Guanosine Triphosphate Analogue Research Articles

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 underlyingmembrane as it progresses through the GTPase cycle to sever vesicles from the plasmamembrane.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 theassembled 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 assembledas a 2-start helix with an inner lumen of 3.4nm, primed for spontaneous fission. Additionally, by cryo-electron tomography, we trapped dynamin helical assemblies within HeLa cells using theGTPase-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.

Computational Modeling of the Neurofibromin-Stimulated Guanosine Triphosphate Hydrolysis by the KRas Protein

We report the results of computational studies of the guanosine triphosphate (GTP) hydrolysis in the active site of the KRas-NF1 protein complex, where KRas stands for the K-isoform of the Ras (ras sarcoma) protein and NF1 (neurofbromin-1) is the activating protein. The model system was constructed using coordinates of heavy atoms from the crystal structure PDB ID 6OB2 with the GTP analog GMPPNP. Large-scale classical molecular dynamics (MD) calculations were performed to analyze conformations of the enzyme-substrate complexes. The Gibbs energy profiles for the hydrolysis reaction were computed using MD simulations with quantum mechanics/molecular mechanics (QM/MM) interaction potentials. The density functional theory DFT(ωB97X-D3/6-31G**) approach was applied in QM and the CHARMM36 force field parameters in MM. The most likely scenario of the chemical step of the GTP hydrolysis in KRas-NF1 corresponds to the water-assisted mechanism of the formation of the inorganic phosphate coupled with the dissociation of GTP to GDP.

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 using in-vitro experiments 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, β)-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.

Structure of metallochaperone in complex with the cobalamin-binding domain of its target mutase provides insight into cofactor delivery

G-protein metallochaperone MeaB in bacteria [methylmalonic aciduria type A (MMAA) in humans] is responsible for facilitating the delivery of adenosylcobalamin (AdoCbl) to methylmalonyl-CoA mutase (MCM), the only AdoCbl-dependent enzyme in humans. Genetic defects in the switch III region of MMAA lead to the genetic disorder methylmalonic aciduria in which the body is unable to process certain lipids. Here, we present a crystal structure of Methylobacterium extorquens MeaB bound to a nonhydrolyzable guanosine triphosphate (GTP) analog guanosine-5'-[(β,γ)-methyleno]triphosphate (GMPPCP) with the Cbl-binding domain of its target mutase enzyme (MeMCMcbl). This structure provides an explanation for the stimulation of the GTP hydrolyase activity of MeaB afforded by target protein binding. We find that upon MCMcbl association, one protomer of the MeaB dimer rotates ~180°, such that the inactive state of MeaB is converted to an active state in which the nucleotide substrate is now surrounded by catalytic residues. Importantly, it is the switch III region that undergoes the largest change, rearranging to make direct contacts with the terminal phosphate of GMPPCP. These structural data additionally provide insights into the molecular basis by which this metallochaperone contributes to AdoCbl delivery without directly binding the cofactor. Our data suggest a model in which GTP-bound MeaB stabilizes a conformation of MCM that is open for AdoCbl insertion, and GTP hydrolysis, as signaled by switch III residues, allows MCM to close and trap its cofactor. Substitutions of switch III residues destabilize the active state of MeaB through loss of protein:nucleotide and protein:protein interactions at the dimer interface, thus uncoupling GTP hydrolysis from AdoCbl delivery.

Molecular Modeling Reveals the Mechanism of Ran-RanGAP-Catalyzed Guanosine Triphosphate Hydrolysis without an Arginine Finger

We report results of a computational study of the reaction mechanism of guanosine triphosphate (GTP) hydrolysis catalyzed by the GTP-binding (GTPase) enzyme rat sarcoma-related nuclear (Ran) in complex with its activating protein RanGAP. According to structural investigations, Ran-RanGAP operates without the so-called arginine finger, an arginine residue from a distinct promoter (GTPase-accelerating protein (GAP)) that completes the active site in many GTPases. In this work, we construct model systems for Ran with and without GAP by motifs of the crystal structure of Ran-RanGAP with the GTP analogue and simulate the GTP hydrolysis reaction in these systems. Enzyme–substrate and enzyme–product complexes and reaction intermediates are obtained in quantum mechanics/molecular mechanics (QM/MM) simulations. Calculations of the free-energy reaction profiles are performed at the molecular dynamics level with the ab initio-type QM(DFT(PBE0-D3)/6-31G**)/MM potentials. We show that the computed activation barriers on the pathways for Ran catalysis with and without GAP are in line with the experimentally estimated rate constants. We demonstrate that mapping the Laplacian of the electron density provides easily visible images of substrate activation, which help distinguish between the reactive and nonreactive enzyme–substrate complexes and explain qualitative features of the enzyme-catalyzed GTP hydrolysis reactions consistent with the computed free-energy profiles. A comparison of reactions in Ran and Ran-RanGAP allows us to characterize the role of GAP operating without an arginine finger.

