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.

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.

Conformational States of the GDP- and GTP-Bound HRAS Affected by A59E and K117R: An Exploration from Gaussian Accelerated Molecular Dynamics.

The HRAS protein is considered a critical target for drug development in cancers. It is vital for effective drug development to understand the effects of mutations on the binding of GTP and GDP to HRAS. We conducted Gaussian accelerated molecular dynamics (GaMD) simulations and free energy landscape (FEL) calculations to investigate the impacts of two mutations (A59E and K117R) on GTP and GDP binding and the conformational states of the switch domain. Our findings demonstrate that these mutations not only modify the flexibility of the switch domains, but also affect the correlated motions of these domains. Furthermore, the mutations significantly disrupt the dynamic behavior of the switch domains, leading to a conformational change in HRAS. Additionally, these mutations significantly impact the switch domain's interactions, including their hydrogen bonding with ligands and electrostatic interactions with magnesium ions. Since the switch domains are crucial for the binding of HRAS to effectors, any alterations in their interactions or conformational states will undoubtedly disrupt the activity of HRAS. This research provides valuable information for the design of drugs targeting HRAS.

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.

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.

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.

Real-time monitoring of the reaction of KRAS G12C mutant specific covalent inhibitor by in vitro and in-cell NMR spectroscopy.

KRAS mutations are major drivers of various cancers. Recently, allele-specific inhibitors of the KRAS G12C mutant were developed that covalently modify the thiol of Cys12, thereby trapping KRAS in an inactive GDP-bound state. To study the mechanism of action of the covalent inhibitors in both in vitro and intracellular environments, we used real-time NMR to simultaneously observe GTP hydrolysis and inhibitor binding. In vitro NMR experiments showed that the rate constant of ARS-853 modification is identical to that of GTP hydrolysis, indicating that GTP hydrolysis is the rate-limiting step for ARS-853 modification. In-cell NMR analysis revealed that the ARS-853 reaction proceeds significantly faster than that in vitro, reflecting acceleration of GTP hydrolysis by endogenous GTPase proteins. This study demonstrated that the KRAS covalent inhibitor is as effective in the cell as in vitro and that in-cell NMR is a valuable validation tool for assessing the pharmacological properties of the drug in the intracellular context.

Homogeneous luminescent quantitation of cellular guanosine and adenosine triphosphates (GTP and ATP) using QT-LucGTP&ATP assay.

Guanosine triphosphate (GTP) and adenosine triphosphate (ATP) are essential nucleic acid building blocks and serve as energy molecules for a wide range of cellular reactions. Cellular GTP concentration fluctuates independently of ATP and is significantly elevated in numerous cancers, contributing to malignancy. Quantitative measurement of ATP and GTP has become increasingly important to elucidate how concentration changes regulate cell function. Liquid chromatography-coupled mass spectrometry (LC-MS) and capillary electrophoresis-coupled MS (CE-MS) are powerful methods widely used for the identification and quantification of biological metabolites. However, these methods have limitations related to specialized instrumentation and expertise, low throughput, and high costs. Here, we introduce a novel quantitative method for GTP concentration monitoring (GTP-quenching resonance energy transfer (QRET)) in homogenous cellular extracts. CE-MS analysis along with pharmacological control of cellular GTP levels shows that GTP-QRET possesses high dynamic range and accuracy. Furthermore, we combined GTP-QRET with luciferase-based ATP detection, leading to a new technology, termed QT-LucGTP&ATP, enabling high-throughput compatible dual monitoring of cellular GTP and ATP in a homogenous fashion. Collectively, GTP-QRET and QT-LucGTP&ATP offer a unique, high-throughput opportunity to explore cellular energy metabolism, serving as a powerful platform for the development of novel therapeutics and extending its usability across a range of disciplines.

Exploration of natural compounds against the human mpox virus DNA-dependent RNA polymerase in silico.

