Nonhydrolyzable GTP Analog Research Articles

Structural analysis of noncanonical translation initiation complexes

Translation initiation is a highly regulated, multi-step process that is critical for efficient and accurate protein synthesis. In bacteria, initiation begins when mRNA, initiation factors, and a dedicated initiator fMet-tRNAfMet bind the small (30S) ribosomal subunit. Specific binding of fMet-tRNAfMet in the peptidyl (P) site is mediated by the inspection of the fMet moiety by initiation factor IF2 and of three conserved G-C base pairs in the tRNA anticodon stem by the 30S head domain. Tandem A-minor interactions form between 16S ribosomal RNA nucleotides A1339 and G1338 and tRNA base pairs G30-C40 and G29-C41, respectively. Swapping the G30-C40 pair of tRNAfMet with C-G (called tRNAfMet M1) reduces discrimination against the noncanonical start codon CUG invitro, suggesting crosstalk between the gripping of the anticodon stem and recognition of the start codon. Here, we solved electron cryomicroscopy structures of Escherichia coli 70S initiation complexes containing the fMet-tRNAfMet M1 variant paired to the noncanonical CUG start codon, in the presence or absence of IF2 and the non-hydrolyzable GTP analog GDPCP, alongside structures of 70S initiation complexes containing this tRNAfMet variant paired to the canonical bacterial start codons AUG, GUG, and UUG. We find that the M1 mutation weakens A-minor interactions between tRNAfMet and 16S nucleotides A1339 and G1338, with IF2 strengthening the interaction of G1338 with the tRNA minor groove. These structures suggest how even slight changes to the recognition of the fMet-tRNAfMet anticodon stem by the ribosome can impact the start codon selection.

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.

Far-reaching effects of tyrosine64 phosphorylation on Ras revealed with BeF3– complexes

Tyrosine phosphorylation on Ras by Src kinase is known to uncouple Ras from upstream regulation and downstream communication. However, the mechanisms by which phosphorylation modulates these interactions have not been detailed. Here, the major mono-phosphorylation level on tyrosine64 is quantified by 31P NMR and mutagenesis. Crystal structures of unphosphorylated and tyrosine64-phosphorylated Ras in complex with a BeF3− ground state analogue reveal “closed” Ras conformations very different from those of the “open” conformations previously observed for non-hydrolysable GTP analogue structures of Ras. They deliver new mechanistic and conformational insights into intrinsic GTP hydrolysis. Phosphorylation of tyrosine64 delivers conformational changes distant from the active site, showing why phosphorylated Ras has reduced affinity to its downstream effector Raf. 19F NMR provides evidence for changes in the intrinsic GTPase and nucleotide exchange rate and identifies the concurrent presence of a major “closed” conformation alongside a minor yet functionally important “open” conformation at the ground state of Ras. This study expands the application of metal fluoride complexes in revealing major and minor conformational changes of dynamic and modified Ras proteins.

Structural insight into G-protein chaperone-mediated maturation of a bacterial adenosylcobalamin-dependent mutase

G-protein metallochaperones are essential for the proper maturation of numerous metalloenzymes. The G-protein chaperone MMAA in humans (MeaB in bacteria) uses GTP hydrolysis to facilitate the delivery of adenosylcobalamin (AdoCbl) to AdoCbl-dependent methylmalonyl-CoA mutase, an essential metabolic enzyme. This G-protein chaperone also facilitates the removal of damaged cobalamin (Cbl) for repair. Although most chaperones are standalone proteins, isobutyryl-CoA mutase fused (IcmF) has a G-protein domain covalently attached to its target mutase. We previously showed that dimeric MeaB undergoes a 180° rotation to reach a state capable of GTP hydrolysis (an active G-protein state), in which so-called switch III residues of one protomer contact the G-nucleotide of the other protomer. However, it was unclear whether other G-protein chaperones also adopted this conformation. Here, we show that the G-protein domain in a fused system forms a similar active conformation, requiring IcmF oligomerization. IcmF oligomerizes both upon Cbl damage and in the presence of the nonhydrolyzable GTP analog, guanosine-5'-[(β,γ)-methyleno]triphosphate, forming supramolecular complexes observable by mass photometry and EM. Cryo-EM structural analysis reveals that the second protomer of the G-protein intermolecular dimer props open the mutase active site using residues of switch III as a wedge, allowing for AdoCbl insertion or damaged Cbl removal. With the series of structural snapshots now available, we now describe here the molecular basis of G-protein-assisted AdoCbl-dependent mutase maturation, explaining how GTP binding prepares a mutase for cofactor delivery and how GTP hydrolysis allows the mutase to capture the cofactor.

