Three-dimensional structure of myosin subfragment-1: a molecular motor.

Directed movement is a characteristic of many living organisms and occurs as a result of the transformation of chemical energy into mechanical energy. Myosin is one of three families of molecular motors that are responsible for cellular motility. The three-dimensional structure of the head portion of myosin, or subfragment-1, which contains both the actin and nucleotide binding sites, is described. This structure of a molecular motor was determined by single crystal x-ray diffraction. The data provide a structural framework for understanding the molecular basis of motility.

The 1.19 A X-ray structure of 2'-O-Me(CGCGCG)(2) duplex shows dehydrated RNA with 2-methyl-2,4-pentanediol in the minor groove.

The crystal and molecular structure of 2'-O-Me(CGCGCG)(2) has been determined at 1.19 A resolution, at 100 K, using synchrotron radiation. The structure in space group P3(2)12 is a half-turn right-handed helix that includes two 2-methyl-2,4-pentanediol (MPD) molecules bound in the minor groove. The structure deviates from A-form RNA. The duplex is overwound with an average value of 9.7 bp per turn, characterised as having a C3'-endo sugar pucker, very low base pair rise and high helical twist and inclination angles. The structure includes 65 ordered water molecules. Only a single row of water molecules is observed in the minor groove due to the presence of hydrophobic 2'-O-methyl groups. As many as five magnesium ions are located in the structure. Two are in the major groove and interact with O(6) and N(7) of guanosine and N(4) of cytidine residues through their hydration spheres. This work provides the first example of molecular interactions of nucleic acids with MPD, which was used as a precipitant, cryo-solvent and resolution enhancing agent. The two MPD molecules intrude into the hydration network in the minor groove, each forming hydrogen bonds between their secondary hydroxyl group and exo-amino functions of guanosine residues. Comparison of the 2'-O-Me(CGCGCG)(2) structure in the P3(2)12 and P6(1)22 crystals delineates stability of the water network within the minor groove to dehydration by MPD and is of interest for evaluating factors governing small molecule binding to RNA. Intrusion of MPD into the minor groove of 2'-O-Me(CGCGCG)(2) is discussed with respect to RNA dehydration, a prerequisite of Z-RNA formation.

A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels.

BACKGROUND: Urease catalyzes the hydrolysis of urea, the final step of organic nitrogen mineralization, using a bimetallic nickel centre. The role of the active site metal ions and amino acid residues has not been elucidated to date. Many pathologies are associated with the activity of ureolytic bacteria, and the efficiency of soil nitrogen fertilization with urea is severely decreased by urease activity. Therefore, the development of urease inhibitors would lead to a reduction of environmental pollution, to enhanced efficiency of nitrogen uptake by plants, and to improved therapeutic strategies for treatment of infections due to ureolytic bacteria. Structure-based design of urease inhibitors would require knowledge of the enzyme mechanism at the molecular level. RESULTS: The structures of native and inhibited urease from Bacillus pasteurii have been determined at a resolution of 2.0 A by synchrotron X-ray cryogenic crystallography. In the native enzyme, the coordination sphere of each of the two nickel ions is completed by a water molecule and a bridging hydroxide. A fourth water molecule completes a tetrahedral cluster of solvent molecules. The enzyme crystallized in the presence of phenylphosphorodiamidate contains the tetrahedral transition-state analogue diamidophosphoric acid, bound to the two nickel ions in an unprecedented mode. Comparison of the native and inhibited structures reveals two distinct conformations of the flap lining the active-site cavity. CONCLUSIONS: The mode of binding of the inhibitor, and a comparison between the native and inhibited urease structures, indicate a novel mechanism for enzymatic urea hydrolysis which reconciles the available structural and biochemical data.

Crystal structure of unliganded phosphofructokinase from Escherichia coli.

In an attempt to characterize the mechanism of co-operativity in the allosteric enzyme phosphofructokinase from Escherichia coli, crystals were grown in the absence of activating ligands. The crystal structure was determined to a resolution of 2.4 A by the method of molecular replacement, using the known structure of the liganded active state as a starting model, and has been refined to a crystallographic R-factor of 0.168 for all data. Although the crystallization solution would be expected to contain the enzyme in its inactive conformation, with a low affinity for the co-operative substrate fructose 6-phosphate, the structure in these crystals does not show the change in quaternary structure seen in the inactive form of the Bacillus stearothermophilus enzyme (previously determined at low resolution), nor does it show any substantial change in the fructose 6-phosphate site from the structure seen in the liganded form. Compared to the liganded form, there are considerable changes around the allosteric effector site, including the disordering of the last 19 residues of the chain. It seems likely that the observed conformation corresponds an active unliganded form, in which the absence of ligand in the effector site induces structural changes that spread through much of the subunit, but cause only minor changes in the active site. It is not clear why the crystals should contain the enzyme in a high-affinity conformation, which presumably represents only a small fraction of the molecules in the crystallizing solution. However, this structure does identify the conformational changes involved in binding of the allosteric effectors.

