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.     

Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life?

Mu transposons carrying the chloramphenicol resistance marker have been inserted into the cloned Escherichia coli genes sodA and sodB coding for manganese superoxide dismutase (MnSOD) and iron superoxide dismutase (FeSOD) respectively, creating mutations and gene fusions. The mutated sodA or sodB genes were introduced into the bacterial chromosome by allelic exchange. The resulting mutants were shown to lack the corresponding SOD by activity measurements and immunoblot analysis. Aerobically, in rich medium, the absence of FeSOD or MnSOD had no major effect on growth or sensitivity to the superoxide generator, paraquat. In minimal medium aerobic growth was not affected, but the sensitivity to paraquat was increased, especially in the sodA mutant. A sodA sodB double mutant completely devoid of SOD was also obtained. It was able to grow aerobically in rich medium, its catalase level was unaffected and it was highly sensitive to paraquat and hydrogen peroxide; the double mutant was unable to grow aerobically on minimal glucose medium. Growth could be restored by removing oxygen, by providing an SOD-overproducing plasmid or by supplementing the medium with the 20 amino acids. It is concluded that the total absence of SOD in E. coli creates a conditional sensitivity to oxygen.     

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.     

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.     

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

Journal of Structural and Functional Genomics -     

Crystal structure of periplasmic catecholate-siderophore binding protein VctP from Vibrio cholerae at 1.7 Å resolution

VctP, one of the two essential siderophore-binding PBPs from the pathogen Vibrio cholerae, plays an important role in the transport of enterobactin and vibriobactin, which have quite different configurations of iron coordination, from the periplasm to the inner membrane. The current study reports the crystal structure of VctP from V. cholerae N16961 at 1.7 Å resolution. A structural comparison of VctP with its homologues and the results of molecular docking indicate that enterobactin and vibriobactin share the same binding pocket. Significantly, a basic triad consisting of Arg137, Arg226 and Arg270 is used to balance the three negative charges of ferric-enterobactin, while a basic dyad consisting of Arg137 and Arg270 is used to balance the two negative charges of ferric-vibriobactin.     

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 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 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 a hemojuvelin-binding fragment of neogenin at 1.8Å

Neogenin is a type I transmembrane glycoprotein with a large ectodomain containing tandem immunoglobulin-like and fibronectin type III (FNIII) domains. Closely related to the tumor suppressor gene DCC, neogenin functions in critical biological processes through binding to various ligands, including netrin, repulsive guidance molecules, and the iron regulatory protein hemojuvelin. We previously reported that neogenin binds to hemojuvelin through its membrane-proximal fifth and sixth FNIII domains (FN5-6), with domain 6 (FN6) contributing the majority of critical binding interactions. Here we present the crystal structure of FN5-6, the hemojuvelin-binding fragment of human neogenin, at 1.8?. The two FNIII domains are orientated nearly linearly, a domain arrangement most similar to that of a tandem FNIII-containing fragment within the cytoplasmic tail of the β4 integrin. By mapping surface-exposed residues that differ between neogenin FN5-6 and the comparable domains from DCC, which does not bind hemojuvelin, we identified a potential hemojuvelin-binding site on neogenin FN6. Neogenin FN5, which does not bind hemojuvelin in isolation, exhibits a highly electropositive surface, which may be involved in interactions with negatively-charged polysaccharides or phospholipids in the membrane bilayer. The neogenin FN5-6 structure can be used to facilitate a molecular understanding of neogenin's interaction with hemojuvelin to regulate iron homeostasis and with hemojuvelin-related repulsive guidance molecules to mediate axon guidance.     

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 the sweet-tasting protein thaumatin II at 1.27Å

Thaumatin, an intensely sweet-tasting protein, elicits a sweet taste sensation at 50 nM. Here the X-ray crystallographic structure of one of its variants, thaumatin II, was determined at a resolution of 1.27 ?. Overall structure of thaumatin II is similar to thaumatin I, but a slight shift of the Cα atom of G96 in thaumatin II was observed. Furthermore, the side chain of residue 67 in thaumatin II is highly disordered. Since residue 67 is one of two residues critical to the sweetness of thaumatin, the present results suggested that the critical positive charges at positions 67 and 82 are disordered and the flexibility and fluctuation of these side chains would be suitable for interaction of thaumatin molecules with sweet receptors.     

