Role Of Nucleotide Binding Research Articles

Solubility and Thermal Stability of Thermotoga maritima MreB.

The basis of MreB research is the study of the MreB protein from the Thermotoga maritima species, since it was the first one whose crystal structure was described. Since MreB proteins from different bacterial species show different polymerisation properties in terms of nucleotide and salt dependence, we conducted our research in this direction. For this, we performed measurements based on tryptophan emission, which were supplemented with temperature-dependent and chemical denaturation experiments. The role of nucleotide binding was studied through the fluorescent analogue TNP-ATP. These experiments show that Thermotoga maritima MreB is stabilised in the presence of low salt buffer and ATP. In the course of our work, we developed a new expression and purification procedure that allows us to obtain a large amount of pure, functional protein.

A biochemical view on the septins, a less known component of the cytoskeleton.

The septins are a conserved family of guanine nucleotide binding proteins, often named the fourth component of the cytoskeleton. They self-assemble into non-polar filaments and further into higher ordered structures. Properly assembled septin structures are required for a wide range of indispensable intracellular processes such as cytokinesis, vesicular transport, polarity establishment and cellular adhesion. Septins belong structurally to the P-Loop NTPases. However, unlike the small GTPases like Ras, septins do not mediate signals to effectors through GTP binding and hydrolysis. The role of nucleotide binding and subsequent GTP hydrolysis by the septins is rather controversially debated. We compile here the structural features from the existing septin crystal- and cryo-EM structures regarding protofilament formation, inter-subunit interface architecture and nucleotide binding and hydrolysis. These findings are supplemented with a summary of available biochemical studies providing information regarding nucleotide binding and hydrolysis of fungal and mammalian septins.

Elucidation of the functional roles of the Q and I motifs in the human chromatin-remodeling enzyme BRG1

The Snf2 proteins, comprising 53 different enzymes in humans, belong to the SF2 family. Many Snf2 enzymes possess chromatin-remodeling activity, requiring a functional ATPase domain consisting of conserved motifs named Q and I-VII. These motifs form two recA-like domains, creating an ATP-binding pocket. Little is known about the function of the conserved motifs in chromatin-remodeling enzymes. Here, we characterized the function of the Q and I (Walker I) motifs in hBRG1 (SMARCA4). The motifs are in close proximity to the bound ATP, suggesting a role in nucleotide binding and/or hydrolysis. Unexpectedly, when substituting the conserved residues Gln758 (Q motif) or Lys785 (I motif) of both motifs, all variants still bound ATP and exhibited basal ATPase activity similar to that of wildtype BRG1 (wtBRG1). However, all mutants lost the nucleosome-dependent stimulation of the ATPase domain. Their chromatin-remodeling rates were impaired accordingly, but nucleosome binding was retained and still comparable with that of wtBRG1. Interestingly, a cancer-relevant substitution, L754F (Q motif), displayed defects similar to the Gln758 variant(s), arguing for a comparable loss of function. Because we excluded a mutual interference of ATP and nucleosome binding, we postulate that both motifs stimulate the ATPase and chromatin-remodeling activities upon binding of BRG1 to nucleosomes, probably via allosteric mechanisms. Furthermore, mutations of both motifs similarly affect the enzymatic functionality of BRG1 in vitro and in living cells. Of note, in BRG1-deficient H1299 cells, exogenously expressed wtBRG1, but not BRG1 Q758A and BRG1 K785R, exhibited a tumor suppressor-like function.

Functional expression, monodispersity and conformational changes in the SBMV virus viral VPg on binding TFE

Southern bean mosaic virus (SBMV) RNA purified from infected plants was used for cloning the viral genome-linked protein (VPg) and was subsequently expressed in Escherichia coli. Circular dichroism (CD), dynamic light scattering (DLS) and saturation transfer difference (STD) by nuclear magnetic resonance (NMR) measurements were employed to determine the degree of monodispersity and to investigate the conformational changes in the absence and presence of trifluoroethanol (TFE) which indicated increased helical content with increasing concentration of TFE. 8-Anilino-1-naphthalenesulfonic acid (ANS) was used as a probe to compare the unfolding regions of the protein before and after addition of TFE. The results indicated that although the TFE concentration influences VPg folding, it does not play a role in nucleotide binding and that the local solvent hydrophobicity causes significant conformational changes.