Abstract PR01: Structural plasticity of KRAS oncogenic mutants – A case of misleading conclusions from GTP analogues

Abstract RAS mutations occur in approximately 20% of human cancers including the majority of pancreatic ductal adenocarcinoma (PDAC), half of colorectal cancers, and a third of all lung cancers. In cases of oncogenic activating RAS mutations, GTP hydrolysis is impaired, and the protein is preferentially held in the active GTP-bound state. When activated, RAS triggers multiple proliferative signaling cascades, including the MAPK/ERK and PI3K pathways, to induce cell growth, division, and differentiation. In solution, RAS exists in two interconverting GTP-bound conformational states, State 1 (inactive) and State 2 (active). State 2 is the active conformation since it binds to RAS Binding Domain (RBD) of effector proteins. Though the State 1 conformation is GTP-bound, it is unable to bind effectors and represents an inactive KRAS conformation. This State 1/State 2 equilibrium is closely coupled to the conformations (γ1 and γ2) exhibited by the γ-phosphate of GTP and its analogues. Solution-state 31P NMR spectroscopy captures and quantifies the γ1 and γ2 peaks representing State 1 and State 2 (Spoerner M et al., JBC 2010). We have initiated solution-state NMR investigation to determine the structural differences between wild-type (WT) and oncogenic mutant KRAS proteins (G12C, G12D, G12V, Q61L, and G13D) when bound to GTP and GppNHp. We quantified the State 1/State 2 equilibrium using 31P NMR. Our results show that GppNHp-bound KRAS proteins harboring oncogenic mutations differentially modulate the State 1/State 2 conformational equilibria resulting in remarkable divergence in conformational populations compared to WT protein; a notion for presence of mutation induced structural plasticity. Much work on the structural biology, biophysics and biochemistry of KRAS proteins is done when bound to the non-hydrolysable GTP analogue, GppNHp. Strikingly, our 31P NMR results show that GTP-bound KRAS elicits significant variations in State 1 and State 2 populations in comparison to GppNHp-bound proteins. These data suggest GppNHp binding induces a significant increase in the State 1 population; an observation that does not translate into GTP-bound KRAS. Our results provide additional insights into the differential binding affinities seen between WT/mutant KRAS and effector proteins (e.g., Hunter JC et al., Mol Cancer Res 2015) as well as interpretation for State 1 conformations in KRAS crystal structures when bound to mostly GppNHp and not GTP. We have also applied this 31P NMR approach to the evaluation of KRAS small molecule inhibitors that shift the population equilibrium to the State 1 (inactive) conformation in the protein-ligand complex. This demonstrates an intriguing therapeutic opportunity and novel mechanism of action that we are further exploring as part of our drug discovery pipeline. Citation Format: Alok K. Sharma, Marcin Dyba, Dominic Esposito, Bin Wang, Pedro J. Beltran, Eli Wallace, Dwight Nissley, Frank McCormick, Anna E. Maciag. Structural plasticity of KRAS oncogenic mutants – A case of misleading conclusions from GTP analogues [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR01.

Vitamin K3 inhibits FtsZ assembly, disrupts the Z-ring in Streptococcus pneumoniae and displays anti-pneumococcal activity.

