apo, GDP-, GTP- and GNP-bound NRAS. Dynamics analyses suggest that binding of three ligands highly affects conformational states of the switch domains from NRAS, which disturbs binding of NRAS to its effectors. The analyses of free energy landscapes (FELs) indicate that binding of GDP, GTP and GNP induces more energetic states of NRAS compared to the apo NRAS but the presence of GNP makes the switch domains more ordered than binding of GDP and GNP. The information of interaction networks of ligands with NRAS reveals that the π-π interaction of residue F28 and the salt bridge interactions of K16 and D119 with ligands stabilize binding of GDP, GTP and GNP to NRAS. Meanwhile magnesium ion plays a bridge role in interactions of ligands with NRAS, which is favourable for associations of GDP, GTP and GNP with NRAS. This work is expected to provide useful information for deeply understanding the function and activity of NRAS.

Structural insights of the elongation factor EF-Tu complexes in protein translation of Mycobacterium tuberculosis.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is the second-deadliest infectious disease worldwide. Emerging evidence shows that the elongation factor EF-Tu could be an excellent target for treating Mtb infection. Here, we report the crystal structures of Mtb EF-Tu•EF-Ts and EF-Tu•GDP complexes, showing the molecular basis of EF-Tu's representative recycling and inactive forms in protein translation. Mtb EF-Tu binds with EF-Ts at a 1:1 ratio in solution and crystal packing. Mutation and SAXS analysis show that EF-Ts residues Arg13, Asn82, and His149 are indispensable for the EF-Tu/EF-Ts complex formation. The GDP binding pocket of EF-Tu dramatically changes conformations upon binding with EF-Ts, sharing a similar GDP-exchange mechanism in E. coli and T. ther. Also, the FDA-approved drug Osimertinib inhibits the growth of M. smegmatis, H37Ra, and M. bovis BCG strains by directly binding with EF-Tu. Thus, our work reveals the structural basis of Mtb EF-Tu in polypeptide synthesis and may provide a promising candidate for TB treatment.

Markov State Models and Molecular Dynamics Simulations Reveal the Conformational Transition of the Intrinsically Disordered Hypervariable Region of K-Ras4B to the Ordered Conformation.

K-Ras4B, the most frequently mutated Ras isoform in human tumors, plays a vital part in cell growth, differentiation, and survival. Its tail, the C-terminal hypervariable region (HVR), is involved in anchoring K-Ras4B at the cellular plasma membrane and in isoform-specific protein-protein interactions and signaling. In the inactive guanosine diphosphate-bound state, the intrinsically disordered HVR interacts with the catalytic domain at the effector-binding region, rendering K-Ras4B in its autoinhibited state. Activation releases the HVR from the catalytic domain, with its ensemble favoring an ordered α-helical structure. The large-scale conformational transition of the HVR from the intrinsically disordered to the ordered conformation remains poorly understood. Here, we deploy a computational scheme that integrates a transition path-generation algorithm, extensive molecular dynamics simulation, and Markov state model analysis to investigate the conformational landscape of the HVR transition pathway. Our findings reveal a stepwise pathway for the HVR transition and uncover several key conformational substates along the transition pathway. Importantly, key interactions between the HVR and the catalytic domain are unraveled, highlighting the pathogenesis of K-Ras4B mild mutations in several congenital developmental anomaly syndromes. Together, these findings provide a deeper understanding of the HVR transition mechanism and the regulation of K-Ras4B activity at an atomic level.

Structural insights into the binding of nanobody Rh57 to active RhoA-GTP.