Identification of Differentially Expressed Genes in Shoot Apex of Garlic (Allium sativum L.) Using Illumina Sequencing

Garlic is widely used as a spice throughout the world. In this study, transcriptional profilings of garlic shoot apex were performed by using the Illumina technology. A total of 45,363 significantly changed expressed transcripts were detected between the dormant and sprouting garlic shoot apex libraries. The expression of 22,836 unigenes was increased by more than 2-fold in sprouting garlic shoot apex as compared with dormant shoot apex (up-regulated unigene), and 22,526 unigenes were identified as having been down-regulated. Gene ontology (GO) annotations indicated that the differentially expressed genes were mainly played role in nucleotide binding, plastid, hydrolase activity, transferase activity, protein metabolic process, nucleic acid binding, protein binding, mitochondrion. A total of 8,725 differentially expressed genes were assigned to five Kyoto Encyclopedia of Genes and Genomes (KEGG) biochemical pathways, including metabolism, genetic information processing, organism system, cellular processes, and environmental information processing. Real-time quantitative RT-PCR (qRT-PCR) was performed to dissect shoot apex sprouting. The differential expression of genes, such as ENHYDROUS, DAG1, DAM, DTH8, indicate that they play a critical role in shoot apex sprouting. These differentially expressed genes comprise related candidates for shoot apex sprouting regulation in Allium species.

Somatotropic gene response to recombinant somatotropin treatment in buffalo leukocytes

The septins are a conserved family of guanine nucleotide binding proteins, often named the fourth component of the cytoskeleton. They self-assemble into non-polar filaments and further into higher ordered structures. Properly assembled septin structures are required for a wide range of indispensable intracellular processes such as cytokinesis, vesicular transport, polarity establishment and cellular adhesion. Septins belong structurally to the P-Loop NTPases. However, unlike the small GTPases like Ras, septins do not mediate signals to effectors through GTP binding and hydrolysis. The role of nucleotide binding and subsequent GTP hydrolysis by the septins is rather controversially debated. We compile here the structural features from the existing septin crystal- and cryo-EM structures regarding protofilament formation, inter-subunit interface architecture and nucleotide binding and hydrolysis. These findings are supplemented with a summary of available biochemical studies providing information regarding nucleotide binding and hydrolysis of fungal and mammalian septins.

Structural Insights into the Mechanism of Phosphoenolpyruvate Carboxykinase Catalysis