The respiratory pathogen, Streptococcus pneumoniae has acquired multiple-drug resistance over the years. An attractive strategy to combat pneumococcal infection is to target cell division to inhibit the proliferation of S. pneumoniae. This work presents Vitamin K3 as a potential anti-pneumococcal drug that targets FtsZ, the master coordinator of bacterial cell division. Vitamin K3 strongly inhibited S. pneumoniae proliferation with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 6 μg/ml. Vitamin K3 disrupted the Z-ring localization in both S. pneumoniae and Bacillus subtilis within 30 min of treatment, while the membrane integrity and nucleoid segregation remain unchanged. Several complementary experiments showed that Vitamin K3 inhibits the assembly of purified S. pneumoniae FtsZ (SpnFtsZ) and induces conformational changes in the protein. Interestingly, Vitamin K3 interfered with GTP binding onto FtsZ and increased the GTPase activity of FtsZ polymers. The intrinsic tryptophan fluorescence of SpnFtsZ revealed that Vitamin K3 delays the nucleation of FtsZ polymers and reduces the rate of polymerization. In the presence of a non-hydrolyzable analog of GTP, Vitamin K3 did not show inhibition of FtsZ polymerization. These results indicated that Vitamin K3 induces conformational changes in FtsZ that increase GTP hydrolysis and thereby, destabilize the FtsZ polymers. Together, our data provide evidence that Vitamin K3 derives its potent anti-pneumococcal activity by inhibiting FtsZ assembly.

Microtubule Growth Is Slowed through Transient Exposure of GDP Tubulin

Microtubule plus-end growth dynamics are determined by a kinetic race dictated by tubulin on- and off-rates at the microtubule tip. The apparent tubulin on- and off-rates can be estimated by measuring the mean plus-end growth rate across a range of free tubulin concentrations. However, when growth phases are studied at higher temporal resolution, fluctuations around the average growth rate are observed, implying more complex dynamics. To understand growth dynamics at the microtubule plus-end, we used Interference Reflection Microscopy (IRM) to quantify time-dependent variations in microtubule growth using GMPCPP, a non-hydrolyzable GTP analog. We found an expected linear dependence of the average growth rate on tubulin concentration. However, there was also a linear relationship between growth variance and tubulin concentration. Utilizing these measured values, we were able to constrain a biochemical model of microtubule growth with an on-rate of 0.7/ μM/pf/s. To determine the degree to which growth in GMPCPP mimics the GTP cap, we repeated the measurements in GTP. We again found a linear dependence of the average growth rate on tubulin; but found a much higher tubulin-dependent growth variance than expected from the GMPCPP experiments. When the growth variance was normalized to growth rate for each condition, there was a 5-fold increase in the relative growth variance in GTP compared to GMPCPP. This result suggests that GTP hydrolysis impacts tubulin binding kinetics at the microtubule plus-end. Consistent with this hypothesis, we then artificially varied the hydrolysis rates from 0 to 0.15 s−1 in the biochemical model. Increasing the hydrolysis rate increased the growth variance, and transient suppression of growth coincided with increasing numbers of exposed GDP tubulin at the plus-end. This work suggests that during growth, exposure of terminal GDP tubulin slows growth until a terminal GTP is restored.

Human GTPBP5 is involved in the late stage of mitoribosome large subunit assembly.

Human mitoribosomes are macromolecular complexes essential for translation of 11 mitochondrial mRNAs. The large and the small mitoribosomal subunits undergo a multistep maturation process that requires the involvement of several factors. Among these factors, GTP-binding proteins (GTPBPs) play an important role as GTP hydrolysis can provide energy throughout the assembly stages. In bacteria, many GTPBPs are needed for the maturation of ribosome subunits and, of particular interest for this study, ObgE has been shown to assist in the 50S subunit assembly. Here, we characterize the role of a related human Obg-family member, GTPBP5. We show that GTPBP5 interacts specifically with the large mitoribosomal subunit (mt-LSU) proteins and several late-stage mitoribosome assembly factors, including MTERF4:NSUN4 complex, MRM2 methyltransferase, MALSU1 and MTG1. Interestingly, we find that interaction of GTPBP5 with the mt-LSU is compromised in the presence of a non-hydrolysable analogue of GTP, implying a different mechanism of action of this protein in contrast to that of other Obg-family GTPBPs. GTPBP5 ablation leads to severe impairment in the oxidative phosphorylation system, concurrent with a decrease in mitochondrial translation and reduced monosome formation. Overall, our data indicate an important role of GTPBP5 in mitochondrial function and suggest its involvement in the late-stage of mt-LSU maturation.

