An intramolecular FRET system monitors fingers subdomain opening in Klentaq1

A major goal of polymerase research is to determine the mechanism through which a nucleotide complementary to a templating DNA base is selected and delivered to the polymerase active site. Structural evidence suggests a large open-to-closed conformational change affecting the fingers subdomain as being crucial to the process. We previously designed a FRET system capable of measuring the rate of fingers subdomain closure in the presence of correct nucleotide. However, this FRET system was limited in that it could not directly measure the rate of fingers subdomain opening by FRET after polymerization or in the absence of DNA. Here we report the development of a new system capable of measuring both fingers subdomain closure and reopening by FRET, and show that the rate of fingers subdomain opening is limited only by the rate of polymerization. We anticipate that this system will scale down to the single molecule level, allowing measurement of fingers subdomain movements in the presence of incorrect nucleotide and in the absence of DNA.     

Enzyme closure and nucleotide binding structurally lock guanylate kinase

We investigate the conformational dynamics and mechanical properties of guanylate kinase (GK) using a multiscale approach combining high-resolution atomistic molecular dynamics and low-resolution Brownian dynamics simulations. The GK enzyme is subject to large conformational changes, leading from an open to a closed form, which are further influenced by the presence of nucleotides. As suggested by recent work on simple coarse-grained models of apo-GK, we primarily focus on GK's closure mechanism with the aim to establish a detailed picture of the hierarchy and chronology of structural events essential for the enzymatic reaction. We have investigated open-versus-closed, apo-versus-holo, and substrate-versus-product-loaded forms of the GK enzyme. Bound ligands significantly modulate the mechanical and dynamical properties of GK and rigidity profiles of open and closed states hint at functionally important differences. Our data emphasizes the role of magnesium, highlights a water channel permitting active site hydration, and reveals a structural lock that stabilizes the closed form of the enzyme.     

A subdomain of MEKK1 that is critical for binding to MKK4

Mitogen-activated protein kinase (MAPK) cascades are central components of signal transduction pathways induced by mitogens and stresses. They consist of a three-kinase module in which a mitogen-activated protein kinase kinase kinase (MAP3K) activates a mitogen-activated protein kinase kinase (MAP2K), which in turn activates MAPK. The molecular determinants that underlie specific MAP3K-MAP2K interactions are poorly understood. In this study, we examined the interaction between the MAP3K MEKK1 and MKK4, a MAP2K of the JNK pathway. Select point mutations in subdomain X of the catalytic domain of MEKK1 (MEKK1delta) were found to impair the ability of MEKK1delta to bind to and activate MKK4. Such mutations were also found to impair MEKK1delta-induced activation of an AP1 reporter gene. These studies point to a critical role for subdomain X in the interaction of MEKK1 with MKK4.     

Carbamoyl phosphate synthetase: closure of the B-domain as a result of nucleotide binding

Carbamoyl phosphate synthetase (CPS) catalyzes the production of carbamoyl phosphate which is subsequently employed in the metabolic pathways responsible for the synthesis of pyrimidine nucleotides or arginine. The catalytic mechanism of the enzyme occurs through three highly reactive intermediates: carboxyphosphate, ammonia, and carbamate. As isolated from Escherichia coli, CPS is an alpha, beta-heterodimeric protein with its three active sites separated by nearly 100 A. In addition, there are separate binding sites for the allosteric regulators, ornithine, and UMP. Given the sizable distances between the three active sites and the allosteric-binding pockets, it has been postulated that domain movements play key roles for intramolecular communication. Here we describe the structure of CPS from E. coli where, indeed, such a domain movement has occurred in response to nucleotide binding. Specifically, the protein was crystallized in the presence of a nonhydrolyzable analogue, AMPPNP, and its structure determined to 2.1 A resolution by X-ray crystallographic analysis. The B-domain of the carbamoyl phosphate synthetic component of the large subunit closes down over the active-site pocket such that some atoms move by more than 7 A relative to that observed in the original structure. The trigger for this movement resides in the hydrogen-bonding interactions between two backbone amide groups (Gly 721 and Gly 722) and the beta- and gamma-phosphate groups of the nucleotide triphosphate. Gly 721 and Gly 722 are located in a Type III' reverse turn, and this type of secondary structural motif is also observed in D-alanine:D-alanine ligase and glutathione synthetase, both of which belong to the "ATP-grasp" superfamily of proteins. Details concerning the geometries of the two active sites contained within the large subunit of CPS are described.     