Crystal structure of reduced bovine erythrocyte superoxide dismutase at 1.9 A resolution.

Cu,Zn superoxide dismutase was investigated crystallographically in the reduced form. Co-ordinate errors were estimated by comparing two independently refined models, based on two different data sets. This gave a detailed error estimation as opposed to the standard sigma A and Luzzati plots, which estimate only the overall error. The high quality of the final model, obtained after scaling together the two data sets, combined with the error estimates allowed a detailed analysis of the protein and solvent structures. An automatic procedure for building and refining solvent structure was tested and found to give reproducible results. Contrary to results obtained from spectroscopic studies, the co-ordination of the metal ions in the catalytic site is preserved in the crystal structure of the reduced enzyme, as compared with the crystal structure of the oxidised form. Analysis of the solvent reveals a well-defined chain of closely packed, hydrogen-bonded water molecules filling the active site groove. This structural feature could serve as a hydrogen bond relay for efficient delivery of protons to the active centre. Analysis of electron density suggests that Glu119 is covalently modified. The modification, if originated in vivo, could have a role in the catalytic mechanism and could affect the overall electrostatic field in the active site. There are significant differences between the active sites of the two crystallographically independent monomers. They are explained in terms of local differences in the crystal environment.

Crystallization and preliminary X-ray analysis of the 12S form of phosphofructokinase from Saccharomyces cerevisiae.

The tetrameric 12S form of yeast phosphofructokinase, obtained by limited proteolytic cleavage of the native enzyme, was crystallized under a variety of conditions. The crystals have been characterized in the X-ray beam and are suitable for crystallographic studies.

Structural consequences of reductive methylation of lysine residues in hen egg white lysozyme: an X-ray analysis at 1.8-A resolution.

Chemical modification of proteins has been and continues to be an important biochemical tool for the study of protein structure and function. One such type of approach has been the reductive methylation of lysine residues. In order to address the consequences of such methylation on the crystallization and structural properties of a protein, the three-dimensional structure of hen egg white lysozyme in which all lysine residues have been alkylated has been determined and refined to a nominal resolution of 1.8 A and a crystallographic R factor of 17.3%. Crystals used in the investigation were grown from 1.5-1.8 M MgSO4 and 50 mM Tris at pH 8.0 and belonged to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 30.6 A, b = 56.3 A, c = 73.2 A, and one molecule per asymmetric unit. It was not possible to grow crystals of the modified lysozyme under the conditions normally employed for the hen egg white protein. Overall, the three-dimensional structures of the native lysozyme and the modified protein are very similar with only two surface loops differing to any significant extent. Specifically, the positions of the alpha-carbons for these two forms of the protein, excluding the surface loops, superimpose with a root-mean-square value of 0.40 A. The magnitude of the structural changes observed between the modified an unmodified forms of lysozyme is similar to that seen when an identical protein structure is solved in two different crystalline lattices.(ABSTRACT TRUNCATED AT 250 WORDS)

The molecular structure of insecticyanin from the tobacco hornworm Manduca sexta L. at 2.6 A resolution.

Insecticyanin, a blue biliprotein isolated from the tobacco hornworm Manduca sexta L., is involved in insect camouflage. Its three-dimensional structure has now been solved to 2.6 A resolution using the techniques of multiple isomorphous replacement, non-crystallographic symmetry averaging about a local 2-fold rotation axis and solvent flattening. All 189 amino acids have been fitted to the electron density map. The map clearly shows that insecticyanin is a tetramer with one of its molecular 2-fold axes coincident to a crystallographic dyad. The individual subunits have overall dimensions of 44 A X 37 A X 40 A and consist primarily of an eight-stranded anti-parallel beta-barrel flanked on one side by a 4.5-turn alpha-helix. Interestingly the overall three-dimensional fold of the insecticyanin subunit shows remarkable similarity to the structural motifs of bovine beta-lactoglobulin and the human serum retinol-binding protein. The electron density attributable to the chromophore is unambiguous and shows that it is indeed the gamma-isomer of biliverdin. The biliverdin lies towards the open end of the beta-barrel with its two propionate side chains pointing towards the solvent and it adopts a rather folded conformation, much like a heme.

