Nucleotide Complexes Research Articles

Granzyme A Deficiency Breaks Immune Tolerance and Promotes Autoimmune Diabetes Through a Type I Interferon-Dependent Pathway.

Granzyme A is a protease implicated in the degradation of intracellular DNA. Nucleotide complexes are known triggers of systemic autoimmunity, but a role in organ-specific autoimmune disease has not been demonstrated. To investigate whether such a mechanism could be an endogenous trigger for autoimmunity, we examined the impact of granzyme A deficiency in the NOD mouse model of autoimmune diabetes. Granzyme A deficiency resulted in an increased incidence in diabetes associated with accumulation of ssDNA in immune cells and induction of an interferon response in pancreatic islets. Central tolerance to proinsulin in transgenic NOD mice was broken on a granzyme A-deficient background. We have identified a novel endogenous trigger for autoimmune diabetes and an in vivo role for granzyme A in maintaining immune tolerance.

A Very Rare Example of a Structurally Characterized 3'-GMP Metal Complex. NMR and Synthetic Assessment of Adducts Formed by Guanine Derivatives with [Pt(Ltri)Cl]Cl Complexes with an N,N',N″ Tridentate Ligand (Ltri) Terminated by Imidazole Rings.

[Pt(N(R)-1,1'-Me2dma)Cl]Cl complexes with tridentate ligands (bis(1-methyl-2-methylimidazolyl)amine, R = H; N-(methyl)bis(1-methyl-2-methylimidazolyl)amine, R = Me) were prepared in order to investigate Pt(N(R)-1,1'-Me2dma)G adducts (G = monodentate N9-substituted guanine or hypoxanthine derivative). Solution NMR spectroscopy is the primary tool for studying metal complexes of nucleosides and nucleotides because such adducts rarely crystallize. However, [Pt(N(H)-1,1'-Me2dma)(3'-GMPH)]NO3·5H2O (5) was crystallized, allowing, to our knowledge, the first crystallographic molecular structure determination for a 3'-GMP platinum complex. The structure is one of only a very few structures of a 3'-GMP complex with any metal. Complex 5 has the syn rotamer conformation, with 3'-GMP bound by N7. All Pt(N(R)-1,1'-Me2dma)G adducts exhibit two new downfield-shifted G H8 signals, consistent with G bound to platinum by N7 and a syn/anti rotamer mixture. Anticancer-active monofunctional platinum(II) complexes have bulky carrier ligands that cause DNA adducts to be distorted. Hence, understanding carrier-ligand steric effects is key in designing new platinum drugs. Ligand bulk can be correlated with the degree of impeded rotation of the G nucleobase about the Pt-N7 bond, as assessed by the observation of rotamers. The signals of syn and anti rotamers are connected by EXSY cross-peaks in 2D ROESY spectra of Pt(N(H)-1,1'-Me2dma)G adducts but not in spectra of Pt(N(H)dpa)G adducts (N(H)dpa = bis(2-picolyl)amine), indicating that rotamer interchange is more facile and carrier-ligand bulk is lower in Pt(N(H)-1,1'-Me2dma)G than in Pt(N(H)dpa)G adducts. The lower steric hindrance is a direct consequence of the greater distance of the G nucleobase from the H4/4' protons in the N(R)-1,1'-Me2dma carrier ligand in comparison to that from the H6/6' protons in the N(H)dpa carrier ligand. Although in 5 the nucleotide is 3'-GMP (not the usual 5'-GMP) and the N(H)-1,1'-Me2dma carrier ligand is very different from those typically present in structurally characterized Pt(II) G complexes, the rocking and canting angles in 5 adhere to long-recognized trends.

The first X-ray structure of a silver–nucleotide complex: interaction of ion Ag(i) with cytidine-5′-monophosphate

The synthesis and X-ray characterization of an unprecedented complex of Ag(i) with cytidine-5′-monophosphate (HCMP) is reported. The coordination of Ag(i) to HCMP is via both the N3 and O2 atoms of two cytosine moieties and the phosphate group, generating a MOF.