RhoA protein is a small GTPase that acts as a molecular switch. When bound to guanosine triphosphate (GTP), RhoA can activate several key signal pathways. Recently, nanobody Rh57 specific binding with GTP bound active RhoA was discovered and developed as a BRET biosensor without cytotoxicity. To further clarify the nanobody Rh57's mechanism of action, we co-expressed, purified, and crystallized the RhoA-Rh57 nanobody complex and solved the structure by X-ray diffraction with a resolution of 2.76 Å. The structure showed that the interaction is mainly through hydrogen bonds, salt bridges, aromatic-aromatic interactions, and hydrophobic interactions. The involved regions include CDR3 and non-hypervariable loop of Rh57, and the SWI switch loops of RhoA, respectively. The different SWI conformation of inactivated RhoA-GDP prevented the Rh57's binding. The possible explanation of Rh57 as a non-cytotoxic BRET intracellular tracer is that Rh57's binding did not overlap with downstream PRK1 and thus did not interfere with the downstream signaling pathway. Our research provides an in-depth understanding of how nanobodies recognize activated RhoA-GTP while not binding inactivated RhoA-GDP. This structural information may also provide critical information for further optimization of relevant nanobodies.

Conformational Dynamics Allows Sampling of an "Active-like" State by Oncogenic K-Ras-GDP.

Mutations in K-Ras GTPase replacing Gly12 with either Asp or Val are common in cancer. These mutations decelerate intrinsic and catalyzed GTP hydrolysis, leading to accumulation of K-Ras-GTP in cells. Signaling cascades initiated by K-Ras-GTP promote cell proliferation, survival, and invasion. Despite functional differences between the most frequent G12D mutation and the most aggressive and chemotherapy resistant G12V mutation, their long-suspected distinct structural features remain elusive. Using NMR, X-ray structures, and computational methods, we found that oncogenic mutants of K-Ras4B, the predominant splice variant of K-Ras, exhibit distinct conformational dynamics when GDP-bound, visiting the "active-like" conformational state similar to the one observed in GTP-bound K-Ras. This behavior distinguishes G12V from wild type and G12D K-Ras4B-GDP. The likely reason is interactions between the aliphatic sidechain of V12 and the Switch II region of K-Ras4BG12V-GDP, which are distinct in K-Ras4BG12D-GDP. In the X-ray structures, crystal contacts reduce the dynamics of the sidechain at position 12 by stabilizing the Switch I region of the protein. This explains why structural differences between G12V and G12D K-Ras have yet not been reported. Together, our results suggest a previously unknown mechanism of K-Ras activation. This mechanism relies on conformational dynamics caused by specific oncogenic mutations in the GDP-bound state. Our findings also imply that the therapeutic strategies decreasing the level of K-Ras-GTP by interfering with nucleotide exchange or by expediting GTP hydrolysis may work differently in different oncogenic mutants.

Probing the Molecular Basis of Cofactor Affinity and Conformational Dynamics of Mycobacterium tuberculosis Elongation Factor Tu: An Integrated Approach Employing Steered Molecular Dynamics and Umbrella Sampling Simulations.

The emergence of multidrug-resistant and extensively drug-resistant tuberculosis strains is the reason that the infectious tuberculosis pathogen is still the most common cause of death. The quest for new antitubercular drugs that can fit into multidrug regimens, function swiftly, and overcome the ever-increasing prevalence of drug resistance continues. The crucial role of MtbEF-Tu in translation and trans-translation processes makes it an excellent target for antitubercular drug design. In this study, the primary sequence of MtbEF-Tu was used to model the three-dimensional structures of MtbEF-Tu in the presence of GDP ("off" state) and GTP ("on" state). The binding free energy computed using both the molecular mechanics/Poisson-Boltzmann surface area and umbrella sampling approaches shows that GDP binds to MtbEF-Tu with an ∼2-fold affinity compared to GTP. The steered molecular dynamics (SMD) and umbrella sampling simulation also shows that the dissociation of GDP from MtbEF-Tu in the presence of Mg2+ is a thermodynamically intensive process, while in the absence of Mg2+, the destabilized GDP dissociates very easily from the MtbEF-Tu. Naturally, the dissociation of Mg2+ from the MtbEF-Tu is facilitated by the nucleotide exchange factor EF-Ts, and this prior release of magnesium makes the dissociation process of destabilized GDP easy, similar to that observed in the umbrella sampling and SMD study. The MD simulations of MtbEF-Tu's "on" state conformation in the presence of GTP reveal that the secondary structure of switch-I and Mg2+ coordination network remains similar to its template despite the absence of identity in the conserved region of switch-I. On the other hand, the secondary structure in the conserved region of the switch-I of MtbEF-Tu unwinds from a helix to a loop in the presence of GDP. The major conformational changes observed in switch-I and the movement of Thr64 away from Mg2+ mainly reflect essential conformational changes to make the shift of MtbEF-Tu's "on" state to the "off" state in the presence of GDP. These obtained structural and functional insights into MtbEF-Tu are pivotal for a better understanding of structural-functional linkages of MtbEF-Tu, and these findings may serve as a basis for the design and development of MtbEF-Tu-specific inhibitors.