Fusarium oxysporum trypsin at atomic resolution at 100 and 283 K: a study of ligand binding.

The X-ray structure of F. oxysporum trypsin has been determined at atomic resolution, revealing electron density in the binding site which was interpreted as a peptide bound in the sites S1, S2 and S3. The structure, which was initially determined at 1.07 A resolution and 283 K, has an Arg in the S1 specificity pocket. The study was extended to 0.81 A resolution at 100 K using crystals soaked in Arg, Lys and Gln to study in greater detail the binding at the S1 site. The electron density in the binding site was compared between the different structures and analysed in terms of partially occupied and overlapping components of peptide, solvent water and possibly other chemical moieties. Arg-soaked crystals reveal a density more detailed but similar to the original structure, with the Arg side chain visible in the S1 pocket and residual peptide density in the S2 and S3 sites. The density in the active site is complex and not fully interpreted. Lys at high concentrations displaces Arg in the S1 pocket, while some main-chain density remains in sites S2 and S3. Gln has been shown not to bind. The free peptide in the S1-S3 sites binds in a similar way to the binding loop of BPTI or the inhibitory domain of the Alzheimer's beta-protein precursor, with some differences in the S1 site.

Crystals of cytochrome c-553 from Bacillus pasteurii show diffraction to 0.97 A resolution.

We report here the purification and characterization of a c-type cytochrome present in the soluble fraction of the gram-positive, alkaliphilic, and highly ureolytic soil bacterium Bacillus pasteurii. The cytochrome is acidic (pI = 3.3), has a molecular mass of 9.5 kDa, and appears to dimerize in 150 mM ionic strength solution. The electronic spectrum is typical of a low-spin hexa-coordinated heme iron. Crystals of the protein in the oxidized state were grown by vapor diffusion at pH 5, by using 3.2 M ammonium sulfate as precipitant. Diffraction data at ultrahigh resolution (0.97 A) and completeness (99.9%) have been collected under cryogenic conditions, by using synchrotron radiation. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with cell constants a = 37.14, b = 39.42, c = 44.02 A, and one protein monomer per asymmetric unit. Attempts to solve the crystal structure by ab initio methods are in progress.

The complex of Bacillus pasteurii urease with acetohydroxamate anion from X-ray data at 1.55 A resolution.

The structure of Bacillus pasteurii urease inhibited with acetohydroxamic acid was solved and refined anisotropically using synchrotron X-ray cryogenic diffraction data (1.55 A resolution, 99.5% completeness, data redundancy = 26, R-factor = 15.1%, PDB code 4UBP). The two Ni ions in the active site are separated by a distance of 3.53 A. The structure clearly shows the binding mode of the inhibitor anion, symmetrically bridging the two Ni ions in the active site through the hydroxamate oxygen and chelating one Ni ion through the carbonyl oxygen. The flexible flap flanking the active site cavity is in the open conformation. The possible implications of the results on structure-based molecular design of new urease inhibitors are discussed.

Evolutionary divergence and conservation of trypsin.

The trypsin sequences currently available in the data banks have been collected and aligned using first the amino acid sequence homology and, subsequently, the superposed crystal structures of trypsins from the cow, the bacterium Streptomyces griseus and the fungus Fusarium oxysporum. The phylogenetic tree constructed according to this multiple alignment is consistent with a continuous evolutionary divergence of trypsin from a common ancestor of both prokaryotes and eukaryotes. Comparison of crystal structures reveals a strict conservation of secondary structure. Similarly, in the alignment of all the sequences, insertions and deletions occur only in regions corresponding to loops between the secondary structure elements in the known crystal structures. The conserved residues cluster around the active site. Almost all conserved residues can be associated with one of the basic functional features of the protein: zymogen activation, catalysis and substrate specificity. In contrast, the residues of the hydrophobic core of the protein and the calcium ion binding sites are generally not conserved. The conserved features of trypsin and the nature of the conservation are discussed in detail.

Crystallization and preliminary X-ray diffraction analysis of cytochrome c' from Rubrivivax gelatinosus at 1.3 A resolution.

Cytochrome c' from the purple non-sulfur phototrophic bacterium Rubrivivax gelatinosus has been crystallized by vapour diffusion at pH 5, 6.3 and 8, in sodium acetate, sodium citrate, and Tris-HCl buffers, respectively. Crystals grown at pH 5 and 6.3 diffract, respectively, to 2.0 A (298 K) and 1.4 A (100 K) using synchrotron radiation. Data up to 1.3 A resolution with 99.8% completeness were collected at 100 K on a crystal grown at pH 8. The space group is P3121 or P3221, and the unit-cell parameters are a = b = 69.63, c = 123.63 A.