Studies on ligand binding to histidine triad nucleotide binding protein 1

Histidine triad nucleotide binding protein (HINT1) is an intracellular protein that binds purine mononucleotides. Strong sequence conservation suggests that these proteins play a fundamental role in cell biology, however its exact cellular function continues to remain elusive. nuclear magnetic resonance (NMR) studies using STD and HSQC were conducted to observe ligand binding to HINT1. These studies were confirmed using fluorescence spectroscopy titrations. We found that AICAR, the first non-phosphate containing ligand, binds to mouse histidine triad nucleotide binding protein 1 (HINT1). Chemical shift perturbations are mapped onto the X-ray structure showing AICAR binds at the same site as GMP. The NMR results demonstrated that this method will be valuable for the future screening of small molecules that can be used to modulate the function of HINT1.     

Shape-specific nucleotide binding of single-stranded RNA by the GLD-1 STAR domain

Proteins containing the STAR RNA-binding domain fulfill vital roles in RNA biogenesis, yet a detailed understanding of STAR domain RNA binding specificity is lacking. In Caenorhabditis elegans, the STAR protein GLD-1 directly binds the 28 nucleotide recognition element TGE within the 3' untranslated region of tra-2 mRNA. The GLD-1:TGE interaction promotes translational silencing of tra-2 mRNA, marking a pivotal event in the spermatogenesis to oogenesis switch in C.elegans hermaphrodites. By measuring the binding affinities of both GLD-1 and TGE mutants, we have explored the molecular determinants of STAR domain specificity. Site-directed GLD-1 mutants were guided by sequence homology with human splicing factor 1 (SF1), for which an RNA:protein complex structure is available in the work done by Liu et al. The RNA binding affinity of 11 mutant GLD-1 proteins was measured, and their binding specificity was assessed with a series of TGE RNAs containing natural or modified nucleotides. This combinatorial analysis of both RNA and protein mutants revealed a diverse array of specificities of individual nucleotide-binding pockets along the interface. At nucleotide position 18, adenosine appears to be specified by the overall shape of a pocket lined with aliphatic side-chains. At position 19, the high preference for cytidine is dependent on both the length of an amino acid side-chain and the identity of terminal functional groups. The nucleotide 21 binding pocket exhibits low discrimination for cytidine, and accommodates most nucleobases. The highly hydrophobic binding interface and apparent small number of hydrogen bonding read-out interactions at these positions is consistent with our finding that few amino acids seem to function individually in establishing binding specificity. Rather, specificity is conferred by the shape of the nucleotide-binding pocket. Our data provide the first detailed, quantitative analysis of the STAR domain, and highlight features of STAR:RNA recognition that are distinct among single-stranded RNA-binding proteins.     

Regulation of Tiam1 nucleotide exchange activity by pleckstrin domain binding ligands

Rho family GTPases play roles in cytoskeletal organization and cellular transformation. Tiam1 is a member of the Dbl family of guanine nucleotide exchange factors that activate Rho family GTPases. These exchange factors have in common a catalytic Dbl homology and adjacent pleckstrin homology domain. Previous structural studies suggest that the pleckstrin domain, a putative phosphoinositide-binding site, may serve a regulatory function. We identified ascorbyl stearate as a compound that binds to the pleckstrin domain of p120 Ras GTPase-activating protein. Furthermore, ascorbyl stearate appears to be a general pleckstrin domain ligand, perhaps by mimicking an endogenous amphiphilic ligand. Tiam1 nucleotide exchange activity was greatly stimulated by ascorbyl stearate. Certain phosphoinositides also stimulated Tiam1 activity but were less potent than ascorbyl stearate. Tiam1 contains an additional N-terminal pleckstrin domain, but only the C-terminal pleckstrin domain was required for activation. Our results suggest that the pleckstrin domains of Dbl-type proteins may not only be involved in subcellular localization but may also directly regulate the nucleotide exchange activity of an associated Dbl homology domain. In addition, this paper introduces ascorbyl stearate as a pleckstrin domain ligand that can modulate the activity of certain pleckstrin domain-containing proteins.     