BACKGROUND: Last year, the human monkeypox virus (hMPXV) emerged as an alarming threat to the community, with a detectable outbreak outside the African continent for the first time. According to The American Centers for Disease Control and Prevention (CDC), the virus is reported globally, with 86,746 confirmed cases (until April 08, 2023). DNA-dependent RNA polymerase (DdRp) is an essential protein for viral replication; hence it is a promising drug target for developing antiviral drugs against DNA viruses. Therefore, this study was conducted to search for natural compounds that could provide scaffolds for RNA polymerase inhibitors. METHODS: In this study, the DdRp structure of hMPXV was modeled and used to screen the natural compounds database (COCONUT). The virtual screening revealed 15 compounds able to tightly bind to the active site of the DdRp (binding energies less than -7.0 kcal/mol) compared to the physiological nucleotide, guanosine triphosphate (GTP). Molecular dynamics simulation was then performed on the top four hits and compared to GTP RESULTS: The results revealed the potential of four compounds (comp289, comp295, comp441, and comp449) in binding the hMPXV DdRp active site with a comparable binding affinity (-17.06 ± 2.96, -11.6 ± 5.34, -14.85 ± 2.66, and -10.79 ± 4.49 kcal/mol) with GTP (-21.03 ± 7.55 kcal/mol) CONCLUSION: These findings may also pave the way for developing new hMPXV inhibitors based on natural product scaffolds. These results need further experimental validation but promising as it was validated by unbiased all-atom MD simulations and binding free energy calculations.

Deoxyguanosine-Linked Bifunctional Inhibitor of SAMHD1 dNTPase Activity and Nucleic Acid Binding.

Sterile alpha motif histidine-aspartate domain protein 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase that exists in monomeric, dimeric, and tetrameric forms. It is activated by GTP binding to an A1 allosteric site on each monomer subunit, which induces dimerization, a prerequisite for dNTP-induced tetramerization. SAMHD1 is a validated drug target stemming from its inactivation of many anticancer nucleoside drugs leading to drug resistance. The enzyme also possesses a single-strand nucleic acid binding function that promotes RNA and DNA homeostasis by several mechanisms. To discover small molecule inhibitors of SAMHD1, we screened a custom ∼69 000-compound library for dNTPase inhibitors. Surprisingly, this effort yielded no viable hits and indicated that exceptional barriers for discovery of small molecule inhibitors existed. We then took a rational fragment-based inhibitor design approach using a deoxyguanosine (dG) A1 site targeting fragment. A targeted chemical library was synthesized by coupling a 5'-phosphoryl propylamine dG fragment (dGpC3NH2) to 376 carboxylic acids (RCOOH). Direct screening of the products (dGpC3NHCO-R) yielded nine initial hits, one of which (R = 3-(3'-bromo-[1,1'-biphenyl]), 5a) was investigated extensively. Amide 5a is a competitive inhibitor against GTP binding to the A1 site and induces inactive dimers that are deficient in tetramerization. Surprisingly, 5a also prevented ssDNA and ssRNA binding, demonstrating that the dNTPase and nucleic acid binding functions of SAMHD1 can be disrupted by a single small molecule. A structure of the SAMHD1-5a complex indicates that the biphenyl fragment impedes a conformational change in the C-terminal lobe that is required for tetramerization.

Difference in Catalytic Loop Repositioning Leads to GMP Variation between Two Human GBP Homologues.

Interferon-gamma-inducible human large GTPases, hGBP1 and hGBP2, have a distinctive feature of hydrolyzing GTP to GDP and GMP through successive phosphate cleavages. In hGBP1, GMP is the major product, which is essential for its anti-pathogenic activities. However, its close homologue hGBP2 produces significantly less GMP, despite having a similar active site architecture. The molecular basis for less GMP formation and catalytic residue(s) in hGBP2 are not fully explored. To address these issues, we performed systematic biochemical, biophysical, and microsecond simulation studies. Our data suggest that the less GMP formation in hGBP2 is due to the lack of H-bond formation between the W79 side-chain (located near the active site) and main-chain carbonyl of K76 (present in the catalytic loop) in the substrate-bound hGBP2. The absence of this H-bond could not redirect the catalytic loop toward the beta phosphate after the cleavage of gamma-phosphate, a step essential for enhanced GMP formation. Furthermore, based on the mutational and structural analyses, this study for the first time indicates that the same residue, T75, mediates both phosphate cleavages in hGBP2 and hGBP1. This suggests the conservation of the catalytic residue in hGBP homologues. These findings emphasize the indispensable role of correct catalytic loop repositioning for efficient beta phosphate cleavage. This led us to propose a new substrate hydrolysis mechanism by hGBP1 and hGBP2, which may also be helpful to understand the GTP hydrolysis in other hGBP homologues. Overall, the study could provide insight into how these two close homologues play crucial roles in host-mediated immunity through different mechanisms.