Analysis of GTP addition in the reverse (3'-5') direction by human tRNAHis guanylyltransferase.

Human tRNAHis guanylyltransferase (HsThg1) catalyzes the 3′–5′ addition of guanosine triphosphate (GTP) to the 5′-end (−1 position) of tRNAHis, producing mature tRNAHis. In human cells, cytoplasmic and mitochondrial tRNAHis have adenine (A) or cytidine (C), respectively, opposite to G−1. Little attention has been paid to the structural requirements of incoming GTP in 3′–5′ nucleotidyl addition by HsThg1. In this study, we evaluated the incorporation efficiencies of various GTP analogs by HsThg1 and compared the reaction mechanism with that of Candida albicans Thg1 (CaThg1). HsThg1 incorporated GTP opposite A or C in the template most efficiently. In contrast to CaThg1, HsThg1 could incorporate UTP opposite A, and guanosine diphosphate (GDP) opposite C. These results suggest that HsThg1 could transfer not only GTP, but also other NTPs, by forming Watson–Crick (WC) hydrogen bonds between the incoming NTP and the template base. On the basis of the molecular mechanism, HsThg1 succeeded in labeling the 5′-end of tRNAHis with biotinylated GTP. Structural analysis of HsThg1 was also performed in the presence of the mitochondrial tRNAHis. Structural comparison of HsThg1 with other Thg1 family enzymes suggested that the structural diversity of the carboxy-terminal domain of the Thg1 enzymes might be involved in the formation of WC base-pairing between the incoming GTP and template base. These findings provide new insights into an unidentified biological function of HsThg1 and also into the applicability of HsThg1 to the 5′-terminal modification of RNAs.

Molecular mechanism of substrate recognition and specificity of tRNAHis guanylyltransferase during nucleotide addition in the 3'-5' direction.

The tRNAHis guanylyltransferase (Thg1) transfers a guanosine triphosphate (GTP) in the 3′–5′ direction onto the 5′-terminal of tRNAHis, opposite adenosine at position 73 (A73). The guanosine at the −1 position (G−1) serves as an identity element for histidyl-tRNA synthetase. To investigate the mechanism of recognition for the insertion of GTP opposite A73, first we constructed a two-stranded tRNAHis molecule composed of a primer and a template strand through division at the D-loop. Next, we evaluated the structural requirements of the incoming GTP from the incorporation efficiencies of GTP analogs into the two-piece tRNAHis. Nitrogen at position 7 and the 6-keto oxygen of the guanine base were important for G−1 addition; however, interestingly, the 2-amino group was found not to be essential from the highest incorporation efficiency of inosine triphosphate. Furthermore, substitution of the conserved A73 in tRNAHis revealed that the G−1 addition reaction was more efficient onto the template containing the opposite A73 than onto the template with cytidine (C73) or other bases forming canonical Watson–Crick base-pairing. Some interaction might occur between incoming GTP and A73, which plays a role in the prevention of continuous templated 3′–5′ polymerization. This study provides important insights into the mechanism of accurate tRNAHis maturation.

K-Ras Populates Conformational States Differently from Its Isoform H-Ras and Oncogenic Mutant K-RasG12D

Structures of wild-type K-Ras from crystals obtained in the presence of guanosine triphosphate (GTP) or its analogs have remained elusive. Of the K-Ras mutants, only K-RasG12D and K-RasQ61H are available in the PDB representing the activated form of the GTPase not in complex with other proteins. We present the crystal structure of wild-type K-Ras bound to the GTP analog GppCH2p, with K-Ras in the state1 conformation. Signatures of conformational states obtained by one-dimensional proton NMR confirm that K-Ras has a more substantial population of state1 in solution than H-Ras, which predominantly favors state 2. The oncogenic mutant K-RasG12D favors state 2, changing the balance of conformational states in favor of interactions with effector proteins. Differences in the population of conformational states between K-Ras and H-Ras, as well as between K-Ras and its mutants, can provide a structural basis for focused targeting of the K-Ras isoform in cancer-specific strategies.

