Zinc stoichiometry in Escherichia coli alkaline phosphatase. Studies by 31P NMR and ion-exchange chromatography.

31P nuclear magnetic resonance spectra and enzymatic activities are compared for alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) species with different zinc contents. The enzyme containing two Zn2+ per protein dimer exists in two forms; one, prepared by dialysis of native enzyme, has full enzymatic activity and a 31P magnetic resonance spectrum similar to but distinguishable from that of the native enzyme containing four or more Zn2+. The other form, prepared by restoring two Zn2+ to apoenzyme, has low enzymatic activity and a 31P magnetic resonance spectrum that indicates stoichiometric binding of phosphate, but otherwise altered properties. Reconstituted enzyme with four Zn2+ is similar to but distinguishable from native enzyme with four Zn2+. Chromatography on DEAE-cellulose can separate apoenzyme and enzyme containing two Zn2+ and suggests that the binding of a pair of Zn2+ is cooperative.     

Aminoacyl-tRNA synthetases from Bacillus stearothermophilus. Asymmetry of substrate binding to tyrosyl-tRNA synthetase.

The interaction of L-tyrosine, L-tyrosyladenylate and tRNA-Tyr with tyrosyl-tRNA synthetase from Bacillus stearothermophilus was studied by equilibrium dialysis, gel filtration and fluorescence spectroscopy. The enzyme, which consists of two identical subunits (mol. wt 2 x 44000), binds only a single molecule of L-tyrosine per dimer with a K-d of 2 x 10-5 M at pH 7.8 and 23 degrees C. The tyrosyl-tRNA synthetase--tyrosyladenylate complex which was isolated by gel filtration also has one adenylate bound per dimeric enzyme molecule. In contrast, two tRNA-Tyr molecules bind per enzyme dimer, but the two binding sites are not equivalent having K-d values of 2 x 10-7 M and 1.3 x 10-6 M respectively at pH 6.5 and 25 degrees C. Since crystallographic analysis of the free enzyme [2] shows that the monomer is the asymmetric unit, the data indicate that substrate binding induces asymmetry in the enzyme.     

Bovine kidney alkaline phosphatase. Purification, subunit structure, and metalloenzyme properties.

Kidney alkaline phosphatase was purified to homogeneity. It is a glycoprotein of about 172,000 molecular weight. Analyses of the subunit structure by sedimentation equilibrium in 6 M guanidine hydrochloride and by gel electrophoresis in sodium dodecyl sulfate indicate that the alkaline phosphatase is a dimer comprising two very similar or identical subunits of about 87,000 molecular weight. The native enzyme contains 4.5 +/- 0.2 g atoms of zinc per mol of protein. Reconstitution experiments from the apophosphatase show that binding of 4 Zn2+ per mol of dimer is essential for full activity. The kinetic data of Zn2+ binding to the apoprotein require at least a two-step mechanism, in which one of the steps corresponds to a conformational change within the enzyme. This paper also presents data concerning amino acid composition, sugar content, enzyme stability, absorbance index, and sedimentation velocity.     

Sequence-specific DNA recognition by basic peptides.

A chiral template with C2 symmetry has been used for modeling a dimeric interface of DNA binding protein. An oligopeptide derived from the basic region of MyoD, a recently described "helix-loop-helix" class of DNA binding protein, has been tethered to the template. Among the four models which differ in chirality and polarity with respect to the arrangement of two subunits, only one dimer model with right-handed and C-terminus to C-terminus arrangement of the peptide subunits binds DNA containing native MyoD binding sequence.     

Subunit interactions in liver alcohol dehydrogenase: A partial alkylation study.

