The crystal structure of cobalt-substituted pseudoazurin from Alcaligenes faecalis

The Cu(II) center at the active site of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by Co(II) via denaturing of the protein, chelation and removal of copper by EDTA and refolding of the apo‐protein, followed by addition of an aqueous solution of CoCl₂. Sitting drop vapour diffusion experiments produced green hexagonal crystals, which belong to space group P6₅, with unit cell dimensions a = b = 50.03, c = 98.80 Å. Diffraction data, collected at 291 K on a copper rotating anode X‐ray source, were phased by the anomalous signal of the cobalt atom. The structure was built automatically, fitted manually and subsequently refined to 1.86 Å resolution. The Co‐substituted protein exhibits similar overall geometry to the native structure with copper. Cobalt binds more strongly to the axial Met86‐Sδ and retains the tetrahedral arrangement with the four ligand atoms, His40‐Nδ₁, Cys78‐Sγ, His81‐Nδ₁, and 86Met‐Sδ, although the structure is less distorted than the native copper protein. The structure reported herein, is the first crystallographic structure of a Co(II)‐substituted pseudoazurin. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 202–207, 2011.     

Comparison of the backbone dynamics of the apo- and holo-carboxy-terminal domain of the biotin carboxyl carrier subunit of Escherichia coli acetyl-CoA carboxylase

The biotin carboxyl carrier protein (BCCP) is a subunit of acetyl-CoA carboxylase, a biotin-dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis. In its functional cycle, this protein engages in heterologous protein-protein interactions with three distinct partners, depending on its state of post-translational modification. Apo-BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the post-translational attachment of biotin to a single lysine residue on BCCP. Holo-BCCP then interacts with the biotin carboxylase subunit of acetyl-CoA carboxylase, which leads to the addition of the carboxylate group of bicarbonate to biotin. Finally, the carboxy-biotinylated form of BCCP interacts with transcarboxylase in the transfer of the carboxylate to acetyl-CoA to form malonyl-CoA. The determinants of protein-protein interaction specificity in this system are unknown. The NMR solution structure of the unbiotinylated form of an 87 residue C-terminal domain fragment (residue 70-156) of BCCP (holoBCCP87) and the crystal structure of the biotinylated form of a C-terminal fragment (residue 77-156) of BCCP from Escherichia coli acetyl-CoA carboxylase have previously been determined. Comparative analysis of these structures provided evidence for small, localized conformational changes in the biotin-binding region upon biotinylation of the protein. These structural changes may be important for regulating specific protein-protein interactions. Since the dynamic properties of proteins are correlated with local structural environments, we have determined the relaxation parameters of the backbone 15N nuclear spins of holoBCCP87, and compared these with the data obtained for the apo protein. The results indicate that upon biotinylation, the inherent mobility of the biotin-binding region and the protruding thumb, with which the biotin group interacts in the holo protein, are significantly reduced.     

A comparative investigation of the thermal unfolding of pseudoazurin in the Cu(II)-holo and apo form

The contribution of the copper ion to the stability and to the unfolding pathway of pseudoazurin was investigated by a comparative analysis of the thermal unfolding of the Cu(II)-holo and apo form of the protein. The unfolding has been followed by calorimetry, fluorescence, optical density, and electron paramagnetic resonance (EPR) spectroscopy. The thermal transition of Cu(II)-holo pseudoazurin is irreversible and occurs between 60.0 and 67.3 degrees C, depending on the scan rate and technique used. The denaturation pathway of Cu(II)-holo pseudoazurin can be described by the Lumry-Eyring model: N --> U --> [corrected] F; the protein reversibly goes from the native (N) to the unfolded (U) state, and then irreversibly to the final (F) state. The simulation of the experimental calorimetric profiles, according to this model, allowed us to determine the thermodynamic and kinetic parameters of the two steps. The DeltaG value calculated for the Cu(II)-holo pseudoazurin is 39.2 kJ.mol(-1) at 25 degrees C. The sequence of events in the denaturation process of Cu(II)-holo pseudoazurin emergence starts with the disruption of the copper site and the hydrophobic core destabilization followed by the global protein unfolding. According to the EPR findings, the native type-1 copper ion shows type-2 copper features after the denaturation. The removal of the copper ion (apo form) significantly reduces the stability of the protein as evidenced by a DeltaG value of 16.5 kJ.mol(-1) at 25 degrees C. Moreover, the apo Paz unfolding occurs at 41.8 degrees C and is compatible with a two-state reversible process N --> [corrected] U.     

The tertiary structure and backbone dynamics of human prolactin

Human prolactin is a 199-residue (23 kDa) protein closely related to growth hormone and placental lactogen with properties and functions resembling both a hormone and a cytokine. As a traditional hormone, prolactin is produced by lactotrophic cells in the pituitary and secreted into the bloodstream where it acts distally to regulate reproduction and promote lactation. Pituitary cells store prolactin in secretory granules organized around large prolactin aggregates, which are produced within the trans layer of the Golgi complex. Extrapituitary prolactin is synthesized by a wide variety of cells but is not stored in secretory granules. Extrapituitary prolactin displays immunomodulatory activities and acts as a growth factor for cancers of the breast, prostate and tissues of the female reproductive system. We have determined the tertiary structure of human prolactin using three-dimensional (3D) and four-dimensional (4D) heteronuclear NMR spectroscopy. As expected, prolactin adopts an "up-up-down-down" four-helical bundle topology and resembles other members of the family of hematopoietic cytokines. Prolactin displays three discrete structural differences from growth hormone: (1) a structured N-terminal loop in contact with the first helix, (2) a missing mini-helix in the loop between the first and second helices, and (3) a shorter loop between the second and third helices lacking the perpendicular mini-helix observed in growth hormone. Residues necessary for functional binding to the prolactin receptor are clustered on the prolactin surface in a position similar to growth hormone. The backbone dynamics of prolactin were investigated by analysis of 15N NMR relaxation phenomena and demonstrated a rigid four-helical bundle with relatively mobile interconnecting loops. Comparison of global macromolecular tumbling at 0.1mM and 1.0mM prolactin revealed reversible oligomerization, which was correlated to dynamic light scattering experiments. The existence of a reversible oligomerization reaction in solution provides insight into previous results describing the in vitro and in vivo aggregation properties of human prolactin.     

Crystallization and preliminary X-ray analysis of electron transfer flavoproteins from human and Paracoccus denitrificans.

Mammalian electron transfer flavoprotein (ETF) is a soluble, heterodimeric flavoprotein responsible for the oxidation of at least nine primary matrix flavoprotein dehydrogenases. Crystals have been obtained for the recombinant human electron transfer flavoprotein (ETFhum) by the sitting-drop vapor diffusion technique using polyethylene glycol (PEG) 1500 at pH 7.0 as the precipitating agent. ETFhum crystallizes in the monoclinic space group P2(1), with unit cell parameters a = 47.46 angstrum, b = 104.10 angstrum, c = 63.79 angstrum, and beta = 110.02 degrees. Based on the assumption of one alpha beta dimer per asymmetric unit, the Vm value is 2.69 angstrum 3/Da. A native data set has been collected to 2.1 angstrum resolution. One heavy-atom derivative has also been obtained by soaking a preformed crystal of ETFhum in 2 mM thimerosal solution for 2h at 19 degrees C. Patterson analysis indicates one major site. The analogous electron transfer flavoprotein from Paracoccus denitrificans (ETFpar) has also been crystallized using PEG 8000 at pH 5.5 as the precipitating agent. ETFpar crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 79.98 angstrum, b = 182.90 angstrum, and c = 70.07 angstrum. The Vm value of 2.33 angstrum 3/Da is consistent with two alpha beta dimers per asymmetric unit. A native data set has been collected to 2.5 angstrum resolution.     

The solution structure and dynamics of the DH‐PH module of PDZRhoGEF in isolation and in complex with nucleotide‐free RhoA

The DH‐PH domain tandems of Dbl‐homology guanine nucleotide exchange factors catalyze the exchange of GTP for GDP in Rho‐family GTPases, and thus initiate a wide variety of cellular signaling cascades. Although several crystal structures of complexes of DH‐PH tandems with cognate, nucleotide free Rho GTPases are known, they provide limited information about the dynamics of the complex and it is not clear how accurately they represent the structures in solution. We used a complementary combination of nuclear magnetic resonance (NMR), small‐angle X‐ray scattering (SAXS), and hydrogen‐deuterium exchange mass spectrometry (DXMS) to study the solution structure and dynamics of the DH‐PH tandem of RhoA‐specific exchange factor PDZRhoGEF, both in isolation and in complex with nucleotide free RhoA. We show that in solution the DH‐PH tandem behaves as a rigid entity and that the mutual disposition of the DH and PH domains remains identical within experimental error to that seen in the crystal structure of the complex, thus validating the latter as an accurate model of the complex in vivo. We also show that the nucleotide‐free RhoA exhibits elevated dynamics when in complex with DH‐PH, a phenomenon not observed in the crystal structure, presumably due to the restraining effects of crystal contacts. The complex is readily and rapidly dissociated in the presence of both GDP and GTP nucleotides, with no evidence of intermediate ternary complexes.     

The TpG chelate of cis(diammineplatinum) forms two head-to-head rotamers in H2O solution

 d(TpG)^– reacts with cis-[Pt(NH₃)₂(H₂O)₂]²⁺ in two steps to yield the platinum chelate cis-[Pt(NH₃)₂{d(TpG)-N3(1),N7(2)}]. In the latter, hindered rotation of the bases leads to an equilibrium between two rotamers interconverting slowly on the NMR time scale. The structure of the two rotameric chelates was studied by means of ¹H NMR and molecular modeling techniques. The major and minor rotamers could be assigned unambiguously to the two head-to-head conformational domains which are characterized by syn/anti and anti/anti sugar-base orientations, respectively. Molecular models derived for both rotamers show that the orientations of the bases are mutually quasi-enantiomeric. The interconversion between the two rotamers (k ≈ 1 s^–1 at 293 K) is approximately 10⁴ times faster than the analogous rotamer interconversion observed in cis-[Pt(NH₃)₂{r(CpG)-N3(1),N7(2)}]⁺ [Girault J-P, Chottard G, Lallemand J-Y, Huguenin F, Chottard J-C (1984) J Am Chem Soc 106 : 7227–7232], suggesting that the steric clash of the exocyclic amino group of the platinum-bound cytosine with the ligands in cis position is more severe than that of the two thymine oxo groups. Received: 23 June 1997 / Accepted: 30 September 1997     

The solution structure of the S.cerevisiae Ste11 MAPKKK SAM domain and its partnership with Ste50

Ste11 is a MAPKKK from Saccharomyces cerevisiae that helps mediate the response to mating pheromone and the ability to thrive in high-salt environments. These diverse functions are facilitated by a direct interaction between the SAM domain of Ste11 with the SAM domain of its regulatory partner, Ste50. We have solved the NMR structure of the Ste11 SAM domain (PDB 1OW5), which reveals a compact, five alpha-helix bundle and a high degree of structural similarity to the Polyhomeotic SAM domain. The combined study of Ste11 SAM rotational correlation times and crosslinking to Ste50-SAM has suggested a mode through which Ste11-SAM oligomerizes and selectively associates with Ste50-SAM. To probe homotypic and heterotypic interations, Ste11-SAM variants each containing a substitution of a surface-exposed hydrophobic residue were constructed. An I59R variant of Ste11-SAM, disrupted binding to Ste50-SAM in vitro. Yeast expressing full-length Ste11-I59R could neither respond to mating pheromone nor thrive in high salt media-demonstrating that the interaction between Ste11 and Ste50 SAM domains is a prerequisite for key signal transduction events.     

The hydration of amides in helices; a comprehensive picture from molecular dynamics,IR, and NMR

We examined the hydration of amides of alpha(3)D, a simple, designed three-helix bundle protein. Molecular dynamics calculations show that the amide carbonyls on the surface of the protein tilt away from the helical axis to interact with solvent water, resulting in a lengthening of the hydrogen bonds on this face of the helix. Water molecules are bonded to these carbonyl groups with partial occupancy ( approximately 50%-70%), and their interaction geometries show a large variation in their hydrogen bond lengths and angles on the nsec time scale. This heterogeneity is reflected in the carbonyl stretching vibration (amide I' band) of a group of surface Ala residues. The surface-exposed amides are broad, and shift to lower frequency (reflecting strengthening of the hydrogen bonds) as the temperature is decreased. By contrast, the amide I' bands of the buried (13)C-labeled Leu residues are significantly sharper and their frequencies are consistent with the formation of strong hydrogen bonds, independent of temperature. The rates of hydrogen-deuterium exchange and the proton NMR chemical shifts of the helical amide groups also depend on environment. The partial occupancy of the hydration sites on the surface of helices suggests that the interaction is relatively weak, on the order of thermal energy at room temperature. One unexpected feature that emerged from the dynamics calculations was that a Thr side chain subtly disrupted the helical geometry 4-7 residues N-terminal in sequence, which was reflected in the proton chemical shifts and the rates of amide proton exchange for several amides that engage in a mixed 3(10)/alpha/pi-helical conformation.     

Conformation and dynamics of the three-helix bundle UBA domain of p62 from experiment and simulation

The ubiquitin associated domain of p62 is a small three-helix bundle of approximately 50 residues that mediates the recognition of polyubiquitin chains and ubiquitylated substrates. The solution structure of a 52 residue construct containing this domain has been characterized using heteronuclear nuclear magnetic resonance (NMR) methods. The resulting ensemble of NMR-derived structures was used in molecular dynamics (MD) simulations to investigate the equilibrium conformation and dynamics of this domain. NOE and (15)N relaxation data have been used to validate the structural ensemble produced by the MD simulations and show a good correlation for residues in regions of secondary structure. A similar approach was taken using an ensemble of structures from the MD simulations to calculate electronic circular dichroism (CD) and IR spectra from first principles with an encouraging correlation with the experimental CD and IR data.     

An inserted Gly residue fine tunes dynamics between mesophilic and thermophilic ribonucleases H

Dynamic processes are inherent properties of proteins and are crucial for a wide range of biological functions. To address how changes in protein sequence and structure affect dynamic processes, a quantitative comparison of microsecond-to-microsecond time scale conformational changes, measured by solution NMR spectroscopy, within homologous mesophilic and thermophilic ribonuclease H (RNase H) enzymes is presented. Kinetic transitions between the observed major state (high population) and alternate (low population) conformational state(s) of the substrate-binding handle region in RNase H from the mesophile Escherichia coli (ecRNH) and thermophile Thermus thermophilus (ttRNH) occur with similar kinetic exchange rate constants, but the difference in stability between exchanging conformers is smaller in ttRNH compared to ecRNH. The altered thermodynamic equilibrium between kinetically exchanging conformers in the thermophile is recapitulated in ecRNH by the insertion of a Gly residue within a putative hinge between alpha-helices B and C. This Gly insertion is conserved among thermophilic RNases H, and allows the formation of additional intrahelical hydrogen bonds. A Gly residue inserted between alpha-helices B and C appears to relieve unfavorable interactions in the transition state and alternate conformer(s) and represents an important adaptation to adjust conformational changes within RNase H for activity at high temperatures.     

Studies on solution NMR structure of brazzein——Secondary structure and molecular scaffold

Brazzein is a sweet-tasting protein isolated from the fruit of West African plantPentadiplandra brazzeana Baillon. It is the smallest and the most water-soluble sweet protein discovered so far and is highly thermostable. The proton NMR study of brazzein at 600 MHz (pH 3.5, 300 K) is presented. The complete sequence specific assignments of the individual backbone and sidechain proton resonances were achieved using through-bond and through-space connectivities obtained from standard two-dimensional NMR techniques. The secondary structure of brazzein contains one alpha-helix (residues 21-29), one short 3(10)-helix (residues 14-17), two strands of antiparallel beta-sheet (residues 34-39, 44-50) and probably a third strand (residues 5-7) near the N-terminus. A comparative analysis found that brazzein shares a so-called 'cysteine-stabilized alpha-beta' (CSalphabeta) motif with scorpion neurotoxins, insect defensins and plant gamma - thionins. The significance of this multi-function motif, the possible active sites and the structural basis of themostability were discussed.     

The Structure of an alternative form of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase

Cytochrome cd(1) nitrite reductase is a bifunctional enzyme, which can catalyze the 1-electron reduction of nitrite to nitric oxide and the 4-electron reduction of dioxygen to water. Here we describe the structure of reduced nitrite reductase, crystallized under anaerobic conditions. The structure reveals substantial domain rearrangements with the c domain rotated by 60 degrees and shifted by approximately 20 A compared with previously known structures from crystals grown under oxidizing conditions. This alternative conformation gives rise to different electron transfer routes between the c and d(1) domains and points to the involvement of elements of very large structural changes in the function in this enzyme. In the present structure, the c heme has a His-69/Met-106 ligation, and this ligation does not change upon oxidation in the crystal. The d(1) heme is penta-coordinated, and the d(1) iron is displaced from the heme plane by 0.5 A toward the proximal ligand, His-200. After oxidation, the iron moves into the d(1) heme plane. A surprising finding is that although reduced nitrite reductase can be readily oxidized by dioxygen in the new crystal, it cannot turnover with its other substrate, nitrite. The results suggest that the rearrangement of the domains affects catalysis and substrate selectivity.     

DNA fine structure and dynamics in crystals and in solution: the impact of BI/BII backbone conformations

Sugar-phosphate backbone conformations are an important structural element for a complete understanding of specific recognition in nucleic acid-protein interactions. They can be involved both in early stages of target discrimination and in structural adaptation upon binding. In the first part of this study, we have analyzed high-resolution structures of double-stranded B-DNA either isolated or bound to proteins, and explored the impact of both the standard BI and the unusual BII phosphate backbone conformations on neighboring sugar puckers and on selected helical parameters. Correlations are found to be similar for free and bound DNA, and in both categories, the possible facing backbone conformations (BI.BI, BI.BII, and BII.BII) define well-characterized substates in the B-DNA conformational space. Notably, BII.BII steps are characterized by specific, and sequence-independent, structural effects involving reduced standard deviations for almost all conformational parameters. In the second part of this work, we analyze four 10 ns molecular dynamics simulations in explicit solvent on the DNA targets of NF-kappaB and bovine papillomavirus E2 proteins, highlighting the multiplicity of backbone dynamical behavior. These results show sequence effects on the percentages of BI and BII conformers, the preferential state of facing backbones, the occurrence of coupled transitions. The backbone states can consequently be seen as a mechanism for transmitting information from the bases to the phosphate groups and thus for modulating the overall structural properties of the target DNA.     

寄生隐丛赤壳菌致病毒素Cp-I的分离纯化和结构分析

寄生隐丛赤壳菌Cryphonectria parasitica菌株经液体培养,石油醚萃取其发酵液获得对板栗带叶嫩枝具有致萎活性的粗提物,以氯仿:石油醚:甲醇(6:2:2)作洗脱剂,粗提物经硅胶色谱分离,共得到3组纯组份,其中第1组份(Cp-I)对板栗幼苗致萎活性较高。质谱、核磁共振和红外光谱测定表明Cp-I分子量为278,化学式为C16H22O4。     

寄生隐丛赤壳菌Cryphonectria parasitica致病毒素Cp-I的分离纯化和结构分析

寄生隐丛赤壳菌Cryphonectria parasitica菌株经液体培养,石油醚萃取其发酵液获得对板栗带叶嫩枝具有致萎活性的粗提物,以氯仿:石油醚:甲醇(6:2:2)作洗脱剂,粗提物经硅胶色谱分离,共得到3组纯组份,其中第1组份(Cp-I)对板栗幼苗致萎活性较高.质谱、核磁共振和红外光谱测定表明Cp-I分子量为278,化学式为C<,16>H<,22>O<,4>.     

The spatial structure of the axially bound methionine in solution conformations of horse ferrocytochrome c and Pseudomonas aeruginosa ferrocytochrome c 551 by 1H NMR

A generally applicable method for the determination of the spatial structure of the heme iron-bound methionine in c-type ferrocytochromes at atomic resolution is presented. It relies primarily on measurements of nuclear Overhauser effects between the individual hydrogen atoms of the axial methionine, and between individual hydrogens of the methionine and the heme group. Four different methionine conformers, corresponding to the four possible stereospecific assignments for the methionine methylene proton resonances, are generated by a structural interpretation of the nuclear Overhauser effects with the use of an interactive computer graphics technique. A unique structure and unique stereospecific resonance assignments are then obtained by discriminating between these four conformers on the basis of van der Waals' constraints and heme ring current effects on the chemical shifts. The use of the method is illustrated with studies of horse ferrocytochrome c and Pseudomonas aeruginosa ferrocytochrome c 551. Comparison with the crystal structures shows close coincidence between the methionine conformations in solution and in single crystals of these proteins.Abbreviations NMR nuclear magnetic resonance - NOE nuclear Overhauser effect - TOE truncated driven nuclear Overhauser effect