Balance of Conformational States Affect the Intrinsic Hydrolysis of NRas When Compared to Other Ras Isoforms

Ras GTPases are small proteins that are involved in various signal transduction pathways that regulate cellular proliferation, survival, migration, and apoptosis. Although the first crystal structure of Ras was determined more than two decades ago, biochemical and structural differences in the G‐domain of the three isoforms, H‐, K‐ and N‐Ras is only now beginning to emerge. Each isoform functions as a molecular switch where guanine nucleotide exchange factors (GEFs) facilitate the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) turning its signal on. Ras signaling is turned off via intrinsic hydrolysis or through the aid of GTPase activating proteins (GAP). Ras mutants are involved in 20% of human cancers, yet there are no inhibitors that target these proteins effectively. Most of the effort was originally focused on HRas as the classical representative of all three isoforms and more recently on KRas, the most frequently mutated in cancers. Thus, the structural biology and biochemistry of NRas have been largely overlooked. We have determined hydrolysis rate constants for NRas and determined the crystal structure of wild‐type NRas bound to GppNHp (GNP), a non‐hydrolyzable analogue of GTP, for comparison with H‐Ras and K‐Ras. Although each of the three isoforms follow a one‐step mechanism of hydrolysis, K‐ and NRas have lower rates than HRas, with structural features that promote disorder in switch II near the active site. N‐Ras mutants at G12 and Q61 are commonly found in melanoma tumors. These conserved residues are found in the phosphate‐binding loop (G12) and switch II (Q61) that help make up the active site of NRas. The structures of NRas mutants reveal distinct mechanisms through which they affect catalysis, consistent with lower hydrolysis rate constants found for G12D, G12S, Q61H, and Q61K compared to wild‐type NRas. Biochemical and structural understanding of NRas mutants will help further influence the development of novel therapeutic targets of NRas mutant melanoma.Support or Funding InformationThis research was funded by NSF Grant Number MCB‐1517295.

Antibiotic Binding Drives Catalytic Activation of Aminoglycoside Kinase APH(2″)-Ia

APH(2″)-Ia is a widely disseminated resistance factor frequently found in clinical isolates of Staphylococcus aureus and pathogenic enterococci, where it is constitutively expressed. APH(2″)-Ia confers high-level resistance to gentamicin and related aminoglycosides through phosphorylation of the antibiotic using guanosine triphosphate (GTP) as phosphate donor. We have determined crystal structures of the APH(2″)-Ia in complex with GTP analogs, guanosine diphosphate, and aminoglycosides. These structures collectively demonstrate that aminoglycoside binding to the GTP-bound kinase drives conformational changes that bring distant regions of the protein into contact. These changes in turn drive a switch of the triphosphate cofactor from an inactive, stabilized conformation to a catalytically competent active conformation. This switch has not been previously reported for antibiotic kinases or for the structurally related eukaryotic protein kinases. This catalytic triphosphate switch presents a means by which the enzyme can curtail wasteful hydrolysis of GTP in the absence of aminoglycosides, providing an evolutionary advantage to this enzyme.

Elongation factor 4 remodels the A-site tRNA on the ribosome

During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5'-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant.

Allosteric role of R97 on the NRas active site

Ras proteins are small GTPases that are involved in signal transduction pathways that controls cellular proliferation, differentiation, and survival. Ras is constitutively activated in roughly 20% of human cancers. The Ras subfamily is made up of H‐, K‐, and NRas, where NRas is the least studied. NRas functions as a molecular switch that exchanges guanosine diphosphate (GDP) for guanosine triphosphate (GTP) rendering activation, and hydrolyzes GTP to GDP for deactivation. Once activated, NRas interacts with Raf, an effector molecule that our lab has hypothesized to allosterically modulate intrinsic hydrolysis. The catalytic G domain of NRas comprises an effector lobe (residues 1–86) and allosteric lobe (residues 87–166). In our allosteric model, switch II of Ras becomes ordered when a negatively charged ligand binds between helix 3 and loop 7 allowing for intrinsic hydrolysis. Molecular dynamics studies of Ras bound to Raf revealed R97 of helix 3 becomes less dynamic upon binding of Raf, coordinating the binding of a negatively charged ligand at the allosteric site. Interestingly, R97G is a rare mutation of NRas, found in a human tumor. Here, we present the structure of NRasR97G bound to the non‐hydrolysable GTP analogue, GppNHp. The structure and hydrolysis rates of NRasR97G are compared to those of wild‐type NRas.Support or Funding InformationThis research was funded by NSF Grant Number MCB‐1244203.

Structures of ribosome-bound initiation factor 2 reveal the mechanism of subunit association.