The transient kinetics of aldehyde reduction by NADH catalyzed by liver alcohol dehydrogenase consist of two kinetic processes. This biphasic rate behavior is consistent with a model in which one of the two identical subunits in the enzyme is inactive during the reaction at the adjacent protomer. Alternatively, enzyme heterogeneity could result in such biphasic behavior. We have prepared liver alcohol dehydrogenase containing a single major isozyme; and the transient kinetics of this purified enzyme are biphasic.Addition of two [¹⁴C]carboxymethyl groups per dimer to the two “reactive” sulfhydryl groups (Cys46) yields enzyme which is catalytically inactive toward alcohol oxidation. Alkylated enzyme, as initially isolated by gel filtration chromatography at pH 7·5, forms an NAD⁺-pyrazole complex. However, the ability to bind NAD⁺-pyrazole is rapidly lost in pH 8·75 buffer; therefore, our alkylated preparations, as isolated by chromatography at pH 8·75, are inactive toward NAD⁺-pyrazole complex formation. We have prepared partially inactivated enzyme by allowing iodoacetic acid to react with liver alcohol dehydrogenase until 50% of the NAD⁺-pyrazole binding capacity remains; under these reaction conditions one [¹⁴C]carboxymethyl group is added per dimer. This partially alkylated enzyme preparation is isolated by gel filtration and has been aged sufficiently to lose NAD⁺-pyrazole binding ability at alkylated subunits. When solutions of native liver alcohol dehydrogenase and partially alkylated liver alcohol dehydrogenase containing the same number of unmodified active sites are allowed to react with substrate under single turnover conditions, partially alkylated enzyme is only half as reactive as native enzyme. This indicates that some molecular species in partially alkylated liver alcohol dehydrogenase that react with pyrazole and NAD⁺ during the active site titration do not react with substrate. These data are consistent with a model in which a subunit adjacent to an alkylated protomer in the dimeric enzyme is inactive toward substrate. In addition, NAD⁺-pyrazole binding at the protomers adjacent to alkylated subunits is slowly lost so that 75% of the enzyme-NAD⁺-pyrazole binding capacity is lost in 50% alkylated enzyme. These data supply strong evidence for subunit interactions in liver alcohol dehydrogenase.Binding experiments performed on partially alkylated liver alcohol dehydrogenase indicate that coenzyme binding is normal at a subunit adjacent to an alkylated protomer even though active ternary complexes cannot be formed. One hypothesis consistent with these results is the unavailability of zinc for substrate binding at the active site in subunits adjacent to alkylated protomers in monoalkylated dimer.     

Brain pyridoxal kinase dissociation of the dimeric structure and catalytic activity of the monomeric species

Reversible dissociation of the dimeric structure of brain pyridoxal kinase into subunits was attained by addition of guanidinium HCl (2 M). The molecular mass of the subunits (40 kDa) was determined by HPLC chromatography. Separation of the processes of refolding and association of the monomeric species was achieved by attaching the protein subunits to a rigid matrix (Affi-gel 15). The matrix-bound monomer is catalytically competent. The reaction of the crosslinking reagent 4,4'-dimaleimidestilbene 2,2'-disulfonate (DMDS), a derivatized stilbene, with the dimeric structure of pyridoxal kinase resulted in the formation of an oligomeric species of 80 kDa detectable by SDS-PAGE. The crosslinked subunits exhibit the same catalytic parameters as the native enzyme. The presence of two nucleotide-binding sites per dimer was determined by fluorimetric titrations using pyridoxyl-ATP, a strong competitive inhibitor with respect to ATP. The ATP analog binds with a Kd = 5 microM to each nucleotide site of the dimeric enzyme. The mode of binding pyridoxyl-ATP to the kinase is discussed in reference to a model which assumes the presence of two binding domains per subunit.     

Direct evidence for the localization of the steroid-binding site of the plasma sex steroid-binding protein (SBP or SHBG) at the interface between the subunits.

Complete dissociation of dimeric plasma sex steroid-binding protein (SBP or SHBG) was obtained in 6 M urea at 10 degrees C. Removal of urea resulted in the refolding of monomers, followed by reformation of dimeric SBP, which migrates with the same mobility as the native protein. Dimerization does not require Ca+2 or steroid. Renatured monomers yield dimers with dissociation constants for 5 alpha-dihydrotesterone (DHT) and 17 beta-estradiol (E2) indistinguishable from those of native human SBP. This phenomenon was also demonstrated by mixing human and rabbit SBPs that, upon renaturation, form a hybrid dimer composed of one human subunit and one rabbit subunit. The hybrid binds both DHT and E2 in contrast to rSBP, which only binds the androgen. Therefore, we conclude that (1) docking of the two subunits creates an asymmetric steroid-binding site located at the interface between the subunits, and (2) only one face of the dimer defines the specificity for binding E2 by encompassing portion of a structural motif that recognizes the flat ring A of E2. The remaining portion, which recognizes the saturated ring A of DHT, is shared by both faces of the dimer. Because native monomers do not exist alone, the often-asked question of whether the SBP monomer binds steroid can be considered meaningless; steroid-binding activity is expressed only in the dimeric state. Finally, formation of the hybrid indicates that SBP dimerization represents a conserved event during the molecular evolution of SBP, suggesting that the structural elements responsible for dimerization will be homologous in SBPs from other species.     