Mutational landscape of K-Ras substitutions at 12th position-a systematic molecular dynamics approach.

K-Ras is a small GTPase and acts as a molecular switch by recruiting GEFs and GAPs, and alternates between the inert GDP-bound and the dynamic GTP-bound forms. The amino acid at position 12 of K-Ras is a hot spot for oncogenic mutations (G12A, G12C, G12D, G12R, G12S, and G12V), disturbing the active fold of the protein, leading to cancer development. This study aimed to investigate the potential conformational changes induced by these oncogenic mutations at the 12th position, impairing GAP-mediated GTP hydrolysis. Comprehensive computational tools (iStable, FoldX, SNPeffect, DynaMut, and CUPSAT) were used to evaluate the effect of these six mutations on the stability of wild type K-Ras protein. The docking of GTP with K-Ras was carried out using AutoDock4.2, followed by molecular dynamics simulations. Furthermore, on comparison of binding energies between the wild type K-Ras and the six mutants, we have demonstrated that the G12A and G12V mutants exhibited the strongest binding efficiency compared to the other four mutants. Trajectory analyses of these mutations revealed that G12A encountered the least deviation, fluctuation, intermolecular H-bonds, and compactness compared to the wildtype, which was supported by the lower Gibbs free energy value. Our study investigates the molecular dynamics simulations of the mutant K-Ras forms at the 12th position, which expects to provide insights about the molecular mechanisms involved in cancer development, and may serve as a platform for targeted therapies against cancer. Communicated by Ramaswamy H. Sarma.

Mechanisms of influence of the microtubule over-stabilizing ligands on the structure and intrinsic dynamics of α,ß-Tubulin.

The intervention into the cell cycle progression by administering microtubule over-stabilizing ligands that arrest the mitotic cell division by preventing spindle dissociation, is a promising strategy to fight against cancers. The building blocks of the microtubules and the spindles, i.e. the α,ß-tubulin dimer, upon binding of such ligands, stay more comfortably in the microtubular multimeric form; the phenomenon of which is the key to the said over-stabilization. Using two such over-stabilizing ligands, Taxol and Taxotere, the present work reports the collective changes that these ligands induce on the structure and dynamics of the α,ß-tubulin dimer which could be reconciled as the molecular basis of the over-stabilization of the microtubules; the trends have been found to be statistically significant across all independent calculations on them. The ligand binding increases the coherence between the residue communities of the two opposite faces of the ß-subunit, which in a periodic arrangement in microtubule are knwon to form intermolecular contact with each other. This is likely to create an indirect cooperativity between those structural regions and this is a consequence of the reshuffling of the internal network of interactions upon ligand binding. Such reorganizations are also complemented by the increased contributions of the softer modes of the intrinsic dynamics more, which is likely to increase the plasticity of the system favourable for making structural adjustments in a multimer. Further, the ligands are able to compensate the drawback of lacking one phosphate group in protein-GDP interactions compared to the same for protein-GTP and this is in agreement with the hints form the earlier reports. The findings form a mechanistic basis of the enhanced capacity of the α,ß-tubulin dimer to get more favourably accommodated into the microtubule superstructure upon binding either of Taxol and Taxotere.

Structure and Energetics of GTP- and GDP-Tubulin Isodesmic Self-Association.