Structural reverse genetics study of the PI5P4Kβ-nucleotide complexes reveals the presence of the GTP bioenergetic system in mammalian cells.

Reverse genetic analysis can connect a gene and its protein counterpart to a biological function(s) by knockout or knockdown of the specific gene. However, when a protein has multiple biochemical activities, the conventional genetics strategy is incapable of distinguishing which biochemical activity of the protein is critical for the particular biological function(s). Here, we propose a structural reverse genetics strategy to overcome this problem. In a structural reverse genetics study, multiple biochemical activities of a protein are segregated by mapping those activities to a structural element(s) in the atomic resolution tertiary structure. Based on the structural mapping, a mutant lacking one biochemical activity of interest can be produced with the other activities kept intact. Expression of the mutant by knockin or ectopic expression in the knockout strain along with the following analysis can connect the single biochemical activity of interest to a biological function. Using the structural reverse genetics strategy, we have dissected the newly identified GTP-dependent activity of a lipid kinase PI5P4Kβ from its ATP-dependent activity. The GTP-insensitive mutant has demonstrated the existence of the GTP bioenergetic sensor system in mammalian cells and its critical role in tumorigenesis. As structural reverse genetics can identify in vivo significance of individual biochemical activity, it is a powerful approach to reveal hidden biological functions, which could be a novel pharmacological target for therapeutic intervention. Given the recent expansion of choices in structural biological methods and advances in genome editing technologies, the time is ripe for structural reverse genetics strategies.

Potential Interactions Between Genetic Polymorphisms of the Transforming Growth Factor-β Pathway and Environmental Factors in Abdominal Aortic Aneurysms

Evidence has accumulated that multiple polymorphisms in the transforming growth factor (TGF)-β pathway and renin–angiotensin system play important roles in determining susceptibility to abdominal aortic aneurysm (AAA). Few studies have considered interactions between these gene polymorphisms and environmental factors. The aim of this study was to evaluate the contribution of single nucleotide polymorphisms (SNPs) and complex gene–environment interactions in AAA. Six SNPs located in TGFB, TGFBR1, TGFBR2 and AGTR1 were selected. Genotyping of blood samples and collection of lifestyle factors were performed in 155 unrelated participants with AAAs and 310 non-AAA controls. Unconditional logistic regression was performed to assess the effects of SNPs on the risk of AAA. Generalized multifactor dimensionality reduction (GMDR) was used to evaluate gene–gene and gene–environment interactions. Participants carrying TGFB1 rs1800469 TT (odds ratio [OR] 1.83, 95% confidence interval [CI] 1.18-2.85) or AGTR1 rs12695895 TT (OR 4.21, 95% CI 1.41-12.53) genotypes had a higher risk of AAA than those with the common CC genotype. The gene–gene interaction of AGTR1 rs5182, TGFBR1 rs1626340, and TGFB1 rs1800469 was found to be the best model according to the results of the GMDR analysis (cross validation consistency [CVC]) 10/10; P = .010). Smoking, dyslipidemia, and rs1800469 together contributed to the risk of AAA, which demonstrated a potential and complex gene–environment interaction among the three variants that might affect AAA risk (CVC 6/10; P = .001). In this study of the Chinese population, homozygosity of TGFB1 rs1800469-T and AGTR1 rs12695895-T might be associated with increased risk of AAA. The complex gene–gene and gene–environment interactions might contribute to the risk of AAA. As a small study, the preliminary results need extensive validation and replication in larger populations.

