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.

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.

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.

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.

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.

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.

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.

ARL15, a GTPase implicated in rheumatoid arthritis, potentially repositions its truncated N-terminus as a function of guanine nucleotide binding.

The ADP ribosylation factor like protein 15 (ARL15) gene encodes for an uncharacterized GTPase associated with rheumatoid arthritis (RA) and other metabolic disorders. Investigation of the structural and functional attributes of ARL15 is important to position the protein as a potential drug target. Using spectroscopy, we demonstrated that ARL15 exhibits properties inherent of GTPases. The Km and Vmax of the enzyme were calculated to be 100 µM and 1.47 µmole/min/µL, respectively. The equilibrium dissociation constant (Kd) of GTP binding with ARL15 was estimated to be about eight-fold higher than that of GDP. Small Angle X-ray Scattering (SAXS) data indicated that in solution, the apo state of monomeric ARL15 adopts a shape characterized by a globe of maximum linear dimension (Dmax) of 6.1 nm, and upon binding to GTP or GDP, the vector distribution profile changes to peak-n-tail shoulder with Dmax extended to 7.6 and 7.7 nm, respectively. Structure restoration using a sequence-based template and experimental SAXS data provided the first visual insight revealing that the folded N-terminal in the unbound state of the protein may toggle open upon binding to guanine nucleotides. The conformational dynamics observed in the N-terminal region offer a scope to develop drugs that target this unique GTPase, potentially providing treatments for a range of metabolic disorders.

Phosphorylation of RhoGDI1, a Rho GDP dissociation inhibitor, regulates root hair development in Arabidopsis under salt stress.

To ensure optimal growth, plants actively regulate their growth and development based on environmental changes. Among these, salt stress significantly influences growth and yield. In this study, we demonstrate that the growth of root hairs of salt-stressed Arabidopsis thaliana seedlings is regulated by the SALT OVERLY SENSITIVE 2 (SOS2)-GUANOSINE NUCLEOTIDE DIPHOSPHATE DISSOCIATION INHIBITOR 1 (RhoGDI1)-Rho GTPASE OF PLANTS 2 (ROP2) module. We show here that the kinase SOS2 is activated by salt stress and subsequently phosphorylates RhoGDI1, a root hair regulator, thereby decreasing its stability. This change in RhoGDI1 abundance resulted in a fine-tuning of polar localization of ROP2 and root hair initiation followed by polar growth, demonstrating how SOS2-regulated root hair development is critical for plant growth under salt stress. Our results reveal how a tissue-specific response to salt stress balances the relationship of salt resistance and basic growth.

Bipolar hemicyanine cationic probe for simultaneous sensing of ATP and GTP.

Adenosine 5'-triphosphate (ATP) and guanosine 5'-triphosphate (GTP) are the most essential energy source in enormous biological processes. Various probes for ATP or GTP sensing, have been widely established, but the probe that could simultaneously monitor ATP and GTP is still rarely reported. Herein, we report a bipolar hemicyanine cationic probe for simultaneous sensing of ATP and GTP via a one-step monitoring process. This probe exhibited strong affinity to ATP and GTP through intramolecular electrostatic and π-π stacking interactions, which the binding constant on each step were determined as 6.15 × 107 M-1 and 1.57 × 106 M-1 for ATP, 3.19 × 107 M-1 and 3.81 × 106 M-1 for GTP. The sensitivity and specificity of this probe toward ATP or GTP over other twelve biological analogues (adenosine 5'-diphosphate (ADP), adenosine 5'-monophosphate (AMP), guanosine 5'-diphosphate (GDP), guanosine 5'-monophosphate (GMP), Etc.) have also been successfully demonstrated. Furthermore, due to the rapid response rate (within 10 s), we also proved that this probe could be employed as a monitor tool during the ATP or GTP-related enzymatic reaction process.

Protocols for Efficient Chemical Synthesis of N2 -Modified Guanosine Phosphate Derivatives: Versatile Probes for mRNA Labeling.

A facile, reliable, and efficient method for the synthesis of N2 -modified guanosine nucleotides such as N2 -[benzyl-N-(propyl)carbamate]-guanosine-5'-O-monophosphate, N2 -[benzyl-N-(propyl)carbamate]-guanosine-5'-O-diphosphate, N2 -[benzyl-N-(propyl)carbamate]-guanosine-5'-O-triphosphate, and N2 -[benzyl-N-(propyl)carbamate]-N7 -methyl-guanosine-5'-O-diphosphate, starting from the corresponding nucleotide is described. The general process entails condensation between the exocyclic amine of guanosine nucleotide and 3-[(benzyloxycarbonyl)amino]propionaldehyde in aqueous methanol, followed by reduction using sodium cyanoborohydride to furnish the corresponding N2 -modified guanosine nucleotide in moderate yield with high purity (>99.5%). © 2023 Wiley Periodicals LLC. Basic Protocol: Synthesis of N2 -modified guanosine derivatives.