Mammalian PEPCK2 catalyzes the reversible formation of PEP from OAA and GTP (or ITP) in a divalent cation-dependent reaction (Scheme 1), as was elegantly discussed in the first minireview of this series on PEPCK (1). SCHEME 1. PEPCK-catalyzed interconversion of OAA and PEP. In this third minireview, high-resolution crystal structures of mammalian PEPCK are examined to gain insights into the mechanism of PEPCK catalysis, including the reaction's reversibility and nucleotide specificity. Regarding reaction reversibility, PEPCK is responsible for regeneration of the high-energy phosphoryl donor PEP from the unstable, activated β-keto acid OAA. When coupled with pyruvate carboxylase, PEPCK reverses the essentially irreversible formation of pyruvate and ATP from PEP and ADP in the glycolytic reaction catalyzed by pyruvate kinase. As illustrated (Fig. 1), PEPCK could achieve this feat by stabilizing the inherently unstable enolate form of pyruvate generated by decarboxylation of OAA (Scheme 1). Stabilization of this intermediate would reduce the energetic cost for phosphoryl transfer by ∼30 kJ mol−1 relative to direct reversal of the pyruvate kinase-catalyzed reaction. An energetic driving force for the pyruvate kinase reaction is the favorable tautomerization of the high-energy enol to its corresponding keto form; in contrast, by stabilizing the enolate, PEPCK could prevent its energetically favorable protonation and tautomerization, allowing phosphoryl transfer to occur. Thus, by stabilizing this intermediate in a high-energy state, the PEPCK reaction would be energetically rendered freely reversible; the crystal structures that will be described indicate that PEPCK does, in fact, stabilize the enolate intermediate. FIGURE 1. Diagram representing the reaction coordinates for the pyruvate kinase-catalyzed (A) and PEPCK-catalyzed (B) reactions. The standard free energy values given are approximate values based upon the average values from a number of literature sources. The ... The recent structures of PEPCK from human, rat, and chicken (2–5), the enzymes from Trypanosoma cruzi (6), Anaerobiospirillum succiniciproducens (7), and Corynebacterium glutamicum (8), and earlier work on the isozyme from Escherichia coli (9–13) illustrate that the active-site residues and architecture are well conserved, despite what is rather poor overall sequence homology when comparing members of the ATP- and GTP-dependent families.3 As detailed in this minireview, the cationic environment of the active site, dominated by the juxtaposition of two divalent metal ions and the positioning of lysine and arginine residues, is well suited to allow for the stabilization of the enolate intermediate discussed above and to facilitate phosphoryl transfer. An informative aspect of the PEPCK-catalyzed reaction revealed by the recent structural data on the GTP-dependent isozyme from rat is the illumination of the previously unappreciated role of conformational changes occurring in the active site during the catalytic cycle (5). The most prevalent mobile feature illustrated by the structural work is a 10-residue Ω-loop lid domain whose closure is potentially capable of protecting the enolate intermediate (Fig. 2) (2–5). A similar domain is present in ATP-dependent PEPCK, as represented by the E. coli enzyme, which was the first PEPCK to be structurally characterized (9). The structural data on PEPCK demonstrate that only upon closure of the lid domain are the substrates positioned correctly for catalysis to occur (5). Furthermore, another loop domain, the ubiquitous P-loop or kinase-1a motif in the GTP-dependent PEPCKs, also shows dynamic behavior, adapting various conformations correlated with substrate binding. The potential role of the dynamic P-loop in catalysis is of interest because it contains a reactive cysteine residue that is conserved in all GTP-dependent PEPCKs and whose specific modification has been known for 2 decades to result in the inactivation of the enzyme (14, 15). As described below, recent structural work characterizing the low-energy conformational states that define the reaction coordinate of the enzyme-catalyzed reaction (2–5, 16), considered together with previous biochemical studies, has allowed a relatively detailed picture of the mechanism of catalysis utilized by PEPCK to emerge. Both the role of the positively charged active site and the important conformational changes occurring within that site are discussed in the context of an integrated mechanism for PEPCK-mediated catalysis. FIGURE 2. Crystallographic images defining the chemical reaction path of PEPCK-mediated conversion of OAA to PEP. A schematic drawing to aid in the interpretation of the structural data is presented on the right-hand side of each panel. In the left-hand images, ...

Role of Nucleotide Binding in Septin-Septin Interactions and Septin Localization in Saccharomyces cerevisiae

Septins are a conserved family of eukaryotic GTP-binding, filament-forming proteins. In Saccharomyces cerevisiae, five septins (Cdc3p, Cdc10p, Cdc11p, Cdc12p, and Shs1p) form a complex and colocalize to the incipient bud site and as a collar of filaments at the neck of budded cells. Septins serve as a scaffold to localize septin-associated proteins involved in diverse processes and as a barrier to diffusion of membrane-associated proteins. Little is known about the role of nucleotide binding in septin function. Here, we show that Cdc3p, Cdc10p, Cdc11p, and Cdc12p all bind GTP and that P-loop and G4 motif mutations affect nucleotide binding and result in temperature-sensitive defects in septin localization and function. Two-hybrid, in vitro, and in vivo analyses show that for all four septins nucleotide binding is important in septin-septin interactions and complex formation. In the absence of complete complexes, septins do not localize to the cortex, suggesting septin localization factors interact only with complete complexes. When both complete and partial complexes are present, septins localize to the cortex but do not form a collar, perhaps because of an inability to form filaments. We find no evidence that nucleotide binding is specifically involved in the interaction of septins with septin-associated proteins.