Potential Interactions Between Genetic Polymorphisms of the Transforming Growth Factor-β Pathway and Environmental Factors in Abdominal Aortic Aneurysms

Evidence has accumulated that multiple polymorphisms in the transforming growth factor (TGF)-β pathway and renin-angiotensin system play important roles in determining susceptibility to abdominal aortic aneurysm (AAA). Few studies have considered interactions between these gene polymorphisms and environmental factors. The aim of this study was to evaluate the contribution of single nucleotide polymorphisms (SNPs) and complex gene-environment interactions in AAA. Six SNPs located in TGFB, TGFBR1, TGFBR2 and AGTR1 were selected. Genotyping of blood samples and collection of lifestyle factors were performed in 155 unrelated participants with AAAs and 310 non-AAA controls. Unconditional logistic regression was performed to assess the effects of SNPs on the risk of AAA. Generalized multifactor dimensionality reduction (GMDR) was used to evaluate gene-gene and gene-environment interactions. Participants carrying TGFB1 rs1800469 TT (odds ratio [OR] 1.83, 95% confidence interval [CI] 1.18-2.85) or AGTR1 rs12695895 TT (OR 4.21, 95% CI 1.41-12.53) genotypes had a higher risk of AAA than those with the common CC genotype. The gene-gene interaction of AGTR1 rs5182, TGFBR1 rs1626340, and TGFB1 rs1800469 was found to be the best model according to the results of the GMDR analysis (cross validation consistency [CVC]) 10/10; p=.010). Smoking, dyslipidemia, and rs1800469 together contributed to the risk of AAA, which demonstrated a potential and complex gene-environment interaction among the three variants that might affect AAA risk (CVC 6/10; p=.001). In this study of the Chinese population, homozygosity of TGFB1 rs1800469-T and AGTR1 rs12695895-T might be associated with increased risk of AAA. The complex gene-gene and gene-environment interactions might contribute to the risk of AAA. As a small study, the preliminary results need extensive validation and replication in larger populations.

Controllable synthesis of nucleotide complex based on pH control: a small-molecule fluorescent probe as an auxiliary ligand to indicate the pre-organization of the nucleotide complex in solution.

Different CMP-bpe-M(ii) (CMP = cytidine monophosphate, bpe = bis(4-pyridyl)ethylene, M(ii) = Mn(2+) and Co(2+)) complexes are synthesized controllably based on pH control and well studied by X-ray single-crystal diffraction analysis. Interestingly, the suitable pH conditions are explored by considering the pre-organization modes of CMP, bpe, and M(2+) in aqueous solution by fluorescence spectroscopy based on acid/basic titration. The organic base bpe serves as a small-molecule fluorescent probe to indicate the changes of pre-organization modes along with the changes of the solution acidity. Furthermore, a perfect self-complementary sugar-base hydrogen bond is first reported based on the crystal structure analysis in this work, and different chiralities and SHG activities of CMP-bpe-Mn(ii) complexes obtained at different pH values are studied.

Disruption of O-GlcNAc Cycling in C. elegans Perturbs Nucleotide Sugar Pools and Complex Glycans.

The carbohydrate modification of serine and threonine residues with O-linked beta- N-acetylglucosamine (O-GlcNAc) is ubiquitous and governs cellular processes ranging from cell signaling to apoptosis. The O-GlcNAc modification along with other carbohydrate modifications, including N-linked and O-linked glycans, glycolipids, and sugar polymers, all require the use of the nucleotide sugar UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP). In this paper, we describe the biochemical consequences resulting from perturbation of the O-GlcNAc pathway in C. elegans lacking O-GlcNAc transferase and O-GlcNAcase activities. In ogt-1 null animals, steady-state levels of UDP-GlcNAc/UDP-GalNAc and UDP-glucose were substantially elevated. Transcripts of genes encoding for key members in the HBP (gfat-2, gna-2, C36A4.4) and trehalose metabolism (tre-1, tre-2, tps-2) were elevated in ogt-1 null animals. While there is no evidence to suggest changes in the profile of N-linked glycans in the ogt-1 and oga-1 mutants, glycans insensitive to PNGase digestion (including O-linked glycans, glycolipids, and glycopolymers) were altered in these strains. Our data support that changes in O-GlcNAcylation alters nucleotide sugar production, overall glycan composition, and transcription of genes encoding glycan processing enzymes. These data along with our previous findings that disruption in O-GlcNAc cycling alters macronutrient storage underscores the noteworthy influence this posttranslational modification plays in nutrient sensing.

Automated nucleic acid chain tracing in real time.