Projection structure of channelrhodopsin-2 at 6 Å resolution by electron crystallography

Channelrhodopsin-2 (ChR2) is the prototype of a new class of light-gated ion channels that is finding widespread applications in optogenetics and biomedical research. We present a  6-Å projection map of ChR2, obtained by cryo-electron microscopy of two-dimensional crystals grown from pure, heterologously expressed protein. The map shows that ChR2 is the same dimer with non-crystallographic 2-fold symmetry in three different membrane crystals. This is consistent with biochemical analysis, which shows a stable dimer in detergent solution. Comparison to the projection map to bacteriorhodopsin indicates a similar structure of seven transmembrane alpha helices. Based on the projection map and sequence alignments, we built a homology model of ChR2 that potentially accounts for light-induced channel gating. Although a monomeric channel is not ruled out, comparison to other membrane channels and transporters suggests that the ChR2 channel is located at the dimer interface on the 2-fold axis, lined by transmembrane helices 3 and 4.     

Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution

A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.     

Crystal structure of Alr1298, a pentapeptide repeat protein from the cyanobacterium Nostoc sp. PCC 7120, determined at 2.1 Å resolution

Nostoc sp. PCC 7120 are filamentous cyanobacteria capable of both oxygenic photosynthesis and nitrogen fixation, with the latter taking place in specialized cells known as heterocysts that terminally differentiate from vegetative cells under conditions of nitrogen starvation. Cyanobacteria have existed on earth for more than 2 billion years and are thought to be responsible for oxygenation of the earth's atmosphere. Filamentous cyanobacteria such as Nostoc sp. PCC 7120 may also represent the oldest multicellular organisms on earth that undergo cell differentiation. Pentapeptide repeat proteins (PRPs), which occur most abundantly in cyanobacteria, adopt a right-handed quadrilateral β-helical structure, also referred to as a repeat five residue (Rfr) fold, with four-consecutive pentapeptide repeats constituting a single coil in the β-helical structure. PRPs are predicted to exist in all compartments within cyanobacteria including the thylakoid and cell-wall membranes as well as the cytoplasm and thylakoid periplasmic space. Despite their intriguing structure and importance to understanding ancient cyanobacteria, the biochemical function of PRPs in cyanobacteria remains largely unknown. Here we report the crystal structure of Alr1298, a PRP from Nostoc sp. PCC 7120 predicted to reside in the cytoplasm. The structure displays the typical right-handed quadrilateral β-helical structure and includes a four-α-helix cluster capping the N-terminus and a single α-helix capping the C-terminus. A gene cluster analysis indicated that Alr1298 may belong to an operon linked to cell proliferation and/or thylakoid biogenesis. Elevated alr1298 gene expression following nitrogen starvation indicates that Alr1298 may play a role in response to nitrogen starvation and/or heterocyst differentiation.     

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.     

Molecular structure of troponin C from chicken skeletal muscle at 3Å resolution

Troponin C is the Ca²⁺-binding subunit of the troponin complex and is involved in the calcium control of muscle contraction. The X-ray structure of chicken TnC has been determined at 3Å resolution using a single heavy atom derivative and application of a novel phase improvement and phase extension procedure. The protein has an unusual dumbbell-shape with a length of about 70A. The N- and C-domains are connected by a single long α-helix of about 9 turns. Two metal binding sites (the Ca²⁺-Mg²⁺ sites) in the C-domain are occupied by metal ions in the crystals and the helix-loop-helix Ca²⁺ -binding folds are very similar to those in other known Ca²⁺ -binding proteins. In contrast, the Ca²⁺ -specific sites in the N-domain appear unoccupied and the two putative Ca²⁺ -binding folds have a vastly different structural arrangement. The conformational rearrangements in the N-domain upon Ca²⁺ binding are believed to be the trigger for a cascade of protein-protein interaction alterations which lead to muscle contraction.