Tubulin self-association is a critical process in microtubule dynamics. The early intermediate structures, energetics, and their regulation by fluxes of chemical energy, associated with guanosine triphosphate (GTP) hydrolysis, are poorly understood. We reconstituted an in vitro minimal model system, mimicking the key elements of the nontemplated tubulin assembly. To resolve the distribution of GTP- and guanosine diphosphate (GDP)-tubulin structures, at low temperatures (∼10 °C) and below the critical concentration for the microtubule assembly, we analyzed in-line size-exclusion chromatography-small-angle X-ray scattering (SEC-SAXS) chromatograms of GTP- and GDP-tubulin solutions. Both solutions rapidly attained steady state. The SEC-SAXS data were consistent with an isodesmic thermodynamic model of longitudinal tubulin self-association into 1D oligomers, terminated by the formation of tubulin single rings. The analysis showed that free dimers coexisted with tetramers and hexamers. Tubulin monomers and lateral association between dimers were not detected. The dimer-dimer longitudinal self-association standard Helmholtz free energies were -14.2 ± 0.4 kBT (-8.0 ± 0.2 kcal mol-1) and -13.1 ± 0.5 kBT (-7.4 ± 0.3 kcal mol-1) for GDP- and GTP-tubulin, respectively. We then determined the mass fractions of dimers, tetramers, and hexamers as a function of the total tubulin concentration. A small fraction of stable tubulin single rings, with a radius of 19.2 ± 0.2 nm, was detected in the GDP-tubulin solution. In the GTP-tubulin solution, this fraction was significantly lower. Cryo-TEM images and SEC-multiangle light-scattering analysis corroborated these findings. Our analyses provide an accurate structure-stability description of cold tubulin solutions.

KARATE: PKA-induced KRAS4B-RHOA-mTORC2 supercomplex phosphorylates AKT in insulin signaling and glucose homeostasis.

AKT is a serine/threonine kinase that plays an important role in metabolism, cell growth, and cytoskeletal dynamics. AKT is activated by two kinases, PDK1 and mTORC2. Although the regulation of PDK1 is well understood, the mechanism that controls mTORC2 is unknown. Here, by investigating insulin receptor signaling in human cells and biochemical reconstitution, we found that insulin induces the activation of mTORC2 toward AKT by assembling a supercomplex with KRAS4B and RHOA GTPases, termed KARATE (KRAS4B-RHOA-mTORC2 Ensemble). Insulin-induced KARATE assembly is controlled via phosphorylation of GTP-bound KRAS4B at S181 and GDP-bound RHOA at S188 by protein kinase A. By developing a KARATE inhibitor, we demonstrate that KRAS4B-RHOA interaction drives KARATE formation. In adipocytes, KARATE controls insulin-dependent translocation of the glucose transporter GLUT4 to the plasma membrane for glucose uptake. Thus, our work reveals a fundamental mechanism that activates mTORC2 toward AKT in insulin-regulated glucose homeostasis.

Structural basis of TRAPPIII-mediated Rab1 activation.

The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo-EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α-helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes.

Cryo-EM structure of metazoan TRAPPIII, the multi-subunit complex that activates the GTPase Rab1.

The TRAPP complexes are nucleotide exchange factors that play essential roles in membrane traffic and autophagy. TRAPPII activates Rab11, and TRAPPIII activates Rab1, with the two complexes sharing a core of small subunits that affect nucleotide exchange but being distinguished by specific large subunits that are essential for activity in vivo. Crystal structures of core subunits have revealed the mechanism of Rab activation, but how the core and the large subunits assemble to form the complexes is unknown. We report a cryo-EM structure of the entire Drosophila TRAPPIII complex. The TRAPPIII-specific subunits TRAPPC8 and TRAPPC11 hold the catalytic core like a pair of tongs, with TRAPPC12 and TRAPPC13 positioned at the joint between them. TRAPPC2 and TRAPPC2L link the core to the two large arms, with the interfaces containing residues affected by disease-causing mutations. The TRAPPC8 arm is positioned such that it would contact Rab1 that is bound to the core, indicating how the arm could determine the specificity of the complex. A lower resolution structure of TRAPPII shows a similar architecture and suggests that the TRAPP complexes evolved from a single ur-TRAPP.