Millisecond Allosteric Dynamics of Activated Ras Reproduced with a Slowly Hydrolyzable GTP Analogue.

The millisecond timescale dynamics of activated Ras transiently sample a low-populated conformational state that has distinct surface property from the major state and represents a promising target for binding of small-molecule compounds. To avoid the complications of hydrolysis, dynamics and other properties of active Ras have so far been routinely investigated by using non-hydrolyzable GTP analogues, which, however, were previously reported to alter both the kinetics and distribution of the conformational exchange. In this study, we quantitatively measured and validated the internal dynamics of Ras complexed with a slowly hydrolyzable GTP analogue, GTPγS, which increases the lifetime of active Ras by 23 times relative to that of native GTP. It was found that GTPγS, in addition to its better mimicking of the exchange kinetics than the commonly used non-hydrolyzable analogues GppNHp and GppCH2 p, can rigorously reproduce the natural dynamics network in active Ras, thus indicating its fitness for use in the development of allosteric inhibitors.

Crystal structure and enzymatic characterization of the putative adenylyl cyclase HpAC1 from Hippeastrum reveal dominant triphosphatase activity

HpAC1, a protein from Hippeastrum hybrid cultivars, was previously suggested to be a plant adenylyl cyclase. We describe a structural and enzymatic characterization of HpAC1. A crystal structure of HpAC1 in complex with a non-hydrolyzable GTP analog confirms a generic CYTH architecture, comprising a β-barrel with an internal substrate site. The structure reveals significant active site differences to AC proteins with CYTH fold, however, and we find that HpAC1 lacks measurable AC activity. Instead, HpAC1 has substantial triphosphatase activity, indicating this protective activity or a related activity as the protein’s physiological function.

A modified method for preparation of fluorescent MantGDP bound CDC42

Small GTPase cycled between the GDP-bound inactive state and GTP-bound active state, catalyzed by guanine nucleotide exchange factors (GEFs). Guanine nucleotide exchange assay was a direct way to investigate the specificity, activity, and kinetics of GEFs. The N-methylanthraniloyl derivative of GDP (mantGDP), which was bound to small GTPase, served as a substitution for labeled small GTPase involved in bioluminescent, colorimetric, or radioactive methods due to its safety and sensitivity. In this study, we present an economical and efficient approach to prepare qualified mantGDP-bound CDC42, a member of the Rho GTPase family. In our protocol, with a Kd value of 0.048 μM, alkaline phosphatase hydrolysis of CDC42 increased mantGDP binding affinity to CDC42, allowing mant-nucleotide associating onto CDC42 more easily. Only 1.5-fold molar excess of mantGDP was required to prepare mantGDP-bound CDC42 without nonhydrolyzable GTP analog and high performance liquid chromatography. The mantGDP-bound CDC42 was verified to be efficient for measuring the guanine nucleotide exchange activity of VAV2.