Crystal structure of the 25 kDa subunit of human cleavage factor I m

Cleavage factor Im is an essential component of the pre-messenger RNA 3′-end processing machinery in higher eukaryotes, participating in both the polyadenylation and cleavage steps. Cleavage factor Im is an oligomer composed of a small 25 kDa subunit (CF Im25) and a variable larger subunit of either 59, 68 or 72 kDa. The small subunit also interacts with RNA, poly(A) polymerase, and the nuclear poly(A)-binding protein. These protein–protein interactions are thought to be facilitated by the Nudix domain of CF Im25, a hydrolase motif with a characteristic α/β/α fold and a conserved catalytic sequence or Nudix box. We present here the crystal structures of human CF Im25 in its free and diadenosine tetraphosphate (Ap4A) bound forms at 1.85 and 1.80 Å, respectively. CF Im25 crystallizes as a dimer and presents the classical Nudix fold. Results from crystallographic and biochemical experiments suggest that CF Im25 makes use of its Nudix fold to bind but not hydrolyze ATP and Ap4A. The complex and apo protein structures provide insight into the active oligomeric state of CF Im and suggest a possible role of nucleotide binding in either the polyadenylation and/or cleavage steps of pre-messenger RNA 3′-end processing.

The role of nucleotide binding and hydrolysis in the function of the fission yeast cdc18(+) gene product.

The fission yeast cdc18(+) gene is required for both initiation of DNA replication and the mitotic checkpoint that normally inhibits mitosis in the absence of DNA replication. The cdc18(+) gene product contains conserved Walker A and B box motifs. Studies of other ATPases have shown that these motifs are required for nucleotide binding and hydrolysis, respectively. We have observed that mutant strains in which either of these motifs is disrupted are inviable. The effects of these mutations were examined by determining the phenotypes of mutant strains following depletion of complementing wild-type Cdc18. In both synchronous and asynchronous cultures, the nucleotide-hydrolysis motif mutant (DE286AA) arrests with a 1C-2C DNA content, and thus exhibits no obvious defects in entry into S phase or in the mitotic checkpoint. In contrast, in cultures synchronized by hydroxyurea arrest and release, the nucleotide-binding motif mutant (K205A) exhibits the null phenotype, with 1C and <1C DNA content, indicating a block in entry into S phase and loss of checkpoint control. In asynchronous cultures this mutant exhibits a mixed phenotype: a percentage of the population displays the null phenotype, while the remaining fraction arrests with a 2C DNA content. Thus, the phenotype exhibited by the K205A mutant is dependent on the cell-cycle position at which wild-type Cdc18 is depleted. These data indicate that both nucleotide binding and hydrolysis are required for Cdc18 function. In addition, the difference in the phenotypes exhibited by the nucleotide-binding and hydrolysis motif mutants is consistent with a two-step model for Cdc18 function in which nucleotide binding and hydrolysis are required for distinct aspects of Cdc18 function that may be executed at different points in the cell cycle.

Involvement of arginine 143 in nucleotide substrate binding at the active site of adenylosuccinate synthetase from Escherichia coli.

Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thio]phosphate (GMPSBDB) at pH 7.1 and 25 degrees C. Reaction of the enzyme with [8-3H]GMPSBDB results in the incorporation of 2 mol of the reagent/mol of subunit; in the presence of active site ligands the incorporation is reduced to 1 mol of reagent/mol of subunit. GMPSBDB reacts with Cys-291 in the initial rapid reaction which is accompanied by loss of 50% of the enzymatic activity; this reaction is not affected by the presence of active site ligands. In the slower reaction, GMPSBDB inactivates the enzyme by reacting with Arg-143. The inactivation kinetics of the slower phase are consistent with the formation of an enzyme--GMPSBDB complex having a Kd of 42 microM. Active site nucleotides, either adenylosuccinate or IMP + GTP, prevent both slower phase inactivation and labeling of Arg-143. Replacement of Arg-143 with a Leu by site-directed mutagenesis does not change the catalytic constant or the K(m) for aspartate but does significantly impair nucleotide binding: the Michaelis constants for IMP and GTP increase by 60-fold and 10-fold, respectively, in the R143L mutant. The crystal structure of the E. coli enzyme [Poland, B.W., Silva, M.M., Serra, M.A., Cho, Y., Kim, K. H., Harris, E.M.S., & Honzatko, R.B. (1993) J. Biol. Chem. 268, 25334--25342] shows that Arg-143 from one subunit projects into the putative active site of the other subunit. These results indicate that both subunits of dimeric adenylosuccinate synthetase contribute to each active site and that Arg-143 plays an important role in nucleotide binding.

Mechanism of human aldehyde reductase: characterization of the active site pocket.

Human aldehyde reductase is a NADPH-dependent aldo-keto reductase that is closely related (65% identity) to aldose reductase, an enzyme involved in the pathogenesis of some diabetic and galactosemic complications. In aldose reductase, the active site residue Tyr48 is the proton donor in a hydrogen-bonding network involving residues Asp43/Lys77, while His110 directs the orientation of substrates in the active site pocket. Mutation of the homologous Tyr49 to phenylalamine or histidine (Y49F or Y49H) and of Lys79 to methionine (K79M) in aldehyde reductase yields inactive enzymes, indicating similar roles for these residues in the catalytic mechanism of aldehyde reductase. A H112Q mutant aldehyde reductase exhibited a substantial decrease in catalytic efficiency (kcat/Km) for hydrophilic (average 150-fold) and aromatic substrates (average 4200-fold) and 50-fold higher IC50 values for a variety of inhibitors than that of the wild-type enzyme. The data suggest that His112 plays a major role in determining the substrate specificity of aldehyde reductase, similar to that shown earlier for the homologous His110 in aldose reductase [Bohren, K. M., et. al. (1994) Biochemistry 33, 2021-2032]. Mutation of Ile298 or Val299 affected the kinetic parameters to a much lesser degree. Unlike native aldose reductase, which contains a thiol-sensitive Cys298, neither the I298C or V299C mutant exhibited any thiol sensitivity, suggesting a geometry of the active site pocket different from that in aldose reductase. Also different from aldose reductase, the detection of a significant primary deuterium isotope effect on kcat (1.48 +/- 0.02) shows that nucleotide exchange is only partially rate-limiting. Primary substrate and solvent deuterium isotope effects on the H112Q mutant suggest that hydride and proton transfers occur in two discrete steps with hydride transfer taking place first. Dissociation constants and spectroscopic and fluorimetric properties of nucleotide complexes with various mutants suggest that, in addition to Tyr49 and His112, Lys79 plays a hitherto unappreciated role in nucleotide binding. The mode of inhibition of aldehyde reductase by aldose reductase inhibitors (ARIs) is generally similar to that of aldose reductase and involves binding to the E:NADP+ complex, as shown by kinetic and direct inhibitor-binding experiments. The order of ARI potency was AL1576 (Ki = 60 nM) > tolrestat > ponalrestat > sorbinil > FK366 > zopolrestat > alrestatin (Ki = 148 microM). Our data on aldehyde reductase suggest that the active site pocket significantly differs from that of aldose reductase, possibly due to the participation of the C-terminal loop in its formation.

