Effects Of Nucleotide Binding Research Articles

Integration of Fourier Transform Infrared Spectroscopy, Fluorescence Spectroscopy, Steady-state Kinetics and Molecular Dynamics Simulations of Gαi1 Distinguishes between the GTP Hydrolysis and GDP Release Mechanism

Gα subunits are central molecular switches in cells. They are activated by G protein-coupled receptors that exchange GDP for GTP, similar to small GTPase activation mechanisms. Gα subunits are turned off by GTP hydrolysis. For the first time we employed time-resolved FTIR difference spectroscopy to investigate the molecular reaction mechanisms of Gαi1. FTIR spectroscopy is a powerful tool that monitors reactions label free with high spatio-temporal resolution. In contrast to common multiple turnover assays, FTIR spectroscopy depicts the single turnover GTPase reaction without nucleotide exchange/Mg(2+) binding bias. Global fit analysis resulted in one apparent rate constant of 0.02 s(-1) at 15 °C. Isotopic labeling was applied to assign the individual phosphate vibrations for α-, β-, and γ-GTP (1243, 1224, and 1156 cm(-1), respectively), α- and β-GDP (1214 and 1134/1103 cm(-1), respectively), and free phosphate (1078/991 cm(-1)). In contrast to Ras · GAP catalysis, the bond breakage of the β-γ-phosphate but not the Pi release is rate-limiting in the GTPase reaction. Complementary common GTPase assays were used. Reversed phase HPLC provided multiple turnover rates and tryptophan fluorescence provided nucleotide exchange rates. Experiments were complemented by molecular dynamics simulations. This broad approach provided detailed insights at atomic resolution and allows now to identify key residues of Gαi1 in GTP hydrolysis and nucleotide exchange. Mutants of the intrinsic arginine finger (Gαi1-R178S) affected exclusively the hydrolysis reaction. The effect of nucleotide binding (Gαi1-D272N) and Ras-like/all-α interface coordination (Gαi1-D229N/Gαi1-D231N) on the nucleotide exchange reaction was furthermore elucidated.

Role of N-Terminal Myristylation in the Structure and Regulation of cAMP-Dependent Protein Kinase

The catalytic (C) subunit of cAMP-dependent protein kinase [protein kinase A (PKA)] is a major target of cAMP signaling, and its regulation is of fundamental importance to biological processes. One mode of regulation is N-myristylation, which has eluded structural and functional characterization so far because most crystal structures are of the non-myristylated enzyme, are phosphorylated on Ser10, and generally lack electron density for the first 13 residues. We crystallized myristylated wild-type (WT) PKA and a K7C mutant as binary (bound to a substrate peptide) and ternary [bound to a substrate peptide and adenosine-5'-(β,γ-imido)triphosphate] complexes. There was clear electron density for the entire N-terminus in the binary complexes, both refined to 2.0 Å, and K7C ternary complex, refined to 1.35 Å. The N-termini in these three structures display a novel conformation with a previously unseen helix from residues 1 to 7. The K7C mutant appears to have a more stable N-terminus, and this correlated with a significant decrease in the B-factors for the N-terminus in the myr-K7C complexes compared to the WT binary complex. The N-terminus of the myristylated WT ternary complex, refined to 2.0 Å, was disordered as in previous structures. In addition to a more ordered N-terminus, the myristylated K7C mutant exhibited a 53% increase in k(cat). The effect of nucleotide binding on the structure of the N-terminus in the WT protein and the kinetic changes in the K7C protein suggest that myristylation or occupancy of the myristyl binding pocket may serve as a site for allosteric regulation in the C-subunit.

Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding.

The ability of electrospray to propel large viruses into a mass spectrometer is established and is rationalized by analogy to the atmospheric transmission of the common cold. Much less clear is the fate of membrane-embedded molecular machines in the gas phase. Here we show that rotary adenosine triphosphatases (ATPases)/synthases from Thermus thermophilus and Enterococcus hirae can be maintained intact with membrane and soluble subunit interactions preserved in vacuum. Mass spectra reveal subunit stoichiometries and the identity of tightly bound lipids within the membrane rotors. Moreover, subcomplexes formed in solution and gas phases reveal the regulatory effects of nucleotide binding on both ATP hydrolysis and proton translocation. Consequently, we can link specific lipid and nucleotide binding with distinct regulatory roles.