Abstract IA02: New structural insights into KRAS biology

Abstract Oncogenic mutations of RAS proteins are responsible for approximately 30% of all human cancers. Among the three RAS isoforms—HRAS, KRAS, and NRAS—KRAS is the most commonly mutated gene (in 86% of RAS-driven cancers) and the mutations are often present in pancreatic, colorectal, and lung cancers. Despite decades of research, no RAS inhibitors have reached the clinic. The NCI RAS Initiative at Frederick National Laboratory for Cancer Research (FNLCR) comprises multidisciplinary teams of scientists that are exploring innovative approaches for designing inhibitors against RAS-driven cancers. Structural biology efforts within the RAS Initiative are focused on solving structures of protein-small-molecule complexes and unraveling new structural insights into KRAS biology. RAS proteins act as binary molecular switches that cycle between active GTP-bound and inactive GDP-bound states. The conversion from inactive to the active form is stimulated by guanine nucleotide exchange factors (GEFs). Conversion back to the inactive form is mediated by GTPase-activating proteins (GAPs). In the active state, RAS proteins interact with a variety of effector proteins, such as RAF kinase, PI3K, and RalGDS, leading to activation of downstream signaling pathways. Oncogenic RAS mutations are predominantly found at amino acid positions G12, G13, and Q61 and impair intrinsic and GAP-mediated GTPase function. In this meeting, I will present our structural work on wild-type and oncogenic mutants of KRAS in the active state, and their complexes with various regulators. We recently solved structures of the most common oncogenic mutants of KRAS in the active state (bound to GMPPNP, a nonhydrolyzable analog of GTP). Structures of KRAS mutants in state 1 conformation show novel pockets that are likely to be amenable to in silico drug discovery approaches. We also solved structures of wild-type and mutant KRAS in complex with two major RasGAP proteins, RASA1/p120GAP and neurofibromin. Structures of RAS-GAP complexes provide insights into impaired GAP-mediated GTP hydrolysis process in the oncogenic mutants of KRAS. SPRED1, which causes Legius syndrome, functions as a negative regulator of the RAS-MAPK pathway and interacts with neurofibromin. Recent studies have shown that the EVH1 domain of SPRED1 binds to the GTPase activating protein-related domain of neurofibromin without interfering with its catalytic activity. Our structural work on a ternary complex consisting of KRAS, neurofibromin, and SPRED1 provides novel insights into the regulation of the RAS-MAPK pathway by neurofibromin and SPRED1. Structural studies on wild-type and oncogenic mutants of KRAS in the active state, and in complex with various regulators, have provided new insights into the KRAS biology as well as new blueprints for structure-based drug discovery approaches. Citation Format: Dhirendra K. Simanshu. New structural insights into KRAS biology [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr IA02.

Abstract PR01: Novel state I structures of oncogenic KRAS4b mutants bound to GTP analog

Abstract The RAS family proteins alternate between active GTP-bound and inactive GDP-bound states. Due to the high affinity of GDP and GTP, and low intrinsic GTPase activity of RAS, switching between the two states is assisted by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Many of the oncogenic RAS mutations result in impaired intrinsic and/or GAP-mediated GTPase function, leading to accumulation of constitutively active RAS. To understand the molecular mechanism behind the impairment, we crystallized and solved the structures of wild-type and six different KRAS4b oncogenic mutants bound to a non-hydrolysable GTP analog GMPPNP. Interestingly, we noticed variations in the conformation of the Switch I region, which contacts the nucleotide and many downstream effector proteins. In three of our mutants (G12C, G13D, Q61L), Switch I adopts an open conformation known as state I, which, similar to the GDP-bound state, does not contact the nucleotide and represents an inactive state of the RAS protein. Importantly, our state I structures revealed a new pocket created by the open Switch I loop that could be targeted in drug discovery. To screen for compounds that bind to this new Switch I pocket or other places of the protein, we started a fragment-screening campaign by X-ray crystallography using the KRAS4b-G13D-GMPPNP crystals. These crystals consistently diffracted to very high (~1.2 Å) resolutions, can be reproduced easily, are tolerant to DMSO soaking, and their putative fragment-binding pockets are unobstructed by lattice contacts, making them ideal for fragment-screening. This abstract is also being presented as Poster A01. Citation Format: Albert H. Chan, Timothy H. Tran, Srisathiyanarayanan Dharmaiah, Dwight V. Nissley, Dhirendra K. Simanshu. Novel state I structures of oncogenic KRAS4b mutants bound to GTP analog [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr PR01.