Probing mutation-induced conformational transformation of the GTP/M-RAS complex through Gaussian accelerated molecular dynamics simulations.

Mutations highly affect the structural flexibility of two switch domains in M-RAS considered an important target of anticancer drug design. Gaussian accelerated molecular dynamics (GaMD) simulations were applied to probe the effect of mutations P40D, D41E, and P40D/D41E/L51R on the conformational transition of the switch domains from the GTP-bound M-RAS. The analyses of free energy landscapes (FELs) not only reveal that three mutations induce less energetic states than the wild-type (WT) M-RAS but also verify that the switch domains are extremely disordered. Principal component analysis (PCA) and dynamics analysis suggest that three mutations greatly affect collective motions and structural flexibility of the switch domains that mostly overlap with binding regions of M-RAS to its effectors, which in turn disturbs the activity of M-RAS. The analyses of the interaction network between GTP and M-RAS show that the high instability in hydrogen bonding interactions (HBIs) of GTP with residue 41 and Y42 in the switch domain I drives the disordered states of the switch domains. This work is expected to provide a molecular mechanism for deeply understanding the function of M-RAS and future drug design towards the treatment of cancers.

Classification of GTP-dependent K-Ras4B active and inactive conformational states.

Classifying reliably active and inactive molecular conformations of wildtype (WT) and mutated oncogenic proteins is a key, ongoing challenge in molecular cancer studies. Here, we probe the GTP-bound K-Ras4B conformational dynamics using long-time atomistic molecular dynamics (MD) simulations. We extract and analyze the detailed underlying free energy landscape of WT K-Ras4B. We use two key reaction coordinates, labeled d1 and d2 (i.e., distances coordinating the Pß atom of the GTP ligand with two key residues, T35 and G60), shown to correlate closely with activities of WT and mutated K-Ras4B. However, our new K-Ras4B conformational kinetics study reveals a more complex network of equilibrium Markovian states. We show that a new reaction coordinate is required to account for the orientation of acidic K-Ras4B sidechains such as D38 with respect to the interface with binding effector RAF1 and rationalize the activation/inactivation propensities and the corresponding molecular binding mechanisms. We use this understanding to unveil how a relatively conservative mutation (i.e., D33E, in the switch I region) can lead to significantly different activation propensities compared with WT K-Ras4B. Our study sheds new light on the ability of residues near the K-Ras4B-RAF1 interface to modulate the network of salt bridges at the binding interface with the RAF1 downstream effector and, thus, to influence the underlying GTP-dependent activation/inactivation mechanism. Altogether, our hybrid MD-docking modeling approach enables the development of new in silico methods for quantitative assessment of activation propensity changes (e.g., due to mutations or local binding environment). It also unveils the underlying molecular mechanisms and facilitates the rational design of new cancer drugs.

GAP positions catalytic H-Ras residue Q61 for GTP hydrolysis in molecular dynamics simulations, complicating chemical rescue of Ras deactivation.