Throughout the four phases of protein biosynthesis-initiation, elongation, termination, and recycling-the ribosome is controlled and regulated by at least one specified translational guanosine triphosphatase (trGTPase). Although the structural basis for trGTPase interaction with the ribosome has been solved for the last three steps of translation, the high-resolution structure for the key initiation trGTPase, initiation factor 2 (IF2), complexed with the ribosome, remains elusive. We determine the structure of IF2 complexed with a nonhydrolyzable guanosine triphosphate analog and initiator fMet-tRNAi (Met) in the context of the Escherichia coli ribosome to 3.7-Å resolution using cryo-electron microscopy. The structural analysis reveals previously unseen intrinsic conformational modes of the 70S initiation complex, establishing the mutual interplay of IF2 and initator transfer RNA (tRNA) with the ribsosome and providing the structural foundation for a mechanistic understanding of the final steps of translation initiation.

Interaction of a novel fluorescent GTP analogue with the small G-protein K-Ras.

A novel fluorescent guanosine 5'-triphosphate (GTP) analogue, 2'(3')-O-{6-(N-(7-nitrobenz-2-oxa-l,3-diazol-4-yl)amino) hexanoic}-GTP (NBD-GTP), was synthesized and utilized to monitor the effect of mutations in the functional region of mouse K-Ras. The effects of the G12S, A59T and G12S/A59T mutations on GTPase activity, nucleotide exchange rates were compared with normal Ras. Mutation at A59T resulted in reduction of the GTPase activity by 0.6-fold and enhancement of the nucleotide exchange rate by 2-fold compared with normal Ras. On the other hand, mutation at G12S only slightly affected the nucleotide exchange rate and did not affect the GTPase activity. We also used NBD-GTP to study the effect of these mutations on the interaction between Ras and SOS1, a guanine nucleotide exchange factor. The mutation at A59T abolished the interaction with SOS1. The results suggest that the fluorescent GTP analogue, NBD-GTP, is applicable to the kinetic studies for small G-proteins.

Tubulin structure-based drug design for the development of novel 4β-sulfur-substituted podophyllum tubulin inhibitors with anti-tumor activity.

The well-characterized anti-tubulin agent, podophyllotoxin (PTOX), with the 4′-position methoxyl group, targets the colchicines domain located between α- and β-tubulin. Two guanosine triphosphate (GTP) analogs of the tubulin-binding region were synthesized from PTOX, where a hydroxyl group was substituted with a carbon-sulfur bond. These compounds, 4-MP-PTOX and 4-TG-PTOX, reduce the dosage and greatly improve the therapeutic effect for microtubule damage in cancer cells. Here we characterize the anti-tubulin properties of these compounds. We found the stronger inhibition of tubulin polymerization (the concentration of 50% growth inhibition, GI50 < 2 μM) for compounds 4-TG-PTOX and 4-MP-PTOX, which were better than that of PTOX or colchicine. The cytotoxicity of two designed compounds on tumor cells was also significantly enhanced by comparing to those of PTOX and colchicines. The ΔH value of 4-MP-PTOX and 4-TG-PTOX binding to tubulin by isothermal titration calorimetry (ITC) was found to be −7.4 and −5.3 kcal·mol−1, respectively. The wide range of enthalpy values across the series may reflect entropy/enthalpy compensation effects. Fragments 6-mercaptopurine (MP) and 6-thioguanine (TG) likely enhance the affinity of 4-MP-PTOX and 4-TG-PTOX binding to tubulin by increasing the number of binding sites. The correctness of rational drug design was strictly demonstrated by a bioactivity test.

Free-energy differences between states with different conformational ensembles

Multiple conformations separated by high-energy barriers represent a challenging problem in free-energy calculations due to the difficulties in achieving adequate sampling. We present an application of thermodynamic integration (TI) in conjunction with the local elevation umbrella sampling (LE/US) method to improve convergence in alchemical free-energy calculations. TI-LE/US was applied to the guanosine triphosphate (GTP) to 8-Br-GTP perturbation, molecules that present high-energy barriers between the anti and syn states and that have inverted preferences for those states. The convergence and reliability of TI-LE/US was assessed by comparing with previous results using the enhanced-sampling one-step perturbation (OSP) method. A linear interpolation of the end-state biasing potentials was sufficient to dramatically improve sampling along the chosen reaction coordinate. Conformational free-energy differences were also computed for the syn and anti states and compared to experimental and theoretical results. Additionally, a coupled OSP with LE/US was carried out, allowing the calculation of conformational and alchemical free energies of GTP and 8-substituted GTP analogs.