Assignment of Ile, Leu, and Val Methyl Correlations in Supra-Molecular Systems: An Application to Aspartate Transcarbamoylase

A number of complementary approaches for the assignment of Ile, Leu, and Val methyl groups in Methyl-TROSY spectra of supra-molecular protein complexes are presented and compared. This includes the transfer of assignments from smaller fragments to the complex using a "divide-and-conquer" approach, assignment transfer via exchange spectroscopy, or, alternatively, generating assignments of the complex through the measurement of pseudocontact shifts, facilitated by the introduction of paramagnetic probes. The methodology is applied to the assignment of the regulatory chains in the 300 kDa enzyme aspartate transcarbamoylase, ATCase. The "divide-and-conquer" method that has proven to be very powerful in applications to other systems produced assignments for approximately 60% of the observed methyl groups in TROSY maps of ATCase. By contrast, the combination of all approaches led to assignments for 86% of the methyls, providing a large number of probes of structure and dynamics. The derived assignments were used to interpret chemical shift changes of ATCase upon titration with the nucleotide ATP. Large shift changes in the N-terminal tails of the regulatory chain provide the first evidence for structural perturbations in a region that is known to play a critical role on the effect of nucleotide binding on distal catalytic sites of this allosteric enzyme.

Probing nucleotide-binding effects on backbone dynamics and folding of the nucleotide-binding domain of the sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase.

In muscle cells, SERCA (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase) plays a key role in restoring cytoplasmic Ca2+ levels to resting concentrations after transient surges caused by excitation-coupling cycles. The mechanism by which Ca2+ is translocated to the lumen of the ER (endoplasmic reticulum) involves major conformational rearrangements among the three cytoplasmic domains: actuator (A), nucleotide-binding (N) and phosphorylation (P) domains; and within the transmembrane Ca2+-binding domain of SERCA. CD, fluorescence spectroscopy and NMR spectroscopy were used in the present study to probe the conformation and stability of the isolated N domain of SERCA (SERCA-N), in the presence and absence of AMP-PNP (adenosine 5'-[beta,gamma-imido]triphosphate). CD and tryptophan fluorescence spectroscopy results established that the effects of nucleotide binding were not readily manifested on the global fold and structural stability of SERCA-N. 15N-backbone-relaxation experiments revealed site-specific changes in backbone dynamics that converge on the central beta-sheet domain. Nucleotide binding produced diverse effects on dynamics, with enhanced mobility observed for Ile369, Cys420, Arg467, Asp568, Phe593 and Gly598, whereas rigidifying effects were found for Ser383, Leu419, Thr484 and Thr532. These results demonstrate that the overall fold and backbone motional properties of SERCA-N remained essentially the same in the presence of AMP-PNP, yet revealing evidence for internal counter-balancing effects on backbone dynamics upon binding the nucleotide, which propagate through the central beta-sheet.

Structure-function relationships in UCP1, UCP2 and chimeras: EPR analysis and retinoic acid activation of UCP2.