Crystal structure of a dimeric form of streptococcal pyrogenic exotoxin A (SpeA1)

Streptococcal pyrogenic exotoxin A (SpeA1) is a bacterial superantigen associated with scarlet fever and streptococcal toxic shock syndrome (STSS). SpeA1 is found in both monomeric and dimeric forms, and previous work suggested that the dimer results from an intermolecular disulfide bond between the cysteines at positions 90 of each monomer. Here, we present the crystal structure of the dimeric form of SpeA1. The toxin crystallizes in the orthorhombic space group P212121, with two dimers in the crystallographic asymmetric unit. The final structure has a crystallographic R-factor of 21.52% for 7248 protein atoms, 136 water molecules, and 4 zinc atoms (one zinc atom per molecule). The implications of SpeA1 dimer on MHC class II and T-cell receptor recognition are discussed.     

Activation of a covalent outer membrane phospholipase A dimer.

The activity of outer membrane phospholipase A (OMPLA) is regulated by reversible dimerization. However, native OMPLA reconstituted in phospholipid vesicles was found to be present as a dimer but nevertheless inactive. To investigate the importance of dimerization for control of OMPLA activity, a covalent OMPLA dimer was constructed and its properties were compared to native OMPLA both in a micellar detergent and after reconstitution in a phospholipid bilayer. Unlike native OMPLA, activity of the covalent OMPLA dimer was independent of type and concentration of detergent in micellar systems. In such systems, the covalent OMPLA dimer invariantly displayed high calcium affinity. In contrast, high calcium concentrations were required to activate a covalent OMPLA dimer when present in intact vesicles. Solubilization of the vesicles increased the affinity for calcium, suggesting that in an intact bilayer the dimer interface is not properly formed. This was supported by the observation that OMPLA variants having an impaired dimeric interface also lacked high affinity calcium binding. A covalent linkage was not able to restore high affinity calcium binding in these variants, demonstrating that a proper dimer interface is essential for optimal catalysis.     

Yeast glyceraldehyde-3-phosphate dehydrogenase. Evidence that subunit cooperativity in catalysis can be controlled by the formation of a complex with phosphoglycerate kinase

Sepharose-bound tetrameric, dimeric and monomeric forms of yeast glyceraldehyde-3-phosphate dehydrogenase were prepared, as well as immobilized hybrid species containing (by selective oxidation of an active center cysteine residue with H2O2) one inactivated subunit per tetramer or dimer. The catalytic properties of these enzyme forms were compared in the forward reaction (glyceraldehyde-3-phosphate oxidation) and reverse reaction (1,3-bisphosphoglycerate reductive dephosphorylation) under steady-state conditions. In the reaction of glyceraldehyde-3-phosphate oxidation, immobilized monomeric and tetrameric forms exhibited similar specific activities. The hybrid-modified dimer contributed on half of the total activity of a native dimer. The tetramer containing one modified subunit possessed 75% of the activity of an unmodified tetramer. In the reaction of 1,3-bisphosphoglycerate reductive dephosphorylation, the specific activity of the monomeric enzyme species was nearly twice as high as that of the tetramer, suggesting that only one-half of the active centers of the oligomer were acting simultaneously. Subunit cooperativity in catalysis persisted in an isolated dimeric species. The specific activity of a monomer associated with a peroxide-inactivated monomer in a dimer was equal to that of an isolated monomeric species and twice as high as that of a native immobilized dimer. The specific activity of subunits associated with a peroxide-inactivated subunit in a tetramer did not differ from that of a native immobilized tetramer; this indicates that interdimeric interactions are involved in catalytic subunit cooperativity. A complex was formed between the immobilized glyceraldehyde-3-phosphate dehydrogenase and soluble phosphoglycerate kinase. Three monomers of phosphoglycerate kinase were bound per tetramer of the dehydrogenase and one per dimer. Evidence is presented that if the reductive dephosphorylation of 1,3-bisphosphoglycerate proceeds in the phosphoglycerate kinase - glyceraldehyde-3-phosphate dehydrogenase complex, all active sites of the latter enzyme act independently, i.e. subunit cooperativity is abolished.     