Isolation of an amino-terminal deleted recombinant ADP-ribosylation factor 1 in an activated nucleotide-free state.

ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that activate cholera toxin ADP-ribosyltransferase in vitro and participate in intracellular vesicular membrane trafficking. ARFs are activated when bound GDP is replaced by GTP and inactivated by hydrolysis of bound GTP to yield ARF-GDP. Usually, ARFs are isolated in an inactive GDP-bound state and require addition of GTP along with detergent or phospholipid for activity. Purified mutant recombinant ARF1 lacking the first 13 amino acids (r delta 13ARF1-P) stimulated cholera toxin activity essentially equally with or without added GTP (and phospholipid or detergent), at least in part due to the presence of bound nucleotides, which later were identified as GTP and GDP. Nucleotide-free r delta 13ARF1 (r delta 13ARF1-F), prepared by dialysis against 7 M urea, was active without added GTP in the absence of SDS but inactive without added GTP in its presence. Renaturation of r delta 13ARF1-F in the presence of GTP, ITP, or GDP yielded, respectively, r delta 13ARF1-GTP and r delta 13ARF1-ITP, which were active, and r delta 13ARF1-GDP, which was inactive. Effects of phospholipids and detergents on nucleotide exchangeability evaluated as effects on activity of rARF1 and r delta 13ARF1-F differed. With r delta 13ARF1-F, 100 microM ITP and 100 microM GTP were essentially equally effective in the presence of cardiolipin or SDS. The finding that r delta 13ARF1 differs from rARF1 in the effects of phospholipids and detergents on nucleotide binding is consistent with the conclusion that the ARF amino terminus plays an important role in nucleotide binding and its specificity as well as the molecular conformation and associated activity.

Mutational analysis of the consensus nucleotide binding sequences in the rat liver mitochondrial ATP synthase beta-subunit.

The coupling step in the biosynthesis of ATP in biological systems is generally believed to involve an energy-requiring release of ATP bound to the beta-subunit of the ATP synthase complex. A molecular description of the ATP binding site on the beta-subunit is, therefore, critical to understanding the mechanism of coupling in the enzyme. Previously, we reported that a purified, bacterially expressed rat liver beta-subunit binds adenine nucleotides tightly and specifically (Garboczi, D. N., Hullihen, J. H., and Pedersen, P. L. (1988) J. Biol. Chem. 263, 15694-15698). In order to assess the contribution of various regions of the isolated beta-subunit to the ATP binding site we have systematically deleted four different regions: the N-terminal region, the Walker A consensus region, the Walker B consensus region (Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. (1982) EMBO J. 1, 945-951), and a "C" region, which, like the A and B regions, bears homology to adenylate kinase. Plasmids directing the expression of double deletions of A and B regions, and B and C regions were also constructed. In addition, 2 residues outside of these regions, His-177 and Tyr-345, which have been predicted to play a central role in nucleotide binding, were mutated. Rabbit antisera to synthetic peptides of the A and C regions verified the identity of the bacterially expressed mutant proteins. Seven of the eight mutant proteins overexpressed in Escherichia coli were resistant to E. coli proteases in the preparative stages, as predicted for compact folded proteins. Furthermore, circular dichroism spectropolarimetry revealed no profound structural alterations in the purified mutant proteins. Relative to the overexpressed full-length beta-subunit, the mutant lacking the A consensus region suffered a 30-fold loss of affinity for ATP and a loss of specificity for 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP) over 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate. The mutant proteins lacking either the N-terminal region or the B region exhibited nucleotide binding properties similar to the full-length beta-subunit, whereas the mutant protein lacking the C region suffered an order of magnitude reduction in affinity for ATP. The affinity of the A and B region double deletion was indistinguishable from the A region deletion in regard to TNP-ATP binding, while the double deletion mutant lacking the B and C regions was not stably expressed in the E. coli SE6004.(ABSTRACT TRUNCATED AT 400 WORDS)