The crystallographic structure solution of nucleotides and nucleotide complexes is now commonplace. The resulting electron-density maps are often poorer than for proteins, and as a result interpretation in terms of an atomic model can require significant effort, particularly in the case of large structures. While model building can be performed automatically, as with proteins, the process is time-consuming, taking minutes to days depending on the software and the size of the structure. A method is presented for the automatic building of nucleotide chains into electron density which is fast enough to be used in interactive model-building software, with extended chain fragments built around the current view position in a fraction of a second. The speed of the method arises from the determination of the 'fingerprint' of the sugar and phosphate groups in terms of conserved high-density and low-density features, coupled with a highly efficient scoring algorithm. Use cases include the rapid evaluation of an initial electron-density map, addition of nucleotide fragments to prebuilt protein structures, and in favourable cases the completion of the structure while automated model-building software is still running. The method has been incorporated into the Coot software package.

Structural analysis of human soluble adenylyl cyclase and crystal structures of its nucleotide complexes-implications for cyclase catalysis and evolution.

The ubiquitous second messenger cAMP regulates a wide array of functions, from bacterial transcription to mammalian memory. It is synthesized by six evolutionarily distinct adenylyl cyclase (AC) families. In mammals, there are two AC types: nine transmembrane ACs (tmACs) and one soluble AC (sAC). Both AC types belong to the widespread cyclase class III, which has members in numerous organisms from archaeons to mammals. Class III also contains all known guanylyl cyclases (GCs), which synthesize the cAMP-related messenger cGMP in many eukaryotes and possibly some prokaryotes. Among mammalian ACs, sAC is uniquely regulated by bicarbonate, and has been proposed to be more closely related to a bacterial AC subfamily than to mammalian ACs, on the basis of sequence comparisons. Here, we used crystal structures of human sAC catalytic domains to analyze its relationships with other class III ACs and GCs, and to study its substrate selection mechanisms. Structural comparisons revealed a similarity within an sAC-like subfamily but no family-specific structure elements, and an unexpected sAC similarity to eukaryotic GCs and a potential bacterial GC. We further solved novel crystal structures of sAC catalytic domains in complex with a substrate analog, unprocessed ATP substrate, and product after soaking with ATP or GTP. The structures show a novel ATP-binding conformation, and suggest mechanisms for substrate association and recognition. Our results could explain the limited substrate specificity of sAC, suggest how specificity is increased in other cyclases, and indicate evolutionary relationships among class III enzymes, with sAC being close to a putative 'ancestor' cyclase. Coordinates and structure factors for the novel sAC-cat structures described have been deposited with the Worldwide PDB (www.pdb.org): ApCpp soak (entry 4usu), ATP + Ca(2+) soak (entry 4usv), GTP + Mg(2+) soak (entry 4ust), ATP soak (entry 4usw).

Metabolism of DNA secondary structures at the eukaryotic replication fork

DNA secondary structures are largely advantageous for numerous cellular processes but can pose specific threats to the progression of the replication machinery and therefore genome duplication and cell division. A number of specialized enzymes dismantle these structures to allow replication fork progression to proceed faithfully. In this review, we discuss the in vitro and in vivo data that has lead to the identification of these enzymes in eukaryotes, and the evidence that suggests that they act specifically at replication forks to resolve secondary structures. We focus on the role of helicases, which catalyze the dissociation of nucleotide complexes, and on the role of nucleases, which cleave secondary structures to allow replication fork progression at the expense of local rearrangements. Finally, we discuss outstanding questions in terms of dismantling DNA secondary structures, as well as the interplay between diverse enzymes that act upon specific types of structures.