Crystallization and preliminary high-resolution X-ray diffraction analysis of native and beta-mercaptoethanol-inhibited urease from Bacillus pasteurii.

Hexagonal crystals of urease from Bacillus pasteurii have been obtained by vapour diffusion at 293 K in 20 mM Tris-HCl, neutral pH, containing 50 mM Na2SO3. Isomorphous crystals of urease inhibited with beta-mercaptoethanol were also obtained by including 4 mM of the inhibitor in the enzyme solution. Crystals of the native and inhibited enzyme diffract respectively to 2.00 A (96.7% completeness) and to 1.65 A (98.7% completeness) using synchrotron X-ray cryogenic (100 K) conditions. The space group is P6322 for both forms, and the unit-cell parameters are a = b = 131.36, c = 189. 76 A for native urease and a = b = 131.34, c = 190.01 A for inhibited urease. Under the same conditions, single crystals of B. pasteurii urease inhibited with acetohydroxamic acid, cisteamine, and phenylphosphorodiamidate were also obtained.

Structure-based rationalization of urease inhibition by phosphate: novel insights into the enzyme mechanism.

The structure of Bacillus pasteurii urease (BPU) inhibited with phosphate was solved and refined using synchrotron X-ray diffraction data from a vitrified crystal (1.85 A resolution, 99.3% completeness, data redundancy 4.6, R-factor 17.3%, PDB code 6UBP). A distance of 3.5 A separates the two Ni ions in the active site. The binding mode of the inhibitor involves the formation of four coordination bonds with the two Ni ions: one phosphate oxygen atom symmetrically bridges the two metal ions (1.9-2.0 A), while two of the remaining phosphate oxygen atoms bind to the Ni atoms at 2.4 A. The fourth phosphate oxygen is directed into the active site channel. Analysis of the H-bonding network around the bound inhibitor indicates that phosphate is bound as the H2PO4- anion, and that an additional proton is present on the Odelta2 atom of Asp(alpha363), an active site residue involved in Ni coordination through Odelta1. The flexible flap flanking the active site cavity is in the open conformation. Analysis of the complex reveals why phosphate is a relatively weak inhibitor and why sulfate does not bind to the nickels in the active site. The implications of the results for the understanding of the urease catalytic mechanism are reviewed. A novel alternative for the proton donor is presented.

Structure of inhibited trypsin from Fusarium oxysporum at 1.55 A.

The structure of trypsin from the fungus Fusarium oxysporum has been refined at 1.55 A resolution by restrained least-squares minimization to an R-factor of 14.4%. The data were recorded from a single-crystal on the X31 beamline at EMBL, Hamburg, using a locally developed image-plate scanner. The final model consists of 1557 protein atoms, 400 water molecules, one molecule of isopropanol and one monoisopropyl phosphoryl inhibitor group covalently bound to the catalytic Ser195. Comparison of the structure with bovine trypsin reveals significant differences in the active site and suggests a possible explanation for the difference in substrate specificity between the two enzymes. In F. oxysporum trypsin the specificity pocket is larger than in bovine trypsin. This explains the preference of F. oxysporum trypsin for the bulkier arginine over lysine and the reverse preference in bovine trypsin. The binding cavity on the C-terminal side of the substrate is more restricted in F. oxysporum trypsin than in mammalian and Streptomyces griseus trypsins, which explains the relative inactivity of F. oxysporum trypsin towards peptide-pNA substrate analogues as an unfavourable steric interaction between the side of the binding cavity and the para-nitroanilino group of peptide-pNA. The observed restriction of the binding cavity does not lead to a reduced catalytic activity compared to other trypsins.

Crystal structure of 2'-O-Me(CGCGCG)2, an RNA duplex at 1.30 A resolution. Hydration pattern of 2'-O-methylated RNA.

The molecular and crystal structure of 2'-O-Me (CGCGCG)2 has been determined using synchrotron radiation at near-atomic resolution (1.30 A), the highest resolution to date in the RNA field. The crystal structure is a half-turn A-type helix with some helical parameters deviating from canonical A-RNA, such as low base pair rise, elevated helical twist and inclination angles. In CG steps, inter-strand guanines are parallel while cytosines are not parallel. In steps GC this motif is reversed. This type of regularity is not seen in other RNA crystal structures. The structure includes 44 water molecules and two hydrated Mg2+ions one of which lies exactly on the crystallographic 2-fold axis. There are distinct patterns of hydration in the major and the minor grooves. The major groove is stabilised by water clusters consisting of fused five- and six-membered rings. Minor groove contains only a single row of water molecules; each water bridges either two self-parallel cytosines or two self-parallel guanines by a pair of hydrogen bonds. The structure provides the first view of the hydration scheme of 2'-O-methylated RNA duplex.