Long-range interdomain communications in eIF5B regulate GTP hydrolysis and translation initiation

Translation initiation controls protein synthesis by regulating the delivery of the first aminoacyl-tRNA to messenger RNAs (mRNAs). In eukaryotes, initiation is sophisticated, requiring dozens of protein factors and 2 GTP-regulated steps. The GTPase eIF5B gates progression to elongation during the second GTP-regulated step. Using electron cryomicroscopy (cryo-EM), we imaged an in vitro initiation reaction which is set up with purified yeast components and designed to stall with eIF5B and a nonhydrolyzable GTP analog. A high-resolution reconstruction of a "dead-end" intermediate at 3.6 Å allowed us to visualize eIF5B in its ribosome-bound conformation. We identified a stretch of residues in eIF5B, located close to the γ-phosphate of GTP and centered around the universally conserved tyrosine 837 (Saccharomyces cerevisiae numbering), that contacts the catalytic histidine of eIF5B (H480). Site-directed mutagenesis confirmed the essential role that these residues play in regulating ribosome binding, GTP hydrolysis, and translation initiation both in vitro and in vivo. Our results illustrate how eIF5B transmits the presence of a properly delivered initiator aminoacyl-tRNA at the P site to the distant GTPase center through interdomain communications and underscore the importance of the multidomain architecture in translation factors to sense and communicate ribosomal states.

Affinity Purification and Functional Characterization of Dynamin-Related Protein 1.

Purification of dynamin-related proteins is complicated by their oligomeric tendencies. In this chapter, we describe an established purification regime to isolate the mitochondrial fission protein Drp1 using bacterial expression. Key attributes of dynamins include their ability to hydrolyze GTP and self-assemble into larger polymers under specific conditions. Therefore, the GTPase activity of Drp1 should be examined to confirm isolation of functional protein, and we describe a conventional colorimetric assay to assess enzyme activity. To determine the ability of Drp1 to self-assemble, we induce Drp1 polymerization through addition of a non-hydrolyzable GTP analogue. A sedimentation assay provides a quantitative measure of polymerization that complements a qualitative assessment through visualization of Drp1 oligomers using negative-stain electron microscopy (EM). Importantly, we highlight the caveats of affinity tags and the influence that these peptide sequences can have on Drp1 function given their proximity to functional domains.

WASP Restricts Active Rac to Maintain Cells' Front-Rear Polarization.

SummaryEfficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif.

Structural insights into the G-loop dynamics of E. coli FtsY NG domain

The bacterial signal recognition particle (SRP) receptor, FtsY, participates with the SRP in co-translation targeting of proteins. Multiple crystal structures of the NG domain of E. coli FtsYNG have been determined at high-resolution (1.22–1.88 Å), in the nucleotide-free (apo) form as well as bound to GDP and non-hydrolysable GTP analogues. The combination of high-resolution and multiple solved structures of FtsYNG in different states revealed a new sensor-relay system of this unique GTPase receptor. A nucleotide sensing function of the P-loop assists FtsYNG in nucleotide-binding and contributes to modulate nucleotide binding properties in SRP GTPases. A reorganization of the other G-loops and the insertion binding domain (IBD) is observed only upon transition from a diphosphate to a triphosphate nucleotide. The role of a magnesium ion during the GDP and GTP-bound states has also been observed. The binding of magnesium in the nucleotide site causes the reorientation of the β- and γ- phosphate groups toward the jaws of the P-loop and stabilizes the binding of the nucleotide, creating a network of hydrogen and water-bridge interactions.

High pressure response of 1H NMR chemical shifts of purine nucleotides

The study of the pressure response by NMR spectroscopy provides information on the thermodynamics of conformational equilibria in proteins and nucleic acids. For obtaining a database for expected pressure effects on free nucleotides and nucleotides bound in macromolecular complexes, the pressure response of 1H chemical shifts and J-coupling constants of the purine 5′-ribonucleotides AMP, ADP, ATP, GMP, GDP, and GTP were studied in the absence and presence of Mg2+-ions. Experiments are supported by quantum-chemical calculations of populations and chemical shift differences in order to corroborate structural interpretations and to estimate missing data for AMP. The preference of the ribose S puckering obtained from the analysis of the experimental J-couplings is also confirmed by the calculations. In addition, the pressure response of the non-hydrolysable GTP analogues GppNHp, GppCH2p, and GTPγS was examined within a pressure range up to 200 MPa. As observed earlier for 31P NMR chemical shifts of these nucleotides the pressure dependence of chemical shifts is clearly non-linear in most cases. In di- and tri-phospho nucleosides, the resonances of the two protons bound to the ribose 5′ carbon are non-equivalent and can be observed separately. The gg-rotamer at C4′- C5′ bond is strongly preferred and the downfield shifted resonance can be assigned to the H5″ proton in the nucleotides. In contrast, in adenosine itself the frequencies of the two resonances are interchanged.