Agonist-bound structure of the human P2Y12 receptor

The P2Y12 receptor (P2Y12R), one of eight members of the P2YR family expressed in humans, has been identified as one of the most prominent clinical drug targets for inhibition of platelet aggregation. Consequently, extensive mutagenesis and modeling studies of the P2Y12R have revealed many aspects of agonist/antagonist binding1-4. However, the details of agonist and antagonist recognition and function at the P2Y12R remain poorly understood at the molecular level. Here, we report the structures of the human P2Y12R in complex with a full agonist 2-methylthio-adenosine-5′-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 Å resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5′-triphosphate (2MeSATP) at 3.1 Å resolution. Analysis of these structures, together with the structure of the P2Y12R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283)5, reveals dramatic conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions, providing the first insight into a different ligand binding landscape in the δ-group of class A G protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y12R, with only partially overlapped binding pockets. The agonist-bound P2Y12R structure answers long-standing ambiguities surrounding P2Y12R-agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example of a GPCR where agonist access to the binding pocket requires large scale rearrangements in the highly malleable extracellular region, the structural studies therefore will provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y12R and potentially for other closely related P2YRs.

Characterization of Complexes of Nucleoside-5′-Phosphorothioate Analogues with Zinc Ions

On the basis of the high affinity of Zn(2+) to sulfur and imidazole, we targeted nucleotides such as GDP-β-S, ADP-β-S, and AP3(β-S)A, as potential biocompatible Zn(2+)-chelators. The thiophosphate moiety enhanced the stability of the Zn(2+)-nucleotide complex by about 0.7 log units. ATP-α,β-CH2-γ-S formed the most stable Zn(2+)-complex studied here, log K 6.50, being ~0.8 and ~1.1 log units more stable than ATP-γ-S-Zn(2+) and ATP-Zn(2+) complexes, and was the major species, 84%, under physiological pH. Guanine nucleotides Zn(2+) complexes were more stable by 0.3-0.4 log units than the corresponding adenine nucleotide complexes. Likewise, AP3(β-S)A-zinc complex was ~0.5 log units more stable than AP3A complex. (1)H- and (31)P NMR monitored Zn(2+) titration showed that Zn(2+) coordinates with the purine nucleotide N7-nitrogen atom, the terminal phosphate, and the adjacent phosphate. In conclusion, replacement of a terminal phosphate by a thiophosphate group resulted in decrease of the acidity of the phosphate moiety by approximately one log unit, and increase of stability of Zn(2+)-complexes of the latter analogues by up to 0.7 log units. A terminal phosphorothioate contributed more to the stability of nucleotide-Zn(2+) complexes than a bridging phosphorothioate.

Catalytic and regulatory roles of species involved in metal–nucleotide equilibriums in human pyridoxal kinase

Pyridoxal 5'-phosphate is the active form of vitamin B6 and its deficiency is directly related with several human disorders, which make human pyridoxal kinase (hPLK) an important pharmacologic target. In spite of this, a carefully kinetic characterization of hPLK including the main species that regulates the enzymatic activity is at date missing. Here we analyse the catalytic and regulatory mechanisms of hPLK as a function of a precise determination of the species involved in metal-nucleotide equilibriums and describe new regulatory mechanisms for this enzyme. hPLK activity is supported by several metals, being Zn(2+) the most effective, although the magnitude of the effect observed is highly dependent on the relative concentrations of metal and nucleotide used. The true substrate for the reaction catalyzed by hPLK is the metal nucleotide complex, while ATP(4-) and HATP(3-) did not affect the activity. The enzyme presents substrate inhibition by both pyridoxal (PL) and ZnATP(2-), although the latter behaves as a weakly inhibitor. Our study also established, for the first time, a dual role for free Zn(2+); as an activator at low concentrations (19 μM optimal concentration) and as a potent inhibitor with a IC50 of 37 μM. These results highlighted the importance of an accurate estimation of the actual concentration of the species involved in metal-nucleotide equilibriums in order to obtain reliable values for the kinetic parameters, and for determine the true regulators of the PLK activity. They also help to explain the dissimilar kinetic parameters reported in the literature for this enzyme.

Revisiting the Nucleotide and Aminoglycoside Substrate Specificity of the Bifunctional Aminoglycoside Acetyltransferase(6′)-Ie/Aminoglycoside Phosphotransferase(2″)-Ia Enzyme

The bifunctional aminoglycoside-modifying enzyme aminoglycoside acetyltransferase(6')-Ie/aminoglycoside phosphotransferase(2″)-Ia, or AAC(6')-Ie/APH(2″)-Ia, is the major source of aminoglycoside resistance in gram-positive bacterial pathogens. In previous studies, using ATP as the cosubstrate, it was reported that the APH(2″)-Ia domain of this enzyme is unique among aminoglycoside phosphotransferases, having the ability to inactivate an unusually broad spectrum of aminoglycosides, including 4,6- and 4,5-disubstituted and atypical. We recently demonstrated that GTP, and not ATP, is the preferred cosubstrate of this enzyme. We now show, using competition assays between ATP and GTP, that GTP is the exclusive phosphate donor at intracellular nucleotide levels. In light of these findings, we reevaluated the substrate profile of the phosphotransferase domain of this clinically important enzyme. Steady-state kinetic characterization using the phosphate donor GTP demonstrates that AAC(6')-Ie/APH(2″)-Ia phosphorylates 4,6-disubstituted aminoglycosides with high efficiency (k(cat)/K(m) = 10(5)-10(7) M(-1) s(-1)). Despite this proficiency, no resistance is conferred to some of these antibiotics by the enzyme in vivo. We now show that phosphorylation of 4,5-disubstituted and atypical aminoglycosides are negligible and thus these antibiotics are not substrates. Instead, these aminoglycosides tend to stimulate an intrinsic GTPase activity of the enzyme. Taken together, our data show that the bifunctional enzyme efficiently phosphorylates only 4,6-disubstituted antibiotics; however, phosphorylation does not necessarily result in bacterial resistance. Hence, the APH(2″)-Ia domain of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme is a bona fide GTP-dependent kinase with a narrow substrate profile, including only 4,6-disubstituted aminoglycosides.

Mn2+–Nucleotide Coordination at the Myosin Active Site As Detected by Pulsed Electron Paramagnetic Resonance

Pulsed electron paramagnetic resonance at the microwave K(a) band (~30 GHz) was used to study the coordination of adenosine nucleotides to Mn(2+) at the active site of myosin ATPase and in solution. We have found that the electron spin echo (ESE) field sweep, electron-nuclear double resonance (ENDOR) and ESE envelope modulation (ESEEM) techniques are not sufficiently specific for reliable differentiation between the solvated and myosin-bound Mn·nucleotide complexes. Therefore, to directly detect binding of the Mn·nucleotide to myosin, we used nonhydrolizable nucleotide analogs, site-directed spin labeling, and pulsed electron-electron double resonance to detect spin probe-manganese dipolar interaction. We found that under substoichiometric conditions, both Mn·AMPPNP and Mn·ADP·AlF(4) form a complex with myosin, and Mn·ADP does not form such a complex. This correlates well with the biological dissociation of Mg·ADP from myosin after the hydrolysis of ATP. The analysis of (31)P ENDOR spectra reveals that in Mn·AMPPNP, Mn·ATP, and Mn·ADP at myosin or in solution, the nucleotide is coordinated to Mn(2+) by two phosphate groups, whereas in Mn·ADP·AlF(4), only one phosphate group is coordinated. The observation of two phosphates and one nitrogen in the coordination sphere of Mn·ADP in solution by ESEEM spectroscopy suggests that a significant population of Mn ions is coordinated by two ADP molecules, one of which is coordinated by phosphates, and the other one, by a nitrogen atom. The developed approach will be generally useful for monitoring the metal-protein binding when such binding does not provide reliable spectroscopic signatures.

Thermodynamic and Mechanistic Insights into Translesion DNA Synthesis Catalyzed by Y‐Family DNA Polymerase Across a Bulky Double‐Base Lesion of an Antitumor Platinum Drug

To determine how the Y-family translesion DNA polymerase η (Polη) processes lesions remains fundamental to understanding the molecular origins of the mutagenic translesion bypass. We utilized model systems employing a DNA double-base lesion derived from 1,2-GG intrastrand cross-links of a new antitumor Pt(II) complex containing a bulky carrier ligand, namely [PtCl(2)(cis-1,4-dach)] (DACH=diaminocyclohexane). The catalytic efficiency of Polη for the insertion of correct dCTP, with respect to the other incorrect nucleotides, opposite the 1,2-GG cross-link was markedly reduced by the DACH carrier ligand. This reduced efficiency of Polη to incorporate the correct dCTP could be due to a more extensive DNA unstacking and deformation of the minor groove induced in the DNA by the cross-link of bulky [PtCl(2)(cis-1,4-dach)]. The major products of the bypass of this double-base lesion produced by [PtCl(2)(cis-1,4-dach)] by Polη resulted from misincorporation of dATP opposite the platinated G residues. The results of the present work support the thesis that this misincorporation could be due to sterical effects of the bulkier 1,4-DACH ligand hindering the formation of the Polη-DNA-incoming nucleotide complex. Calorimetric analysis suggested that thermodynamic factors may contribute to the forces that governed enhanced incorporation of the incorrect dATP by Polη as well.

Structure of a topoisomerase II–DNA–nucleotide complex reveals a new control mechanism for ATPase activity

Type IIA topoisomerases control DNA supercoiling and disentangle chromosomes by a complex, ATP-dependent strand passage mechanism. Although a general framework exists for type IIA topoisomerase function, the architecture of the full-length enzyme has remained undefined. Here we present the first structure of a fully-catalytic Saccharomyces cerevisiae topoisomerase II homodimer, complexed with DNA and a nonhydrolyzable ATP analog. The enzyme adopts a domain-swapped configuration wherein the ATPase domain of one protomer sits atop the nucleolytic region of its partner subunit. This organization produces an unexpected interaction between the bound DNA and a conformational transducing element in the ATPase domain, which we show is critical for both DNA-stimulated ATP hydrolysis and global topoisomerase activity. Our data indicate that the ATPase domains pivot about each other to ensure unidirectional strand passage and that this state senses bound DNA to promote ATP turnover and enzyme reset.

Coordination Tendency of Some Biologically Important Zwitterionic Buffers toward Metal Ion Nucleotide Complexes at Different Temperatures

The formation of binary and ternary complexes of Cu(II), Ni(II), Co(II), Mn(II), Zn(II), Ca(II), Mg(II), and Pd(II) with 4-morpholinoethane sulfonic acid (MES), 2-(cyclohexylamino)ethane sulfonic a...

Nucleotide recognition by the initiation factor aIF5B: Free energy simulations of a neoclassical GTPase

The GTPase aIF5B is a universally conserved initiation factor that assists ribosome assembly. Crystal structures of its nucleotide complexes, X-ray(GTP) and X-ray(GDP), are similar in the nucleotide vicinity, but differ in the orientation of a distant domain IV. This has led to two, contradictory, mechanistic models. One postulates that X-ray(GTP) and X-ray(GDP) are, respectively, the active, "ON" and the inactive, "OFF" states; the other postulates that both structures are OFF, whereas the ON state is still uncharacterized. We study GTP/GDP binding using molecular dynamics and a continuum electrostatic free energy method. We predict that X-ray(GTP) has a ≈ 3 kcal/mol preference to bind GDP, apparently contradicting its assignment as ON. However, the preference arises mainly from a single, nearby residue from the switch 2 motif: Glu81, which becomes protonated upon GTP binding, with a free energy cost of about 4 kcal/mol. We then propose a different model, where Glu81 protonation/deprotonation defines the ON/OFF states. With this model, the X-ray(GTP):GTP complex, with its protonated Glu81, is ON, whereas X-ray(GTP):GDP is OFF. The model postulates that distant conformational changes such as domain IV rotation are "uncoupled" from GTP/GDP exchange and do not affect the relative GTP/GDP binding affinities. We analyze the model using a general thermodynamic framework for GTPases. It yields rather precise predictions for the nucleotide specificities of each state, and the state specificities of each nucleotide, which are roughly comparable to the homologues IF2 and aIF2, despite the lack of any conformational switching in the model.