Functional interaction of Ras signaling proteins with upstream, negative regulatory GTPase activating proteins (GAPs) represents a crucial step in cellular decision making related to growth and survival. Key components of the catalytic transition state for Ras deactivation by GAP-accelerated hydrolysis of Ras-bound guanosine triphosphate (GTP) are thought to include an arginine residue from the GAP (the arginine finger), a glutamine residue from Ras (Q61), and a water molecule that is likely coordinated by Q61 to engage in nucleophilic attack on GTP. Here, we use in-vitro fluorescence experiments to show that 0.1-100 mM concentrations of free arginine, imidazole, and other small nitrogenous molecule fail to accelerate GTP hydrolysis, even in the presence of the catalytic domain of a mutant GAP lacking its arginine finger (R1276A NF1). This result is surprising given that imidazole can chemically rescue enzyme activity in arginine-to-alanine mutant protein tyrosine kinases (PTKs) that share many active site components with Ras/GAP complexes. Complementary all-atom molecular dynamics (MD) simulations reveal that an arginine finger GAP mutant still functions to enhance Ras Q61-GTP interaction, though less extensively than wild-type GAP. This increased Q61-GTP proximity may promote more frequent fluctuations into configurations that enable GTP hydrolysis as a component of the mechanism by which GAPs accelerate Ras deactivation in the face of arginine finger mutations. The failure of small molecule analogs of arginine to chemically rescue catalytic deactivation of Ras is consistent with the idea that the influence of the GAP goes beyond the simple provision of its arginine finger. However, the failure of chemical rescue in the presence of R1276A NF1 suggests that the GAPs arginine finger is either unsusceptible to rescue due to exquisite positioning or that it is involved in complex multivalent interactions. Therefore, in the context of oncogenic Ras proteins with mutations at codons 12 or 13 that inhibit arginine finger penetration toward GTP, drug-based chemical rescue of GTP hydrolysis may have bifunctional chemical/geometric requirements that are more difficult to satisfy than those that result from arginine-to-alanine mutations in other enzymes for which chemical rescue has been demonstrated.

Catalytic site mutations confer multiple states of G protein activation.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) that function as molecular switches for cellular growth and metabolism are activated by GTP and inactivated by GTP hydrolysis. In uveal melanoma, a conserved glutamine residue critical for GTP hydrolysis in the G protein α subunit is often mutated in Gαq or Gα11 to either leucine or proline. In contrast, other glutamine mutations or mutations in other Gα subtypes are rare. To uncover the mechanism of the genetic selection and the functional role of this glutamine residue, we analyzed all possible substitutions of this residue in multiple Gα isoforms. Through cell-based measurements of activity, we showed that some mutants were further activated and inactivated by G protein-coupled receptors. Through biochemical, molecular dynamics, and nuclear magnetic resonance-based structural studies, we showed that the Gα mutants were functionally distinct and conformationally diverse, despite their shared inability to hydrolyze GTP. Thus, the catalytic glutamine residue contributes to functions beyond GTP hydrolysis, and these functions include subtype-specific, allosteric modulation of receptor-mediated subunit dissociation. We conclude that G proteins do not function as simple on-off switches. Rather, signaling emerges from an ensemble of active states, a subset of which are favored in disease and may be uniquely responsive to receptor-directed ligands.

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.

A RhoA structure with switch II flipped outward revealed the conformational dynamics of switch II region.

Small GTPase RhoA switches from GTP-bound state to GDP-bound state by hydrolyzing GTP, which is accelerated by GTPases activating proteins (GAPs). However, less study of RhoA structural dynamic changes was conducted during this process, which is essential for understanding the molecular mechanism of GAP dissociation. Here, we solved a RhoA structure in GDP-bound state with switch II flipped outward. Because lacking the intermolecular interactions with guanine nucleotide, we proposed this conformation of RhoA could be an intermediate after GAP dissociation. Further molecular dynamics simulations found the conformational changes of switch regions are indeed existing in RhoA and involved in the regulation of GAP dissociation and GEF recognition. Besides, the guanine nucleotide binding pocket extended to switch II region, indicating a potential "druggable" cavity for RhoA. Taken together, our study provides a deeper understanding of the dynamic properties of RhoA switch regions and highlights the direction for future drug development.

Molecular dynamics simulation study on Bacillus subtilis EngA: the presence of Mg2+ at the active-sites promotes the functionally important conformation.

EngA, a GTPase contains two GTP binding domains [GD1, GD2], and the C-terminal KH domain shown to be involved in the later stages of ribosome maturation. Association of EngA to the ribosomal subunit in the intermediate stage of maturation is essential for complete ribosome maturation. However, this association was shown to be dependent on the nucleotide bound combinations. This nucleotide dependent association tendency is attributed to the conformational changes that occur among different nucleotide bound combinations. Therefore, to explore the conformational changes, all-atom molecular dynamics simulations for Bacillus subtilis EngA in different nucleotide bound combinations along with the presence or absence of Mg2+ in the active-sites were carried out. The presence of Mg2+ along with the bound nucleotide at the GD2 active-site dictates the GD2-Sw-II mobility, but the GD1-Sw-II mobility has not shown any nucleotide or Mg2+ dependent movement. However, the GD1-Sw-II secondary conformations are shown to be influenced by the GD2 nucleotide bound state. This allosteric connection between the GD2 active-site and the GD1-Sw-II is also observed through the dynamic network analysis. Further, the exploration of the GD1-KH interface interactions exhibited a more attractive tendency when GD1 is bound to GTP-Mg2+. In addition, the presence of Mg2+ stabilizes active-site water and also increases the distances between the α- and γ- phosphates of the bound GTP. Curiously, three water molecules in the GD1 active-site and only one water molecule in the GD2 active-site are stabilized. This indicates that the probability of GTP hydrolysis is more in GD1 compared to GD2.Communicated by Ramaswamy H. Sarma.

The role of GTP hydrolysis by EF-G in ribosomal translocation.

Translocation of transfer RNA (tRNA) and messenger RNA (mRNA) through the ribosome is catalyzed by the GTPase elongation factor G (EF-G) in bacteria. Although guanosine-5'-triphosphate (GTP) hydrolysis accelerates translocation and is required for dissociation of EF-G, its fundamental role remains unclear. Here, we used ensemble Förster resonance energy transfer (FRET) to monitor how inhibition of GTP hydrolysis impacts the structural dynamics of the ribosome. We used FRET pairs S12-S19 and S11-S13, which unambiguously report on rotation of the 30S head domain, and the S6-L9 pair, which measures intersubunit rotation. Our results show that, in addition to slowing reverse intersubunit rotation, as shown previously, blocking GTP hydrolysis slows forward head rotation. Surprisingly, blocking GTP hydrolysis completely abolishes reverse head rotation. We find that the S13-L33 FRET pair, which has been used in previous studies to monitor head rotation, appears to report almost exclusively on intersubunit rotation. Furthermore, we find that the signal from quenching of 3'-terminal pyrene-labeled mRNA, which is used extensively to follow mRNA translocation, correlates most closely with reverse intersubunit rotation. To account for our finding that blocking GTP hydrolysis abolishes a rotational event that occurs after the movements of mRNA and tRNAs are essentially complete, we propose that the primary role of GTP hydrolysis is to create an irreversible step in a mechanism that prevents release of EF-G until both the tRNAs and mRNA have moved by one full codon, ensuring productive translocation and maintenance of the translational reading frame.

The Importance of Mg2+ -Free State in Nucleotide Exchange of Oncogenic K-Ras Mutants.

For efficient targeting of oncogenic K-Ras interaction sites, a mechanistic picture of the Ras-cycle is necessary. Herein, we used NMR relaxation techniques and molecular dynamics simulations to decipher the role of slow dynamics in wild-type and three oncogenic P-loop mutants of K-Ras. Our measurements reveal a dominant two-state conformational exchange on the ms timescale in both GDP- and GTP-bound K-Ras. The identified low-populated higher energy state in GDP-loaded K-Ras has a conformation reminiscent of a nucleotide-bound/Mg2+ -free state characterized by shortened ß2/ß3-strands and a partially released switch-I region preparing K-Ras for the interaction with the incoming nucleotide exchange factor and subsequent reactivation. By providing insight into mutation-specific differences in K-Ras structural dynamics, our systematic analysis improves our understanding of prolonged K-Ras signaling and may aid the development of allosteric inhibitors targeting nucleotide exchange in K-Ras.