Uncoupling proteins (UCPs) are composed of three repeated domains of approximately 100 amino acids each. We have used chimeras of UCP1 and UCP2, and electron paramagnetic resonance (EPR), to investigate domain specific properties of these UCPs. Questions include: are the effects of nucleotide binding on proton transport solely mediated by amino acids in the third C-terminal domain, and are the amino acids in the first two domains involved in retinoic or fatty acid activation? We first confirmed that our reconstitution system produced UCP1 that exhibited known properties, such as activation by fatty acids and inhibition of proton transport by purine nucleotides. Our results confirm the observations reported for recombinant yeast that retinoic acid, but not fatty acids known to activate UCP1, activates proton transport by UCP2 and that this activation is insensitive to nucleotide inhibition. We constructed chimeras in which the last domains of UCP1 or UCP2 were switched and tested for activation by fatty acids or retinoic acid and inhibition by nucleotides. U1U2 is composed of mUCP1 (amino acids 1-198) and hUCP2 (amino acids 211-309). Fatty acids activated proton transport of U1U2 and GTP mediated inhibition. In the other chimeric construct U2U1, hUCP2 (amino acids 1-210) and mUCP1 (amino acids 199-307), retinoic acid still acted as an activator, but no inhibition was observed with GTP. Using EPR, a method well suited to the analysis of the structure of membrane proteins such as UCPs, we confirmed that UCP2 binds nucleotides. The EPR data show large structural changes in UCP1 and UCP2 on exposure to ATP, implying that a putative nucleotide-binding site is present on UCP2. EPR analysis also demonstrated changes in conformation of UCP1/UCP2 chimeras following exposure to purine nucleotides. These data demonstrate that a nucleotide-binding site is present in the C-terminal domain of UCP2. This domain was able to inhibit proton transport only when fused to the N-terminal part of UCP1 (chimera U1U2). Thus, residues involved in nucleotide inhibition of proton transport are located in the two first carrier motifs of UCP1. While these results are consistent with previously reported effects of the C-terminal domain on nucleotide binding, they also demonstrate that interactions with the N-terminal domains are necessary to inhibit proton transport. Finally, the results suggest that proteins such as UCP2 may transport protons even though they are not responsible for basal or cold-induced thermogenesis.

Functional Conformation Changes in the TF 1-ATPase β Subunit Probed by 12 Tyrosine Residues

The effect of nucleotide binding on the structure of the F 1-ATPase β subunit from thermophilic bacillus PS-3 (TF 1 β) was investigated by monitoring the NMR signals of the 12 tyrosine residues. The 3,5-proton resonances of 12 tyrosine residues could be observed for the specifically deuterated β subunit. The assignment of 3,5-proton resonances of all of the tyrosine residues was accomplished using 14 mutant proteins, in each of which one or two tyrosine residues were replaced by phenylalanine. Binding of Mg·ATP induced an upfield shift of Tyr 341 resonance, suggesting that their aromatic rings are stacked to each other. Besides Tyr 341, the signal shift observed on Mg·ATP binding was restricted to the resonances of Tyr 148, Tyr 199, Tyr 238, and Tyr 307, suggesting that Mg·ATP induces a conformational change in the hinge region. This can be correlated to the change from the open to closed conformations as implicated in the crystal structure. Mg·ADP induced a similar but distinctly different conformational change. Therefore, the intrinsic conformational change in the β subunit induced by the nucleotide binding is proposed to be one of the essential driving forces for the F 1 rotation. Reconstitution experiments showed that Tyr 277, one of the four conserved tyrosines, is essential to the formation of the α 3 β 3 γ complex.

The mobile loop region of the NAD(H) binding component (dI) of proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum: complete NMR assignment and effects of bound nucleotides

The dI component of transhydrogenase binds NAD + and NADH. A mobile loop region of dI plays an important role in the nucleotide binding process, and mutations in this region result in impaired hydride transfer in the complete enzyme. We have previously employed one-dimensional 1H-NMR spectroscopy to study wild-type and mutant dI proteins of Rhodospirillum rubrum and the effects of nucleotide binding. Here, we utilise two- and three-dimensional NMR experiments to assign the signals from virtually all of the backbone and side-chain protons of the loop residues. The mobile loop region encompasses 17 residues: Asp 223-Met 239. The assignments also provide a much strengthened basis for interpreting the structural changes occurring upon nucleotide binding, when the loop closes down onto the surface of the protein and loses mobility. The role of the mobile loop region in catalysis is discussed with particular reference to a newly-developed model of the dI protein, based on its homology with alanine dehydrogenase.

Influence of nucleotides on the secondary structure and on the thermal stability of mitochondrial F1 visualized by infrared spectroscopy

We have studied the secondary structure of mitochondrial F1 using infrared spectroscopy. Our results show that in the absence of added nucleotides this complex contains similar percentages of α-helices, β-structures and reverse turns (30%, 28% and 31%, respectively). The influence of ADP and ATP on the different types of secondary structure was determined; when all the nucleotide-binding sites were occupied, small but reproducible changes were observed, corresponding to a decrease in β-structure and an increase in α-helix and reverse turns. The effect of nucleotide binding on the thermal stability of F1 was also studied; the thermal denaturation temperature, 55°C, was increased by 11°C and 7°C by ATP and ADP, respectively. These results indicate that nucleotide binding affects the secondary structure of F1, stabilizing the complex.