Characterization of a Nucleotide Kinase Encoded by Bacteriophage T7

Gene 1.7 protein is the only known nucleotide kinase encoded by bacteriophage T7. The enzyme phosphorylates dTMP and dGMP to dTDP and dGDP, respectively, in the presence of a phosphate donor. The phosphate donors are dTTP, dGTP, and ribo-GTP as well as the thymidine and guanosine triphosphate analogs ddTTP, ddGTP, and dITP. The nucleotide kinase is found in solution as a 256-kDa complex consisting of ~12 monomers of the gene 1.7 protein. The two molecular weight forms co-purify as a complex, but each form has nearly identical kinase activity. Although gene 1.7 protein does not require a metal ion for its kinase activity, the presence of Mg(2+) in the reaction mixture results in either inhibition or stimulation of the rate of kinase reactions depending on the substrates used. Both the dTMP and dGMP kinase reactions are reversible. Neither dTDP nor dGDP is a phosphate acceptor of nucleoside triphosphate donors. Gene 1.7 protein exhibits two different equilibrium patterns toward deoxyguanosine and thymidine substrates. The K(m) of 4.4 × 10(-4) M obtained with dTTP for dTMP kinase is ~3-fold higher than that obtained with dGTP for dGMP kinase (1.3 × 10(-4) M), indicating that a higher concentration of dTTP is required to saturate the enzyme. Inhibition studies indicate a competitive relationship between dGDP and both dGTP, dGMP, whereas dTDP appears to have a mixed type of inhibition of dTMP kinase. Studies suggest two functions of dTTP, as a phosphate donor and a positive effector of the dTMP kinase reaction.

Conformational changes in tubulin in GMPCPP and GDP-taxol microtubules observed by cryoelectron microscopy

Microtubules are dynamic polymers that stochastically switch between growing and shrinking phases. Microtubule dynamics are regulated by guanosine triphosphate (GTP) hydrolysis by β-tubulin, but the mechanism of this regulation remains elusive because high-resolution microtubule structures have only been revealed for the guanosine diphosphate (GDP) state. In this paper, we solved the cryoelectron microscopy (cryo-EM) structure of microtubule stabilized with a GTP analogue, guanylyl 5'-α,β-methylenediphosphonate (GMPCPP), at 8.8-Å resolution by developing a novel cryo-EM image reconstruction algorithm. In contrast to the crystal structures of GTP-bound tubulin relatives such as γ-tubulin and bacterial tubulins, significant changes were detected between GMPCPP and GDP-taxol microtubules at the contacts between tubulins both along the protofilament and between neighboring protofilaments, contributing to the stability of the microtubule. These findings are consistent with the structural plasticity or lattice model and suggest the structural basis not only for the regulatory mechanism of microtubule dynamics but also for the recognition of the nucleotide state of the microtubule by several microtubule-binding proteins, such as EB1 or kinesin.

Immobilization of Biotinylated hGBP1 in a Defined Orientation on Surfaces Is Crucial for Uniform Interaction with Analyte Proteins and Catalytic Activity

Guanylate binding proteins (GBPs) belong to the dynamin superfamily of large GTP binding proteins. A biochemical feature common to these proteins is guanosine-triphosphate (GTP) binding leading to self-assembly of the proteins, and this in turn results in higher catalytic GTP hydrolysis activity. In the case of human guanylate binding protein 1 (hGBP1) homodimer formation is observed after binding of nonhydrolyzable GTP analogs like GppNHp. hGBP1 is one of seven GBP isoforms identified in human. While cellular studies suggest heterocomplex formation of various isoforms biochemical binding studies in quantitative terms are lacking. In this work we established a method to study hGBP1 interactions by attaching this protein in a defined orientation to a surface allowing for interaction with molecules from the solution. Briefly, specifically biotinylated hGBP1 is attached to a streptavidin layer on a self-assembled monolayer (SAM) surface allowing for characterization of the packing density of the immobilized protein by surface plasmon resonance (SPR) technology and atomic force microscopy (AFM), respectively. In addition, the enzymatic activity of immobilized hGBP1 and the kinetics of interaction with binding partners in solution are quantified. We present a procedure for attaching an enzyme in a defined orientation to a surface which exposes its active end, the GTPase domain to the solution resulting in a homogeneous population of this enzyme in terms of enzymatic activity and of interaction with soluble proteins.

Response to Comment on “The Mechanism for Activation of GTP Hydrolysis on the Ribosome”

Our report of the crystal structure of elongation factor Tu (EF-Tu) and aminoacyl–transfer RNA bound to the ribosome with a guanosine triphosphate (GTP) analog included a proposed mechanism of GTP hydrolysis by EF-Tu involving histidine-84. Liljas et al . summarize experimental evidence against this mechanism and propose a substrate-assisted catalytic model. However, these experiments and the model are also problematic. Further study is required to definitively determine the mechanism of GTP hydrolysis by EF-Tu.