Crystal structure of oxidized Bacillus pasteurii cytochrome c553 at 0.97-A resolution.

This article reports the first X-ray structure of the soluble form of a c-type cytochrome isolated from a Gram-positive bacterium. Bacillus pasteurii cytochrome c(553), characterized by a low reduction potential and by a low sequence homology with cytochromes from Gram-negative bacteria or eukaryotes, is a useful case study for understanding the structure-function relationships for this class of electron-transfer proteins. Diffraction data on a single crystal of cytochrome c(553) were obtained using synchrotron radiation at 100 K. The structure was determined at 0.97-A resolution using ab initio phasing and independently at 1.70 A in an MAD experiment. In both experiments, the structure solution exploited the presence of a single Fe atom as anomalous scatterer in the protein. For the 0.97-A data, the phasing was based on a single data set. This is the most precise structure of a heme protein to date. The crystallized cytochrome c(553) contains only 71 of the 92 residues expected from the intact protein sequence, lacking the first 21 amino acids at the N-terminus. This feature is consistent with previous evidence that this tail, responsible for anchoring the protein to the cytoplasm membrane, is easily cleaved off during the purification procedure. The heme prosthetic group in B. pasteurii cytochrome c(553) is surrounded by three alpha-helices in a compact arrangement. The largely exposed c-type heme group features a His-Met axial coordination of the Fe(III) ion. The protein is characterized by a very asymmetric charge distribution, with the exposed heme edge located on a surface patch devoid of net charges. A structural search of a representative set of protein structures reveals that B. pasteurii cytochrome c(553) is most similar to Pseudomonas cytochromes c(551), followed by cytochromes c(6), Desulfovibrio cytochrome c(553), cytochromes c(552) from thermophiles, and cytochromes c from eukaryotes. Notwithstanding a low sequence homology, a structure-based alignment of these cytochromes shows conservation of three helical regions, with different additional secondary structure motifs characterizing each protein. In B. pasteurii cytochrome c(553), these motifs are represented by the shortest interhelix connecting fragments observed for this group of proteins. The possible relationships between heme solvent accessibility and the electrochemical reduction potential are discussed.

The sequence and X-ray structure of the trypsin from Fusarium oxysporum.

The trypsin from Fusarium oxysporum is equally homologous to trypsins from Streptomyces griseus, Streptomyces erythraeus and to bovine trypsin. A DFP (diisopropylfluorophosphate) inhibited form of the enzyme has been crystallized from 1.4 M Na2SO4, buffered with citrate at pH 5.0-5.5. The crystals belong to space group P2(1) with cell parameters a = 33.43 A, b = 67.65 A, c = 39.85 A and beta = 107.6 degrees. There is one protein molecule in the asymmetric unit. X-ray diffraction data to a resolution of 1.8 A were collected on film using synchrotron radiation. The structure was solved by molecular replacement using models of bovine and S. griseus trypsins and refined to an R-factor of 0.141. The overall fold is similar to other trypsins, with some insertions and deletions. There is no evidence of the divalent cation binding sites seen in other trypsins. The covalently bound inhibitor molecule is clearly visible.

Crystallization and structure determination to 2.5-A resolution of the oxidized [2Fe-2S] ferredoxin isolated from Anabaena 7120.

The molecular structure of the oxidized form of the [2Fe-2S] ferredoxin isolated from the cyanobacterium Anabaena species strain PCC 7120 has been determined by X-ray diffraction analysis to a nominal resolution of 2.5 A and refined to a crystallographic R factor of 18.7%. Crystals used in this investigation belong to the space group P2(1)2(1)2(1) with unit cell dimensions of a = 37.42 A, b = 38.12 A, and c = 147.12 A and two molecules in the asymmetric unit. The three-dimensional structure of this ferredoxin was solved by a method that combined X-ray data from one isomorphous heavy-atom derivative with noncrystallographic symmetry averaging and solvent flattening. As in other plant-type [2Fe-2S] ferredoxins, the iron-sulfur cluster is located toward the outer edge of the molecule, and the irons are tetrahedrally coordinated by both inorganic sulfurs and sulfurs provided by protein cysteine residues. The main secondary structural elements include four strands of beta-pleated sheet and three alpha-helical regions.