Regulation of KATP channel expression and activity by the SUR1 nucleotide binding fold 1

ATP-sensitive K(+) (K(ATP)) channels are oligomeric complexes of pore-forming Kir6 subunits and regulatory Sulfonylurea Receptor (SUR) subunits. SUR, an ATP-Binding Cassette (ABC) transporter, confers Mg-nucleotide stimulation to the channel via nucleotide interactions with its two cytoplasmic domains (Nucleotide Binding Folds 1 and 2; NBF1 and NBF2). Regulation of K(ATP) channel expression is a complex process involving subunit assembly in the ER, SUR glycosylation in the Golgi, and trafficking to the plasma membrane. Dysregulation can occur at different steps of the pathway, as revealed by disease-causing mutations. Here, we have addressed the role of SUR1 NBF1 in gating and expression of reconstituted channels. Deletion of NBF1 severely impairs channel expression and abolishes MgADP stimulation. Total SUR1 protein levels are decreased, suggestive of increased protein degradation, but they are not rescued by treatment with sulfonylureas or the proteasomal inhibitor MG-132. Similar effects of NBF1 deletion are observed in recombinant K(ATP) channels obtained by "splitting" SUR1 into two separate polypeptides (a N-terminal "half" and a C-terminal "half"). Interestingly, the location of the "splitting point" in the vicinity of NBF1 has marked effects on the MgADP stimulation of resulting channels. Finally, ablation of the ER retention motif upstream of NBF1 (in either "split" or full-length SUR1) does not rescue expression of channels lacking NBF1. These results indicate that, in addition to NBF1 being required for MgADP stimulation of the channel, it plays an important role in the regulation of channel expression that is independent of the ER retention checkpoint and the proteasomal degradation pathway.     

Cross-talk in the A1-ATPase from Methanosarcina mazei Go1 due to nucleotide binding

Changes in the A(3)B(3)CDF-complex of the Methanosarcina mazei G?1 A(1)-ATPase in response to ligand binding have been studied by small-angle x-ray scattering, protease digestion, fluorescence spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and CuCl(2)-induced disulfide formation. The value of the radius of gyration, R(g), increases slightly when MgATP, MgADP, or MgADP + P(i) (but not MgAMP-PNP) is present. The nucleotide-binding subunits A and B were reacted with N-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide, and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP-PNP, MgATP, MgADP + P(i), or MgADP. Trypsin treatment of A(1) resulted in cleavage of the stalk subunits C and F, which was rapid in the presence of MgAMP-PNP but slow when MgATP or MgADP were added to the enzyme. When A(1) was supplemented with CuCl(2) a clear nucleotide dependence of an A-A-D cross-linking product was generated in the presence of MgADP and MgATP but not when MgAMP-PNP or MgADP + P(i) was added. The site of cross-link formation was located in the region of the N and C termini of subunit D. The data suggest that the stalk subunits C, D, and F in A(1) undergo conformational changes during ATP hydrolysis.     

Syntaxin-1A inhibits cardiac KATP channels by its actions on nucleotide binding folds 1 and 2 of sulfonylurea receptor 2A