Second distinct conformation of the phosphohistidine loop in succinyl-CoA synthetase.

Succinyl-CoA synthetase (SCS) catalyzes a reversible reaction that is the only substrate-level phosphorylation in the citric acid cycle. One of the essential steps for the transfer of the phosphoryl group involves the movement of the phosphohistidine loop between active site I, where CoA, succinate and phosphate bind, and active site II, where the nucleotide binds. Here, the first crystal structure of SCS revealing the conformation of the phosphohistidine loop in site II of the porcine GTP-specific enzyme is presented. The phosphoryl transfer bridges a distance of 29 Šbetween the binding sites for phosphohistidine in site I and site II, so these crystal structures support the proposed mechanism of catalysis by SCS. In addition, a second succinate-binding site was discovered at the interface between the α- and ß-subunits of SCS, and another magnesium ion was found that interacts with the side chains of Glu141ß and Glu204ß via water-mediated interactions. These glutamate residues interact with the active-site histidine residue when it is bound in site II.

Atomistic Basis of Microtubule Dynamic Instability Assessed Via Multiscale Modeling.

Microtubule "dynamic instability," the abrupt switching from assembly to disassembly caused by the hydrolysis of GTP to GDP within the ß subunit of the αß-tubulin heterodimer, is necessary for vital cellular processes such as mitosis and migration. Despite existing high-resolution structural data, the key mechanochemical differences between the GTP and GDP states that mediate dynamic instability behavior remain unclear. Starting with a published atomic-level structure as an input, we used multiscale modeling to find that GTP hydrolysis results in both longitudinal bond weakening (~ 4 kBT) and an outward bending preference (~ 1.5 kBT) to both drive dynamic instability and give rise to the microtubule tip structures previously observed by light and electron microscopy. More generally, our study provides an example where atomic level structural information is used as the sole input to predict cellular level dynamics without parameter adjustment.

GTP-dependent formation of straight tubulin oligomers leads to microtubule nucleation.

Nucleation of microtubules (MTs) is essential for cellular activities, but its mechanism is unknown because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. Here, combining rapid flush negative stain electron microscopy (EM) and kinetic analysis, we demonstrate that the formation of straight oligomers of critical size is essential for nucleation. Both GDP and GTP tubulin form single-stranded oligomers with a broad range of curvatures, but upon nucleation, the curvature distribution of GTP oligomers is shifted to produce a minor population of straight oligomers. With tubulin having the Y222F mutation in the ß subunit, the proportion of straight oligomers increases and nucleation accelerates. Our results support a model in which GTP binding generates a minor population of straight oligomers compatible with lateral association and further growth to MTs. This study suggests that cellular factors involved in nucleation promote it via stabilization of straight oligomers.

Conformational switch that induces GDP release from Gi.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are composed of α, ß, and γ subunits. Gα switches between guanosine diphosphate (GDP)-bound inactive and guanosine triphosphate (GTP)-bound active states, and Gßγ interacts with the GDP-bound state. The GDP-binding regions are composed of two sites: the phosphate-binding and guanine-binding regions. The turnover of GDP and GTP is induced by guanine nucleotide-exchange factors (GEFs), including G protein-coupled receptors (GPCRs), Ric8A, and GIV/Girdin. However, the key structural factors for stabilizing the GDP-bound state of G proteins and the direct structural event for GDP release remain unclear. In this study, we investigated structural factors affecting GDP release by introducing point mutations in selected, conserved residues in Gαi3. We examined the effects of these mutations on the GDP/GTP turnover rate and the overall conformation of Gαi3 as well as the binding free energy between Gαi3 and GDP. We found that dynamic changes in the phosphate-binding regions are an immediate factor for the release of GDP.