Farnesylation of human guanylate-binding protein 1 as safety mechanism preventing structural rearrangements and uninduced dimerization.

Human guanylate-binding protein 1 (hGBP1) belongs to the family of dynamin-like proteins and is activated by addition of nucleotides, leading to protein oligomerization and stimulated GTPase activity. Invivo, hGBP1 is post-translationally modified by attachment of a farnesyl group yielding farn-hGBP1. In this study, hydrodynamic differences in farn-hGBP1 and unmodified hGBP1 were investigated using dynamic light scattering (DLS), analytical ultracentrifugation (AUC) and analytical size-exclusion chromatography (SEC). In addition, we performed small-angle X-ray scattering (SAXS) experiments coupled with a SEC setup (SEC-SAXS) to investigate structural properties of nonmodified hGBP1 and farn-hGBP1 in solution. SEC-SAXS measurements revealed that farnesylation keeps hGBP1 in its inactive monomeric and crystal-like conformation in nucleotide-free solution, whereas unmodified hGBP1 forms a monomer-dimer equilibrium both in the inactive ground state in nucleotide-free solution as well as in the activated state that is trapped by addition of the nonhydrolysable GTP analogue GppNHp. Nonmodified hGBP1 is structurally perturbed as compared to farn-hGBP. In particular, GppNHp binding leads to large structural rearrangements and higher conformational flexibility of the monomer and the dimer. Structural changes observed in the nonmodified protein are prerequisites for further oligomer assemblies of farn-hGBP1 that occur in the presence of nucleotides. DATABASE: All SEC-SAXS data, corresponding fits to the data and structural models are deposited in the Small Angle Scattering Biological Data Bank [SASBDB (Nucleic Acids Res, 43, 2015, D357)] with project IDs: SASDEE8, SASDEF8, SASDEG8, SASDEH8, SASDEJ8, SASDEK8, SASDEL8 and SASDEM8.

Bimodal Nickel-Binding Site on Escherichia coli [NiFe]-Hydrogenase Metallochaperone HypA.

[NiFe]-hydrogenase enzymes catalyze the reversible oxidation of hydrogen at a bimetallic cluster and are used by bacteria and archaea for anaerobic growth and pathogenesis. Maturation of the [NiFe]-hydrogenase requires several accessory proteins to assemble and insert the components of the active site. The penultimate maturation step is the delivery of nickel to a primed hydrogenase enzyme precursor protein, a process that is accomplished by two nickel metallochaperones, the accessory protein HypA and the GTPase HypB. Recent work demonstrated that nickel is rapidly transferred to HypA from GDP-loaded HypB within the context of a protein complex in a nickel selective and unidirectional process. To investigate the mechanism of metal transfer, we examined the allosteric effects of nucleotide cofactors and partner proteins on the nickel environments of HypA and HypB by using a combination of biochemical, microbiological, computational, and spectroscopic techniques. We observed that loading HypB with either GDP or a nonhydrolyzable GTP analogue resulted in a similar nickel environment. In addition, interaction with a mutant version of HypA with disrupted nickel binding, H2Q-HypA, does not induce substantial changes to the HypB G-domain nickel site. Instead, the results demonstrate that HypB modifies the acceptor site of HypA. Analysis of a peptide maquette derived from the N-terminus of HypA revealed that nickel is predominately coordinated by atoms from the N-terminal Met-His motif. Furthermore, HypA is capable of two nickel-binding modes at the N-terminus, a HypB-induced mode and a binding mode that mirrors the peptide maquette. Collectively, these results reveal that HypB brings about changes in the nickel coordination of HypA, providing a mechanism for the HypB-dependent control of the acquisition and release of nickel by HypA.