Interaction of phosphorylated elongation factor EF-2 with nucleotides and ribosomes

The intrinsic fluorescence emission spectrum of elongation factor EF-2 due to the 7 Trp residues was not modified after complete phosphorylation of the factor by the specific Ca 2+/Calmodulin-dependent kinase III. The effect of nucleotide binding on this fluorescence revealed differences between phosphorylated and unmodified EF-2. Low concentrations of GTP had a smaller quenching effect on the fluorescence of phosphorylated EF-2 than on the fluorescence of unmodified EF-2, whereas GDP had exactly the same quenching effect on the fluorescence of both samples. These results suggest that phosphorylation of EF-2 decreased its affinity for GTP but not for GDP. Ability of phosphorylated EF-2 to form a ternary complex with ribosomes in the presence of a non-hydrolysable GTP analog and its ability to protect ribosomes against ricin-inactivation were both decreased to the same extent. The lower affinity of phosphorylated EF-2 for GTP could be responsible for a weaker and/or incorrect interaction of the factor with the ribosome, in particular with the ricin-site of the 28-S rRNA assumed to be involved in translocation initiation.

Effects of nucleotide binding on thermal transitions and domain structure of myosin subfragment 1.

The thermal unfolding and domain structure of myosin subfragment 1 (S1) from rabbit skeletal muscles and their changes induced by nucleotide binding were studied by differential scanning calorimetry. The binding of ADP to S1 practically does not influence the position of the thermal transition (maximum at 47.2 degrees C), while the binding of the non-hydrolysable analogue of ATP, adenosine 5'-[beta, gamma-imido]triphosphate (AdoPP[NH]P) to S1, or trapping of ADP in S1 by orthovanadate (Vi), shift the maximum of the heat adsorption curve for S1 up to 53.2 and 56.1 degrees C, respectively. Such an increase of S1 thermostability in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is confirmed by results of turbidity and tryptophan fluorescence measurements. The total heat adsorption curves for S1 and its complexes with nucleotides were decomposed into elementary peaks corresponding to the melting of structural domains in the S1 molecule. Quantitative analysis of the data shows that the domain structure of S1 in the complexes S1-AdoPP[NH]P and S1-ADP-Vi is similar and differs radically from that of nucleotide-free S1 and S1 in the S1-ADP complex. These data are the first direct evidence that the S1 molecule can be in two main conformations which may correspond to different states during the ATP hydrolysis: one of them corresponds to nucleotide-free S1 and to the complex S1-ADP, and the other corresponds to the intermediate complexes S1-ATP and S1-ADP-Pi. Surprisingly it turned out that the domain structure of S1 with ADP trapped by p-phenylene-N, N'-dimaleimide (pPDM) thiol cross-linking almost does not differ from that of the nucleotide-free S1. This means that pPDM-cross-linked S1 in contrast to S1-AdoPP[NH]P and S1-ADP-Vi can not be considered a structural analogue of the intermediate complexes S1-ATP and S1-ADP-Pi.

Nucleutide- and temperature- induced changes in myosin subfragment-1 structure

The effects of nucleotide binding and temperature on the internal structural dynamics of myosin subfragment 1 (S1) were monitored by intrinsic tryptophan phosphorescence lifetime and fluorescence anisotropy measurements. Changes in the global conformation of S1 were monitored by measuring its rate of rotational diffusion using transient electric birefringence techniques. At 5°C, the binding of MgADP, MgADP,P and MgADP,V (vanadate) progressively reduce the rotational freedom of S1 tryptophans, producing what appear to be increasingly more rigidified S1-nucleotide structures. The changes in the luminescence properties of the tryptophans suggest that at least one is located at the interface of two S1 subdomains. Increasing the temperature from 0 to 25°C increases the apparent internal mobility of S1 tryptophans in all cases and, in addition, a reversible temperature-dependent transition centered near 15°C was observed for S1,S1-MgADP and S1-MgADP,P, but not for S1-MgADP,V. The rotational diffusion constants of S1 and S1-MgADP were measured at temperatures between 0 and 25°C. After adjusting for the temperature and viscosity of the solvent, the data indicate that the thermally induced transition at 15°C comprises local conformational changes, but no global conformational change. Structural features of S1-MgADP,P, which may relate to its role in force generation while bound to actin, are presented.