ATP-sensitive potassium (KATP) channels couple the metabolic status of the cell to its membrane potential to regulate a number of cell actions, including secretion (neurons and neuroendocrine cells) and muscle contractility (skeletal, cardiac, and vascular smooth muscle). KATP channels consist of regulatory sulfonylurea receptors (SUR) and pore-forming (Kir6.X) subunits. We recently reported (Pasyk, E. A., Kang, Y., Huang, X., Cui, N., Sheu, L., and Gaisano, H. Y. (2004) J. Biol. Chem. 279, 4234-4240) that syntaxin-1A (Syn-1A), known to mediate exocytotic fusion, was capable of binding the nucleotide binding folds (NBF1 and C-terminal NBF2) of SUR1 to inhibit the KATP channels in insulin-secreting pancreatic islet beta cells. This prompted us to examine whether Syn-1A might modulate cardiac SUR2A/KATP channels. Here, we show that Syn-1A is present in the plasma membrane of rat cardiac myocytes and binds the SUR2A protein (of rat brain, heart, and human embryonic kidney 293 cells expressing SUR2A/Kir6. 2) at its NBF1 and NBF2 domains to decrease KATP channel activation. Unlike islet beta cells, in which Syn-1A inhibition of the channel activity was apparently mediated only via NBF1 and not NBF2 of SUR1, both exogenous recombinant NBF1 and NBF2 of SUR2A were found to abolish the inhibitory actions of Syn-1A on K(ATP) channels in rat cardiac myocytes and HEK293 cells expressing SUR2A/Kir6.2. Together with our recent report, this study suggests that Syn-1A binds both NBFs of SUR1 and SUR2A but appears to exhibit distinct interactions with NBF2 of these SUR proteins in modulating the KATP channels in islet beta cells and cardiac myocytes.     

Cytochrome c promotes caspase-9 activation by inducing nucleotide binding to Apaf-1

We report here the biochemical analysis of the reconstituted de novo procaspase-9 activation using highly purified cytochrome c, recombinant apoptotic protease-activating factor-1 (Apaf-1), and recombinant procaspase-9. Using a nucleotide binding assay, we found that Apaf-1 alone bound dATP poorly and the nucleotide binding to Apaf-1 was significantly stimulated by cytochrome c. The binding of dATP to Apaf-1 induces the formation of a multimeric Apaf-1. cytochrome c complex, apoptosome. Procaspase-9 also synergistically promotes dATP binding to Apaf-1 in a cytochrome c-dependent manner. The dATP bound to apoptosome remained as dATP, not dADP. A nonhydrolyzable ATP analog, ADPCP (beta,gamma-methylene adenosine 5'-triphosphate), was able to support apoptosome formation and caspase activation in place of dATP or ATP. These data indicate that the key event in Apaf-1-mediated caspase-9 activation is cytochrome c-induced dATP binding to Apaf-1.     

A single nucleotide incorporation step limits human telomerase repeat addition activity

Human telomerase synthesizes telomeric DNA repeats (GGTTAG)_n onto chromosome ends using a short template from its integral telomerase RNA (hTR). However, telomerase is markedly slow for processive DNA synthesis among DNA polymerases. We report here that the unique template‐embedded pause signal restricts the first nucleotide incorporation for each repeat synthesized, imparting a significantly greater K_M. This slow nucleotide incorporation step drastically limits repeat addition processivity and rate under physiological conditions, which is alleviated with augmented concentrations of dGTP or dGDP, and not with dGMP nor other nucleotides. The activity stimulation by dGDP is due to nucleoside diphosphates functioning as substrates for telomerase. Converting the first nucleotide of the repeat synthesized from dG to dA through the telomerase template mutation, hTR‐51U, correspondingly shifts telomerase repeat addition activity stimulation to dATP‐dependent. In accordance, telomerase without the pause signal synthesizes DNA repeats with extremely high efficiency under low dGTP concentrations and lacks dGTP stimulation. Thus, the first nucleotide incorporation step of the telomerase catalytic cycle is a potential target for therapeutic enhancement of telomerase activity.     

Crystal structures of a ddATP-, ddTTP-, ddCTP, and ddGTP- trapped ternary complex of Klentaq1: insights into nucleotide incorporation and selectivity

The mechanism by which DNA polymerase I enzymes function has been the subject of extensive biochemical and structural studies. We previously determined the structure of a ternary complex of the large fragment of DNA polymerase I from Thermus aquaticus (Klentaq1) bound to a primer/template DNA and a dideoxycytidine 5'-triphosphate (ddCTP). In this report, we present the details of the 2.3-A resolution crystal structures of three additional ternary complexes of Klentaq1 bound to a primer/template DNA and a dideoxyguanosine 5'-triphosphate (ddGTP), a dideoxythymidine 5'-triphosphate (ddTTP), or a dideoxyadenosine 5'-triphosphate (ddATP). Comparison of the active site of the four ternary complexes reveals that the protein residues around the nascent base pair (that formed between the incoming dideoxynucleoside triphosphate [ddNTP] and the template base) form a snug binding pocket into which only a correct Watson-Crick base pair can fit. Except in the ternary complex bound to dideoxyguanosine 5'-triphosphate, there are no sequence specific contacts between the protein side chains and the nascent base pair, suggesting that steric constraints imposed by the protein onto the nascent base pair is the major contributor to nucleotide selectivity at the polymerase active site. The protein around the polymerase active site also shows plasticity, which may be responsible for the substrate diversity of the enzyme. Two conserved side chains, Q754 and R573, form hydrogen bonds with the N3 atom in the purine base and O2 atom in the pyrimidine base at the minor groove side of the base pair formed by the incorporated ddNMP and the corresponding template base in all the four ternary complexes. These hydrogen-bonding interactions may provide a means of detecting misincorporation at this position.     

A dynamic model of long-range conformational adaptations triggered by nucleotide binding in GroEL-GroES

The molecular chaperone, GroEL, essential for correct protein folding in E. coli, is composed of 14 identical subunits organized in two interacting rings, each providing a folding chamber for non‐native substrate proteins. The oligomeric assembly shows positive cooperativity within each ring and negative cooperativity between the rings. Although it is well known that ATP and long‐range allosteric interactions drive the functional cycle of GroEL, an atomic resolution view of how ligand binding modulates conformational adaptations over long distances remains a major challenge. Moreover, little is known on the relation between equilibrium dynamics at physiological temperatures and the allosteric transitions in GroEL. Here we present multiple all‐atom molecular dynamics simulations of the GroEL‐GroES assemblies at different stages of the functional cycle. Combined with an extensive analysis of the complete set of experimentally available structures, principal component analysis and conformer plots, we provide an explicit evaluation of the accessible conformational space of unliganded GroEL. Our results suggest the presence of pre‐existing conformers at the equatorial domain level, and a shift of the conformational ensemble upon ATP‐binding. At the inter‐ring interface the simulations capture a remarkable offset motion of helix D triggered by ATP‐binding to the folding active ring. The reorientation of helix D, previously only observed upon GroES association, correlates with a change of the internal dynamics in the opposite ring. This work contributes to the understanding of the molecular mechanisms in GroEL and highlights the ability of all‐atom MD simulations to model long‐range structural changes and allosteric events in large systems. Proteins 2012;. © 2012 Wiley Periodicals, Inc.     

A lysine residue in the fingers subdomain of T7 DNA polymerase modulates the miscoding potential of 8-oxo-7,8-dihydroguanosine

8-oxo-7,8-dihydroguanosine (8oG) is a highly mutagenic DNA lesion that stably pairs with adenosine, forming 8oG(syn).dA(anti) Hoogsteen base pairs. DNA polymerases show different propensities to insert dCMP or dAMP opposite 8oG, but the molecular mechanisms that determine faithful or mutagenic bypass are poorly understood. Here, we report kinetic and structural data providing evidence that, in T7 DNA polymerase, residue Lys536 is responsible for attenuating the miscoding potential of 8oG. The Lys536Ala polymerase shows a significant increase in mutagenic 8oG bypass versus wild-type polymerase, and a crystal structure of the Lys536Ala mutant reveals a closed complex with an 8oG(syn).dATP mismatch in the polymerase active site, in contrast to the unproductive, open complex previously obtained by using wild-type polymerase. We propose that Lys536 acts as a steric and/or electrostatic filter that attenuates the miscoding potential of 8oG by normally interfering with the binding of 8oG in a syn conformation that pairs with dATP.