The C6 zinc cluster dictates asymmetric binding by HAP1.

Unlike other C6 zinc cluster proteins such as GAL4 and PPR1, HAP1 binds selectively to asymmetric DNA sites containing a direct repeat of two CGG triplets. Here, we show that the HAP1 zinc cluster is solely responsible for asymmetric binding by HAP1. An asymmetric interaction between two zinc clusters of a HAP1 dimer must position the zinc clusters in a directly repeated orientation, and enable them to recognize two CGG triplets in a direct repeat. Further, our data suggest that this asymmetric interaction acts cooperatively with the interaction between dimerization elements to promote HAP1 dimerization, and locks HAP1-DNA complexes in a stable, dimeric conformation.     

Interaction of the glucocorticoid receptor DNA-binding domain with DNA as a dimer is mediated by a short segment of five amino acids

We have previously shown that protein-protein interactions mediate cooperative binding of the glucocorticoid receptor DNA-binding domain to a glucocorticoid response element (Dahlman-Wright, K., Siltala-Roos, H., Carlstedt-Duke, J., and Gustafsson, J.-A. (1990) J. Biol. Chem. 265, 14030-14035). The cooperativity of DNA binding is lost when the distance between the two half-sites constituting a glucocorticoid responsive element is altered or when their relative orientation is changed. We show here that mutations in the responsive element which interfere with cooperative DNA binding by the glucocorticoid receptor DNA-binding domain in vitro also abolish transactivation by the full length glucocorticoid receptor in vivo. We also identify a short segment in the proximity of one of the bound zinc ions that is required for cooperative binding of the glucocorticoid receptor DNA-binding domain to a glucocorticoid response element. We suggest that this segment is involved in dimer formation of the native glucocorticoid receptor and that it is important for correct positioning of the dimeric molecule on the double helix of DNA.     

Mapping of the dimer interface of the Escherichia coli mannitol permease by cysteine cross-linking

A cysteine cross-linking approach was used to identify residues at the dimer interface of the Escherichia coli mannitol permease. This transport protein comprises two cytoplasmic domains and one membrane-embedded C domain per monomer, of which the latter provides the dimer contacts. A series of single-cysteine His-tagged C domains present in the native membrane were subjected to Cu(II)-(1,10-phenanthroline)(3)-catalyzed disulfide formation or cysteine cross-linking with dimaleimides of different length. The engineered cysteines were at the borders of the predicted membrane-spanning alpha-helices. Two residues were found to be located in close proximity of each other and capable of forming a disulfide, while four other locations formed cross-links with the longer dimaleimides. Solubilization of the membranes did only influence the cross-linking behavior at one position (Cys(73)). Mannitol binding only effected the cross-linking of a cysteine at the border of the third transmembrane helix (Cys(134)), indicating that substrate binding does not lead to large rearrangements in the helix packing or to dissociation of the dimer. Upon mannitol binding, the Cys(134) becomes more exposed but the residue is no longer capable of forming a stable disulfide in the dimeric IIC domain. In combination with the recently obtained projection structure of the IIC domain in two-dimensional crystals, a first proposal is made for alpha-helix packing in the mannitol permease.     

Interaction of the dimeric form of retroviral RNA with paromonycin and magnesium as revealed using 2-aminopurine fluorescence

Studying the dimeric RNA structural organization is a step toward the understanding of retroviral genomic RNA dimerization. A kissing loop dimer is rearranged into an extended dimer during maturation of the virus particle. The extended dimer formation may be inhibited by ligands interacting with the RNA kissing loop dimer. A study was made of the interaction of dimeric RNA with paromomycin and magnesium ions. RNA dimers were formed from two hairpin RNAs having complementary sequences in the loop. The structural features of RNA dimers and the influence of the ligands were inferred from the fluorescence of 2-aminopurine (2-AP) incorporated in one of the two RNA hairpin sequences. As dimeric RNA interacted with paromomycin, 2-AP fluorescence increased. The increase was explained by a flipping of the fluorescent base out of the RNA structure. The binding constants and stoichiometry were estimated for dimeric RNA binding with paromomycin. An RNA dimer was found to interact with two paromomycin molecules; the binding constant was approximately the same (about 3 × 10⁵ M⁻¹) for both types of dimers. It was observed that the antibiotic and Mg²⁺ ions compete for binding to the hairpin RNA dimer and that one paromomycin molecule is displaced by one Mg²⁺ ion.     

The regulatory subunit monomer of cAMP-dependent protein kinase retains the salient kinetic properties of the native dimeric subunit

Monomeric regulatory subunit (R) fragments of type II cAMP-dependent protein kinase were compared with the parent dimeric R. The monomeric fragments were generated by either endogenous proteolysis of rabbit muscle R or by trypsin treatment of bovine heart R in the holoenzyme form. During isolation of pure R from rabbit muscle, carboxyl-terminal fragments of Mr = 42,000 (42 K) and Mr = 37,000 by denaturing gels are generated by endogenous proteolysis. Although the autophosphorylation site is retained, the 42 K is not dimeric (as is its native 56 K precursor) but, in contrast to the monomeric 37 K product, actively reassociates with purified catalytic subunit (C). Several lines of evidence indicate a type II R origin of the 42 K. N-terminal sequence analysis of the 42 K shows some homology with known bovine RI, RII, and cGMP-dependent protein kinase sequences. Both cyclic nucleotide-binding sites (two/42 K or 37 K) and the site selectivity of cAMP analogs are retained in the monomeric fragments. When purified bovine heart holoenzyme, which contains a dimeric Mr = 56,000 R (denaturing gel analysis) and two C subunits, is treated with trypsin followed by separation procedures, the product is a fully recovered active enzyme with an unaltered ratio of cAMP binding to catalytic activity. From Mr considerations, the product is a dimer containing one intact C and a proteolyzed R of Mr = 48,000 on denaturing gels. This dimeric enzyme is not significantly different from the parent tetramer in cAMP concentration dependence (Hill constant = 1.63), [3H]cAMP dissociation behavior (both intrasubunit cAMP-binding sites are present), stimulation of [3H]cIMP binding by site-selective cAMP analogs, and synergism between two analogs in kinase activation. The data indicate that 1) proteolytic cleavage of the native R dimer can cause monomerization without appreciably affecting the inhibition of C and 2) essentially all of the cAMP binding cooperativity is an intrasubunit interaction.     

Bovine kidney alkaline phosphatase. Catalytic properties, subunit interactions in the catalytic process, and mechanism of Mg2+ stimulation.

Kidney alkaline phosphatase is an enzyme which requires two types of metals for maximal activity: zinc, which is essential, and magnesium, which is stimulatory. The main features of the Mg2+ stimulation have been analyzed. The stimulation is pH-dependent and is observed mainly between pH 7.5 and 10.5. Mg2+ binding to native alkaline phosphatase is characterized by a dissociation constant of 50 muM at pH 8.5,25 degrees. Binding of Zn2+ is an athermic process. Both the rate constants of association, ka, and of dissociation, kd, have low values. Typical values are 7 M(-1) at pH 8.0, 25 degrees, for ka and 4.10(-4) S(-1) at pH 8.0, 25 degrees, for kd. The on and off processes have high activation energies of 29 kcal mol (-1). Mg2+ can be replaced at its specific site by Mn2+, Co2+, Ni2+, and Zn2+. Zinc binding to the Mg2+ site inhibits the native alkaline phosphatase. Mn2+, Co2+, and Ni2+ also bind to the Mg2+ site with a stimulatory effect which is nearly identic-al with that of Mg2+, Mn2+ is the stimulatory cation which binds most tightly to the Mg2+ site; the dissociation constant of the Mn2+ kidney phosphatase complex is 2 muM at pH 8.5. The stoichiometry of Mn2+ binding has been found to be 1 eq of Mn2+ per mol of dimeric kidney phosphatase. The native enzyme displays absolute half-site reactivity for Mn2+ binding. Mg2+ binding site and the substrate binding sites are distinct sites. The Mg2+ stimulation corresponds to an allosteric effect. Mg2+ binding to its specific sites does not affect substrate recognition, it selectively affects Vmax values. Quenching of the phosphoenzyme formed under steady state conditions with [32P]AMP as a substrate as well as stopped flow analysis of the catalyzed hydrolysis of 2,4-dinitrophenyl phosphate or p-nitrophenyl phosphate have shown that the two active sites of the native and of the Mg2+-stimulated enzyme are not equivalent. Stopped flow analysis indicated that one of the two active sites was phosphorylated very rapidly whereas the other one was phosphorylated much more slowly at pH 4.2. Half of the sites were shown to be reactive at pH 8.0. Quenching experiments have shown that only one of the two sites is phosphorylated at any instant; this result was confirmed by the stopped flow observation of a burst of only 1 mol of nitrophenol per mol of dimeric phosphatase in the pre-steady state hydrolysis of p-nitrophenyl phosphate. The half-of-the-sites reactivity observed for the native and for the Mg2+-stimulated enzyme indicates that the same type of complex, the monophosphorylated complex, accumulates under steady state conditions with both types of enzymes. Mg2+ binding to the native enzyme at pH 8.0 increases considerably the dephosphorylation rate of this monophosphorylated intermediate. A possible mechanism of Mg2+ stimulation is discussed.     

A synthetic peptide mimic of plasma apolipoprotein E that binds the LDL receptor.

Human plasma apolipoprotein E (apoE) is a low density lipoprotein (LDL) receptor ligand. It targets cholesterol-rich lipoproteins to LDL receptors on both hepatic and peripheral cells. The region of apoE responsible for its binding to the LDL receptor has been localized to amino acids 140-160. An apoE 141-155 monomeric peptide and a dimeric 141-155 tandem peptide were synthesized and tested for their inhibition of 125I-LDL degradation by human fibroblasts and human monocytic-like cells, THP-1. The monomer had no activity at 250 microM, but the dimer inhibited 125I-LDL degradation by 50% at 5 microM. The inhibition was specific for the LDL receptor because the dimer did not inhibit the degradation of 125I-acetylated LDL by scavenger receptors expressed by phorbol ester-stimulated THP-1 cells. As reported for native apoE, amino acid substitutions of Lys-143----Ala, Leu-144----Pro, and Arg-150----Ala decreased the inhibitory effectiveness of the dimer. Furthermore, a trimer of the 141-155 sequence had a 20-fold greater inhibitory activity than the dimer. Studies with a radioiodinated dimer indicated that some of the inhibitory activity could be a result of the interaction of the dimer with LDL. However, direct binding of the 125I-dimeric peptide to THP-1 cells was observed as well. This binding was time-dependent, linear with increasing cell number, Ca(2+)- but not Mg(2+)-dependent, saturable, inhibited by lipoproteins, and increased by preculture of the cells in lipoprotein-depleted medium. Therefore, a synthetically prepared dimeric repeat of amino acid residues 141-155 of apoE binds the LDL receptor.     

Interaction of phenosafranine with nucleic acids and model polyphosphates: II. Characterisation of phenosafranine binding to DNA

The binding of phenosafranine (PS) to DNA was studied by a combination of spectroscopic methods (absorption and fluorescence) together with hydrodynamic measurements (sedimentation and viscosity), Analysis of spectroscopic binding curves revealed that the strength of binding of PS to DNA is generally lower than that of proflavine. These measurements enabled recognition of several modes of interaction between PS and native DNA: strong monomer binding prevailing at high DNA phosphate/dye ratios (p) comprising binding outside the DNA helix as well as intercalation; two modes of dimer binding at lower values of p; and probably also weak surface-binding of monomers as p approaches unity. Longer surface-bound aggregates of PS were not detected because of the low tendency of the dye to form aggregates, though the presence of dimeric species distinct frorn pure surface-stacked PS dimer was indicated by various observations. It occurs over a broad range of p values Starting at p ≈110 for ionic strengths 10^?3–10^?1. Thermal denaturation data indicate that this species is bound more strongly than pure surface-bound stacked dimer. Its dimeric character may be explained in terms of interaction of an intercalated dye molecule with an adjacent outside-bound one as suggested for acridines by Armstrong et al. Various properties of this species are discussed. Both strong and weak modes or binding of PS to DNA are sensitive to the presence of organic solvents. The effectiveness of solvents to destabilise the complexes substantially coincides with their capacity to alter the water activity. Viscometric investigations reveal that in the region of strongest bindins (p ? 15) the elongation of the DNA helix by approximately 0.18 nm per bound PS molecule is accompanied by a strong negative change in persistence length, i.e. bending. Similar bending is also found at higher levels of binding (p ? 15) induced by less lightly bound PS molecules, in which region, however, the unusually high elongation of approximately 0.34 nm per bound PS molecule is observed.     

Crystal structure of the tetrameric cytidine deaminase from Bacillus subtilis at 2.0 A resolution

Cytidine deaminases (CDA, EC 3.5.4.5) are zinc-containing enzymes in the pyrimidine salvage pathway that catalyze the formation of uridine and deoxyuridine from cytidine and deoxycytidine, respectively. Two different classes have been identified in the CDA family, a homodimeric form (D-CDA) with two zinc ions per dimer and a homotetrameric form (T-CDA) with four zinc ions per tetramer. We have determined the first structure of a T-CDA from Bacillus subtilis. The active form of T-CDA is assembled of four identical subunits with one active site apiece. The subunit of D-CDA is composed of two domains each exhibiting the same fold as the T-CDA subunits, but only one of them contains zinc in the active site. The similarity results in a conserved structural core in the two CDA forms. An intriguing difference between the two CDA structures is the zinc coordinating residues found at the N-terminal of two alpha-helices: three cysteine residues in the tetrameric form and two cysteine residues and one histidine residue in the dimeric form. The role of the zinc ion is to activate a water molecule and thereby generate a hydroxide ion. How the zinc ion in T-CDA surrounded with three negatively charged residues can create a similar activity of T-CDA compared to D-CDA has been an enigma. However, the structure of T-CDA reveals that the negative charge caused by the three ligands is partly neutralized by (1) an arginine residue hydrogen-bonded to two of the cysteine residues and (2) the dipoles of two alpha-helices.     

Assessing Structural Determinants of Zn2+ Binding to Human HV1 via Multiple MD Simulations

Voltage-gated proton channels (H_V1) are essential for various physiological tasks but are strongly inhibited by Zn²⁺ cations. Some determinants of Zn²⁺ binding have been elucidated experimentally and in computational studies. However, the results have always been interpreted under the assumption that Zn²⁺ binds to monomeric H_V1 despite evidence that H_V1 expresses as a dimer and that the dimer has a higher affinity for zinc than the monomer and experimental data that suggest coordination in the dimer interface. The results of former studies are also controversial, e.g., supporting either one single or two binding sites. Some structural determinants of the binding are still elusive. We performed a series of molecular dynamics simulations to address different structures of the human proton channel, the monomer and two plausible dimer conformations, to compare their respective potential to interact with and bind Zn²⁺ via the essential histidines. The series consisted of several copies of the system to generate independent trajectories and increase the significance compared to a single simulation. The amount of time simulated totals 29.9 μs for 126 simulations of systems comprising ∼59,000 to ∼187,000 atoms. Our approach confirms the existence of two binding sites in monomeric and dimeric human H_V1. The dimer interface is more efficient for attracting and binding Zn²⁺ via the essential histidines than the monomer or a dimer with the histidines in the periphery. The higher affinity is due to the residues in the dimer interface that create an attractive electrostatic potential funneling the zinc cations toward the binding sites.