ΔpH-activation of the thiol-modified chloroplast ATP hydrolase: Nucleotide binding effects

The activation of the membrane-bound ATP hydrolase by the electrochemical proton gradient Δ \\ ̃ gm H + was studied with dithiothreitol-treated, dark-adapted lettuce thylakoids. After reactivation, carried out at different light intensities, hydrolysis of ATP or GTP was measured upon fast membrane deenergization and substrate addition. The following results were obtained. (1) The initial rate of ATP hydrolysis depends on both the ΔpH attained during the light-reactivation stage and on the external pH (ΔpH was measured by the 9-aminoacridine technique, Δψ being collapsed by valinomycin). (2) When hexylamine is present the initial rate of ATP hydrolysis, as that of GTP hydrolysis measured in the presence of 50 μM GDP, only depends on the ΔpH. (3) ADP addition is able to inhibit a fraction of the ATPases within less than a few seconds. The affinity for the inhibitor is increased if ADP is added 10 s after complete membrane deenergization. From the results obtained, with either ATP or GTP in delocalized conditions (hexylamine present), it is proposed that the number of active thiol-reduced ATPases is a simple function of the ΔpH, independent of the external pH. The results without hexylamine are interpreted as being due to an incomplete delocalization of the proton gradient. The mechanistic implications of this ΔpH-activation are discussed. The rapid deactivation of only a fraction of active ATPases by ADP binding, and more especially the decrease in affinity for this inhibitor by ΔpH, suggests the existence of different active states which are discriminated by their deactivation and not their catalytic properties.

Staphylococcal nuclease reviewed: A Prototypic study in contemporary enzymology. II. Solution studies of the nucleotide binding site and the effects of nucleotide binding.

This is the second of a series of four articles in which the chemical, enzymological and crystallographic work on Ribonucleate (deoxyribonucleate)-3'-nucleotidohydrolase, EC 3.1.4.4. (staphylococcal nuclease, micrococcal nuclease) will be reviewed and correlated. This article discusses studies in solution delineating the extent of the binding site of the enzyme and identifying some of the particular amino acid residues that form this site. In addition, the effects of the very potent inhibitory combination of thymidine-3',5'-diphosphate and Ca2+ on the conformation of the enzyme and its physical, chemical and enzymological properties will be reviewed.

Effect of nucleotide binding on the proximity of the essential sulfhydryl groups of myosin. Chemical probing of movement of residues during conformational transitions.

The reaction of myosin with three bifunctional sulfhydryl reagents of differing cross-linking span is reported. In the absence of nucleotide only p-N,N'-phenylenedimaleimide with a cross-linking span of 12-14 A can bridge between the two essential sulfhydryls of myosin. The other two reagents, 2,4-dinitro-1,5-difluorobenzene and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone with cross-linking spans of 3-5 and 7-10 A, respectively, react under identical conditions with the SH1 sulfhydryl but do not bridge to the SH2 group. In the presence of MgADP, both p-N,N'-phenylenedimaleimide and 4,4'-difluoro-3,3'-dinitrodiphenyl sulfone bridge across the SH1 and SH2 groups indicating a closer proximity of these two sulfhydryls in the presence of bound nucleotide. These results are discussed in relation to the conformational change induced in myosin by binding of the nucleotide.

Spin-labeling studies of aspartate transcarbamylase. I. Effects of nucleotide binding and subunit separation.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSpin-labeling studies of aspartate transcarbamylase. I. Effects of nucleotide binding and subunit separationTrent BuckmanCite this: Biochemistry 1970, 9, 16, 3255–3265Publication Date (Print):August 4, 1970Publication History Published online1 May 2002Published inissue 4 August 1970https://doi.org/10.1021/bi00818a020RIGHTS & PERMISSIONSArticle Views22Altmetric-Citations42LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts