Crystal structure analysis of sea lamprey hemoglobin at 2 Å resolution

The crystal structure of the predominant hemoglobin component of blood from the sea lamprey, Petromyzon marinus, has been determined by X-ray diffraction analysis. Crystals for this analysis were grown from cyanide methemoglobin V as crystal type D₂. These crystals are in space group P2₁2₁2₁ and have unit cell dimensions of $\text{a = 44.57}\text{A}\text{?}\text{, b = 96.62}\text{A}\text{?}\text{and}\text{c = 31.34}\text{A}\text{?}$. Isomorphous heavyatom derivatives were prepared by soaking crystals in solutions of Hg(CN)₂, K₂Hg(CNS)₄ and KAu(CN)₂. Diffracted intensities to as far as 2 Å spacings were measured on a diffractometer. Phases were found by means of the isomorphous replacements and anomalous scattering, with supplementary information provided by the tangent formula. An atomic model was fitted to the final electron density map in a Richards optical comparator. The lamprey hemoglobin molecule is generally similar in structure to other globins, but differs in many details. Each molecule is in contact with ten neighboring molecules in the crystal lattice. The nature of the binding of the heavy atoms to lamprey hemoglobin has been interpreted.     

Structure analysis and molecular model of the selenoenzyme glutathione peroxidase at 2.8 Å resolution

The three-dimensional structure of bovine erythrocyte glutathione peroxidase, a tetrameric enzyme containing 4 gram atoms of selenium per mole (M_r = 84,000), has been determined at 2.8 Å resolution using the multiple isomorphous replacement method. By correlation calculations in Patterson space the tetramers were shown to exhibit molecular [222] symmetry, proving the monomers to be identical or at least very similar.The monomer consists of a single polypeptide chain of 178 amino acid residues. Its shape is nearly spherical with a radius of $\text{r ≈ 19}\text{A}\text{?}$. A tentative sequence corresponding to a partially refined model (R = 0.38) is given. Each subunit is built up from a central core of two parallel and two anti-parallel strands of pleated sheet surrounded by four α-helices. One of the helices runs antiparallel to the neighbouring β-strands giving rise to a βαβ substructure, an architecture that has been found in several other proteins e.g. flavodoxin, thioredoxin, rhodanese and dehydrogenases. A comparison of the glutathione peroxidase subunit structure with thioredoxin-S₂ revealed large regions of structural resemblance. The central four-stranded β structure together with two parallel α-helices resembles nearly 80% of the thioredoxin fold.The active sites of glutathione peroxidase are located in flat depressions on the molecular surface. Probably each active centre is built up by segments from two subunits. The catalytically active selenocysteines were found at the N-terminal ends of long α-helices and are surrounded by an accumulation of aromatic side-chains. A difference Fourier map between oxidized and substrate-reduced glutathione peroxidase as well as heavy-atom binding led to the conclusion that the two-electron redox-cycle involves a reversible transition of the active-site selenium from a selenenic acid (RSeOH) to a seleninic acid (RSeOOH).     

Crystal structure of rubredoxin from Pyrococcus furiosus at 0.95 Å resolution, and the structures of N-terminal methionine and formylmethionine variants of Pf Rd. Contributions of N-terminal interactions to thermostability

The high-resolution crystal structure of the small iron-sulfur protein rubredoxin (Rd) from the hyperthermophilic archeon Pyrococcus furiosus (Pf) is reported in this paper, together with those of its methionine ([_0M]Pf Rd) and formylmethionine (f[_0M]Pf Rd) variants. These studies were conducted to assess the consequences of the presence or absence of a salt bridge between the amino terminal nitrogen of Ala1 and the side chain of Glu14 to the structure and stability of this rubredoxin. The structure of wild-type Pf Rd was solved to a resolution of 0.95?Å and refined by full-matrix least-squares techniques to a crystallographic agreement factor of 12.8% [F>2σ(F) data, 25?617 reflections], while those of the [_0M]Pf and f[_0M]Pf Rd variants were solved at slightly lower resolutions (1.1?Å, R=11.5%, 17?213 reflections; 1.2?Å, R=13.7%, 12?478 reflections, respectively). The quality of the data was such that about half of the hydrogen atoms of the protein were clearly visible. All three structures were ultimately refined using the program SHELXL-93 with anisotropic atomic displacement parameters for all non-hydrogen protein atoms, and calculated hydrogen positions included but not refined. In this paper we also report thermostability data for all three forms of Pf Rd, and show that they follow the sequence wild-type >[_0M]Pf>formyl[_0M]Pf. Comparison of the three Pf Rd structures in the N-terminal region show that the structures of wild-type Pf Rd and f[_0M]Pf are rather similar, while that of [_0M]Pf Rd shows a number of additional hydrogen bonds involving the extra methionine group. While the salt bridge between the Ala1 amino group and the Glu14 carboxylate is not the primary determinant of the thermostability of Pf Rd, alterations to the amino terminus do have a moderate influence on the thermostability of this protein.     

X-ray structure of recombinant horse L-chain apoferritin at 2.0 Å resolution: implications for stability and function

The X-ray structure of recombinant horse L-chain (rL) apoferritin, solved at 2.0?Å resolution with a final R factor of 17.9%, gives evidence that the residue at position 93 in the sequence is a proline and not a leucine, as found in earlier sequencing studies. The structure is isomorphous with other apoferritin structures, and we thus draw particular attention to those structural features which can be related to the stability and function of the protein. Analysis of hydrogen bonding and salt bridge interactions shows that dimers and tetramers are the most stable molecular entities within the protein shell: a result confirming earlier biophysical experiments. The stability of horse rL apoferritin to both dissociation into subunits at acidic pH values and to complete unfolding in guanidine chloride solutions is compared with that of other apoferritins. This emphasizes the role played by the salt bridge in the stability of this protein family. The horse rL apoferritin is significantly more resistant to denaturation than horse spleen ferritin, which in turn is more resistant than any human rH apoferritins, even those for which a salt bridge is restored. Finally, this structure determination not only establishes that a preformed pocket exists in L-chain apoferritin, at a site known to be able to bind porphyrin, but also underlines the particular function of a cluster of glutamic acids (E53, E56, E57 and E60) located at the entrance of this porphyrin-binding pocket.     

The structure of the heterodimer reaction center from Rhodobacter sphaeroides at 2.55 å resolution

Crystals have been obtained of reaction centers of the heterodimer mutant that has significantly different properties than wild type due to the primary donor being formed from both a bacteriochlorophyll and bacteriopheophytin rather than two bacteriochlorophylls as found for wild type. The crystals belong to the trigonal space group P3(1)21 and the structure has been refined to a resolution limit of 2.55 A with an R factor of 19.0%. The electron density maps confirm that a primary donor does indeed contain a bacteriopheophytin due to the His to Leu substitution at M202 that coordinates the corresponding bacteriochlorophyll in wild-type. Other structural changes compared to wild type are relatively minor with the relative orientation and positioning of the two tetrapyrroles forming the primary donor being unchanged within the error. Compared to wild type, the only significant alterations are small shifts of residues M196 to M206, a rotation of the side chain of Ile M206, and the loss of a bound water molecule that in wild-type is hydrogen-bonded to both His M202 and the bacteriochlorophyll monomer on the active branch. Since hydrogen-bonding interactions strongly influence the energies of tetrapyrroles, the loss of the water molecule should result in changes in the energies of the bacteriochlorophyll monomer that contributes to the observed functional differences with wild-type.     

Refinement of human lysozyme at 1.5 Å resolution analysis of non-bonded and hydrogen-bond interactions

The structure of human lysozyme has been crystallographically refined at 1.5 Å resolution by difference map and restrained least-squares procedures to an R factor of 0.187. A comprehensive analysis of the non-bonded and hydrogen-bonded contacts in the lysozyme molecule, which were not restrained, revealed by the refinement has been carried out. The non-bonded CC contacts begin at ~3.45 Å, and the shorter contacts are dominated, as expected, by interactions between trigonal and tetrahedral carbon atoms. The CO contact distances have a “foot” at 3.05 Å. The CN distance plot shows a significant peak at 3.25 Å, which results from close contact between peptide NHs and carbonyl carbons involved in N_iC′_{i ? 2} interactions in α-helices and reverse turns. The distances involving sulphur atoms discriminate SC trigonal interactions at 3.4 to 3.6 Å from SC tetrahedral interactions at 3.7 Å. All these types of non-bonded interactions show minimum distances close to standard van der Waals' separations.Analysis of hydrogen-bond distances has been carried out by using standard geometry to place hydrogen atoms and measuring the XHO distances. On this basis, there are 130 intramolecular hydrogens: 111 NHO bonds, of which 69 are between main-chain atoms, 13 between side-chain atoms and 29 between mainchain and side-chain atoms. If a cluster of four well-defined internal water molecules is included in the protein structure, there is a total of 19 OHO hydrogen bonds. The mean NO, NHO distances and HN?O angles are 2.96 ± 0.17 Å, 2.05 ± 0.18 Å and 18.5 ± 9.6 °, and the mean OO, OHO distances and HÔO angles are 2.83 ± 0.19 Å, 1.98 ± 0.26 Å and 23.8 ± 13.4 °. The distances agree well with standard values, although the hydrogen bonds are consistently more non-linear than in equivalent small molecules. An analysis of the hydrogen-bond angles at the receptor atom indicates that the α-helix, β-sheet and reverse turn have characteristic angular values. A detailed analysis of the regularity of the α-helices and reverse turns shows small but consistent differences between the α-helices in lysozyme and the current standard model, which may now need revision. Of the 21 reverse turns that include a hydrogen bond, the conformations of 19 agree very closely with four of the five standard types. We conclude that the restrained least-squares method of refinement has been validated by these analyses.     

Crystal and molecular structure of the sulfhydryl protease calotropin DI at 3·2 Å resolution

The three-dimensional structure of the sulfhydryl protease calotropin DI from the madar plant, Calotropis gigantea, has been determined at 3·2 Å resolution using the multiple isomorphous replacement method with five heavy atom derivatives. A Fourier synthesis based on protein phases with a mean figure of merit of 0·857 was used for model building. The polypeptide backbone of calotropin DI is folded to form two distinct lobes, one of which is comprised mainly of α-helices, while the other is characterized by a system of all antiparallel pleated sheets. The overall molecular architecture closely resembles those found in the sulfhydryl proteases papain and actinidin.Despite the unknown amino acid sequence of calotropin DI a number of residues around its active center could be identified. These amino acid side-chains were found in a similar arrangement as the corresponding ones in papain and actinidin. The polypeptide chain between residues 1 and 18 of calotropin DI folds in a unique manner, providing a possible explanation for the unusual inability of calotropin DI to hydrolyze those synthetic substrates that papain and actinidin act upon.     

Crystal structure of Turkey egg-white lysozyme: Results of the molecular replacement method at 5 Å resolution

Turkey egg-white lysozyme differs from hen egg-white lysozyme in its primary structure in 7 of the 129 residues. We have determined the rotational and translational parameters relating the known co-ordinates of hen egg-white lysozyme molecule to the turkey lysozyme. The rotational parameters were determined using the rotation function, the translational parameters were determined by placing the properly rotated molecule systematically at all positions within the unit cell and searching for those positions producing few intermolecular contacts between the α-carbon atoms of one molecule and all its neighbors. These parameters were refined by minimizing the conventional R factor between observed and calculated structure amplitudes. The final rotational and translational parameters give an R value of 46.7% for reflections with d spacings between 6 Å and 12 Å and have 7 intermolecular contacts closer than 5 Å between the a carbon atoms of one molecule and all its neighbors. An electron density map has been calculated at 5 Å resolution; the packing of the molecules in this form appears to present the entire length of the active cleft in the vicinity of the crystallographic 6-fold axis and does not appear to be blocked by neighboring molecules.     

Crystal structure of AcrB complexed with linezolid at 3.5 Å resolution

AcrB is an inner membrane resistance-nodulation-cell division efflux pump and is part of the AcrAB–TolC tripartite efflux system. We have determined the crystal structure of AcrB with bound Linezolid at a resolution of 3.5 Å. The structure shows that Linezolid binds to the A385/F386 loops of the symmetric trimer of AcrB. A conformational change of a loop in the bottom of the periplasmic cleft is also observed.     

Crystal structure of the C-terminal globular domain of oligosaccharyltransferase from Archaeoglobus fulgidus at 1.75 Å resolution

Protein N-glycosylation occurs in the three domains of life. Oligosaccharyltransferase (OST) transfers glycan to asparagine in the N-glycosylation sequon. The catalytic subunit of OST is called STT3 in eukaryotes, AglB in archaea, and PglB in eubacteria. The genome of a hyperthermophilic archaeon, Archaeoglobus fulgidus, encodes three AglB paralogs. Two of them are the shortest AglBs across all domains of life. We determined the crystal structure of the C-terminal globular domain of the smallest AglB to identify the minimal structural unit. The Archaeoglobus AglB lacked a β-barrel-like structure, which had been found in other AglB and PglB structures. In agreement, the deletion in a larger Pyrococcus AglB confirmed its dispensability for the activity. By contrast, the Archaeoglobus AglB contains a kinked helix bearing a conserved motif, called DK/MI motif. The lysine and isoleucine residues in the motif participate in the Ser/Thr recognition in the sequon. The Archaeoglobus AglB structure revealed that the kinked helix contained an unexpected insertion. A revised sequence alignment based on this finding identified a variant type of the DK motif with the insertion. A mutagenesis study of the Archaeoglobus AglB confirmed the contribution of this particular type of the DK motif to the activity. When taken together with our previous results, this study defined the classification of OST: one group consisting of eukaryotes and most archaea possesses the DK-type Ser/Thr pocket, and the other group consisting of eubacteria and the remaining archaea possesses the MI-type Ser/Thr pocket. This classification provides a useful framework for OST studies.     

Crystal structure determination at 1.4 Å resolution of ferredoxin from the green alga Chlorella fusca

Background: [2Fe–2S] ferredoxins, also called plant-type ferredoxins, are low-potential redox proteins that are widely distributed in biological systems. In photosynthesis, the plant-type ferredoxins function as the central molecule for distributing electrons from the photolysis of water to a number of ferredox-independent enzymes, as well as to cyclic photophosphorylation electron transfer. This paper reports only the second structure of a [2Fe–2S] ferredoxin from a eukaryotic organism in its native form.Results: Ferredoxin from the green algae Chlorella fusca has been purified, characterised, crystallised and its structure determined to 1.4 Å resolution – the highest resolution structure published to date for a plant-type ferredoxin. The structure has the general features of the plant-type ferredoxins already described, with conformational differences corresponding to regions of higher mobility. Immunological data indicate that a serine residue within the protein is partially phosphorylated. A slightly electropositive shift in the measured redox potential value, -325 mV, is observed in comparison with other ferredoxins.Conclusions: This high-resolution structure provides a detailed picture of the hydrogen-bonding pattern around the [2Fe–2S] cluster of a plant-type ferredoxin; for the first time, it was possible to obtain reliable error estimates for the geometrical parameters. The presence of phosphoserine in the protein indicates a possible mechanism for the regulation of the distribution of reducing power from the photosynthetic electron-transfer chain.     

Crystal structure of the his-tagged saccharopine reductase from Saccharomyces cerevisiae at 1.7-Å resolution

The three-dimensional structure of the saccharopine reductase enzyme from the budding yeast Saccharomyces cerevisiae was determined to 1.7-A resolution in the apo form by using molecular replacement. The enzyme monomer consists of three domains: domain I is a variant of the Rossmann fold, domain II folds into a alpha/beta structure containing a mixed seven-stranded beta-sheet as the central core, and domain III has an all-helical fold. Comparative fold alignment with the enzyme from Magnaporthe grisea suggests that domain I binds to NADPH, and domain II binds to saccharopine and is involved in dimer formation. Domain III is involved in closing the active site of the enzyme once substrates are bound. Structural comparison of the saccharopine reductase enzymes from S. cerevisiae and M. grisea indicates that domain II has the highest number of conserved residues, suggesting that it plays an important role in substrate binding and in spatially orienting domains I and III.     

Novel features in the structure of P-glycoprotein (ABCB1) in the post-hydrolytic state as determined at 7.9 Å resolution

Background

P-glycoprotein (ABCB1) is an ATP-binding cassette transporter that plays an important role in the clearance of drugs and xenobiotics and is associated with multi-drug resistance in cancer. Although several P-glycoprotein structures are available, these are either at low resolution, or represent mutated and/or quiescent states of the protein.

Results

In the post-hydrolytic state the structure of the wild-type protein has been resolved at about 8?Å resolution. The cytosolic nucleotide-binding domains (NBDs) are separated but ADP remains bound, especially at the first NBD. Gaps in the transmembrane domains (TMDs) that connect to an inner hydrophilic cavity are filled by density emerging from the annular detergent micelle. The NBD-TMD linker is partly resolved, being located between the NBDs and close to the Signature regions involved in cooperative NBD dimerization. This, and the gap-filling detergent suggest steric impediment to NBD dimerization in the post-hydrolytic state. Two central regions of density lie in two predicted drug-binding sites, implying that the protein may adventitiously bind hydrophobic substances even in the post-hydrolytic state. The previously unresolved N-terminal extension was observed, and the data suggests these 30 residues interact with the headgroup region of the lipid bilayer.

Conclusion

The structural data imply that (i) a low basal ATPase activity is ensured by steric blockers of NBD dimerization and (ii) allocrite access to the central cavity may be structurally linked to NBD dimerization, giving insights into the mechanism of drug-stimulation of P-glycoprotein activity.

     

The X-ray structure of human serum ceruloplasmin at 3.1 Å: nature of the copper centres

The X-ray structure of human serum ceruloplasmin has been solved at a resolution of 3.1?Å. The structure reveals that the molecule is comprised of six plastocyanin-type domains arranged in a triangular array. There are six copper atoms; three form a trinuclear cluster sited at the interface of domains 1 and 6, and there are three mononuclear sites in domains 2, 4 and 6. Each of the mononuclear coppers is coordinated to a cysteine and two histidine residues, and those in domains 4 and 6 also coordinate to a methionine residue; in domain 2, the methionine is replaced by a leucine residue which may form van der Waals type contacts with the copper. The trinuclear centre and the mononuclear copper in domain 6 form a cluster essentially the same as that found in ascorbate oxidase, strongly suggesting an oxidase role for ceruloplasmin in the plasma.     

Crystal structure of Escherichia coli manganese superoxide dismutase at 2.1-Å resolution

The three-dimensional structure of the manganese-dependent superoxide dismutase (MnSOD) from Escherichia coli has been determined by X-ray crystallography at 2.1?Å resolution. The protein crystallizes with two homodimers in the asymmetric unit, and a model comprising 6528 protein atoms (residues 1–205 of all four monomers), four manganese ions and 415 water molecules has been refined to an R factor of 0.188 (R _free 0.218). The structure shows a high degree of similarity with other MnSOD and FeSOD enzymes. The Mn centres are 5-coordinate, trigonal bipyramidal, with His26 and a solvent molecule, probably a hydroxide ion, as apical ligands, and His81, Asp167 and His171 as equatorial ligands. The coordinated solvent molecule is linked to a network of hydrogen bonds involving the non-coordinated carboxylate oxygen of Asp167 and a conserved glutamine residue, Gln146. The MnSOD dimer is notable for the way in which the two active sites are interconnected and a "bridge" comprising His171 of one monomer and Glu170 of the other offers a route for inter-site communication. Comparison of E. coli MnSOD and FeSOD (a) reveals some differences in the dimer interface, (b) yields no obvious explanation for their metal specificities, and (c) provides a structural basis for differences in DNA binding, where for MnSOD the groove formed by dimerization is complementary in charge and surface contour to B-DNA.     

Crystal structure of chicken liver basic fatty acid-binding protein at 2.7 Å resolution

The three-dimensional structure of chicken liver basic fatty acid-binding protein has been determined at 2.7 Å resolution by X-ray crystallography. Phases were calculated using the multiple isomorphous replacement procedure and a preliminary model was built. This model, with an initial R-factor of 0.57, was then improved by a cycle of refinement by simulated annealing which brought the R factor down to 0.32. The protein is structured as a compact 10-stranded--barrel which encapsulates a residual electron density that can be interpreted as a fatty acid molecule. The NH₂-terminus portion of the molecule contains two short -helices. The structure of this liver protein appears very similar to that of the Escherichia coli derived rat intestinal FABP recently determined by X-ray diffraction methods.     

Improving the accuracy of macromolecular structure refinement at 7 Å resolution

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Highlights? Improved models by DEN refinement at 7 Å ? Best method is DEN refinement with initial segmented rigid-body refinement ? R_free has predictive power at 7 Å     

Crystal structure of a stress inducible protein from Mycoplasma pneumoniae at 2.85 Å resolution

Journal of Structural and Functional Genomics -