Excited-state observation of active K-Ras reveals differential structural dynamics of wild-type versus oncogenic G12D and G12C mutants.

Despite the prominent role of the K-Ras protein in many different types of human cancer, major gaps in atomic-level information severely limit our understanding of its functions in health and disease. Here, we report the quantitative backbone structural dynamics of K-Ras by solution nuclear magnetic resonance spectroscopy of the active state of wild-type K-Ras bound to guanosine triphosphate (GTP) nucleotide and two of its oncogenic P-loop mutants, G12D and G12C, using a new nanoparticle-assisted spin relaxation method, relaxation dispersion and chemical exchange saturation transfer experiments covering the entire range of timescales from picoseconds to milliseconds. Our combined experiments allow detection and analysis of the functionally critical Switch I and Switch II regions, which have previously remained largely unobservable by X-ray crystallography and nuclear magnetic resonance spectroscopy. Our data reveal cooperative transitions of K-Ras·GTP to a highly dynamic excited state that closely resembles the partially disordered K-Ras·GDP state. These results advance our understanding of differential GTPase activities and signaling properties of the wild type versus mutants and may thus guide new strategies for the development of therapeutics.

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.

Computer simulation reveals the effect of severing enzymes on dynamic and stabilized microtubules.

Microtubule (MT) severing enzymes Katanin and Spastin cut the MT into smaller fragments and are being studied extensively usingin-vitroexperiments due to their crucial role in different cancers and neurodevelopmental disorders. It has been reported that the severing enzymes are either involved in increasing or decreasing the tubulin mass. Currently, there are a few analytical and computational models for MT amplification and severing. However, these models do not capture the action of MT severing explicitly, as these are based on partial differential equations in one dimension. On the other hand, a few discrete lattice-based models were used earlier to understand the activity of severing enzymes only on stabilized MTs. Hence, in this study, discrete lattice-based Monte Carlo models that included MT dynamics and severing enzyme activity have been developed to understand the effect of severing enzymes on tubulin mass, MT number, and MT length. It was found that the action of severing enzyme reduces average MT length while increasing their number; however, the total tubulin mass can decrease or increase depending on the concentration of GMPCPP (Guanylyl-(α,ß)-methylene-diphosphonate)-which is a slowly hydrolyzable analogue of GTP (Guanosine triphosphate). Further, relative tubulin mass also depends on the detachment ratio of GTP/GMPCPP and Guanosine diphosphate tubulin dimers and the binding energies of tubulin dimers covered by the severing enzyme.

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.

RABL4/IFT27 in a nucleotide-independent manner promotes phospholipase D ciliary retrieval via facilitating BBSome reassembly at the ciliary tip.

Certain ciliary transmembrane and membrane-associated signaling proteins export from cilia as intraflagellar transport (IFT) cargoes in a BBSome-dependent manner. Upon reaching the ciliary tip via anterograde IFT, the BBSome disassembles before being reassembled to form an intact entity for cargo phospholipase D (PLD) coupling. During this BBSome remodeling process, Chlamydomonas Rab-like 4 GTPase IFT27, by binding its partner IFT25 to form the heterodimeric IFT25/27, is indispensable for BBSome reassembly. Here, we show that IFT27 binds IFT25 in an IFT27 nucleotide-independent manner. IFT25/27 and the IFT subcomplexes IFT-A and -B are irrelevant for maintaining the stability of one another. GTP-loading onto IFT27 enhances the IFT25/27 affinity for binding to the IFT-B subcomplex core IFT-B1 entity in cytoplasm, while GDP-bound IFT27 does not prevent IFT25/27 from entering and cycling through cilia by integrating into IFT-B1. Upon at the ciliary tip, IFT25/27 cycles on and off IFT-B1 and this process is irrelevant with the nucleotide state of IFT27. During BBSome remodeling at the ciliary tip, IFT25/27 promotes BBSome reassembly independent of IFT27 nucleotide state, making postremodeled BBSomes available for PLD to interact with. Thus, IFT25/27 facilitates BBSome-dependent PLD export from cilia via controlling availability of intact BBSomes at the ciliary tip, while IFT27 nucleotide state does not participate in this regulatory event.