Structure and refinement of penicillopepsin at 1.8 A resolution

Penicillopepsin, the aspartyl protease from the mould Penicillium janthinellum, has had its molecular structure refined by a restrained-parameter least-squares procedure at 1.8 Å resolution to a conventional R-factor of 0.136. The estimated co-ordinate accuracy for the majority of the 2363 atoms of the enzyme is better than 0.12 Å. The average atomic thermal vibration parameter, B, for the atoms of the enzyme is 14.5 Ų. One determining factor of this low average B value is the large central hydrophobic core, in which there are two prominent clusters of aromatic residues, one of nine, the other of seven residues. The N and C-terminal domains of penicillopepsin display an approximate 2-fold symmetry: 70 residue pairs are topologically equivalent, related by a rotation of 177 ° and a translation of 1.2 Å. The analysis of the secondary structural features of the molecule reveals non-linear hydrogen bonding. In penicillopepsin, there is no difference in the mean hydrogen-bond parameters for the elements of α-helix, parallel or antiparallel β-pleated sheet. The mean values for these structural elements are: NO, 2.90 Å; NHO, 1.95 Å; N?O, 160 °. The average hydrogen-bond parameters of the reverse β-turns and the 3₁₀ helices are distinctly different from the above values. The analysis of sidechain conformational angles χ¹ and χ² penicillopepsin and other enzyme structures refined in this laboratory shows much narrower distributions as compared with those compiled from unrefined protein structures. The close proximity of the carboxyl groups of Asp33 and Asp213 suggests that they share a proton in a tight hydrogen-bonded environment (Asp33OD2 to Asp213OD1 is 2.87 Å). There are several solvent molecules in the active site region and, in particular, O39 forms hydrogen-bonded interactions with both aspartate residues. The disposition of the two carboxyl groups suggests that neither is likely to be involved in a direct nucleophilic attack on the scissile bond of a substrate. The average atomic B-factors of the residues in this region of the molecule are between 5 and 8 Ų, confirming the proposal that conformational mobility of the active site residues has no role in the enzymatic mechanism. However, conformational mobility of neighbouring regions of the molecule e.g. the “flap” containing Tyr75, is verified by the high B-factors for those residues. The positions of 319 solvent sites per asymmetric unit have been selected from difference electron density maps and refined. Thirteen have been classified as internal, and several of these may have key roles during catalysis. The positively charged N^ζ atom of Lys304 forms hydrogen bonds to the carboxylate of Asp14 (internal ion pair) and to two internal water molecules O5 and O25. The protonated side-chain of Asp300 forms a hydrogen bond to Thr214O, 2.78 Å, and is the recipient of a hydrogen bond from a surface pocket water molecule O46. There is no possibility for direct interaction between Asp300 and Lys304 without large conformational changes of their environment. The intermolecular packing involves many protein-protein contacts (66 residues) with a large number of solvent molecules involved in bridging between polar residues at the contact surface. The penicillopepsin molecules resemble an approximate hexagonal close-packing of spheres with each molecule having 12 “nearest” neighbours.     

Crystallization,crystal structure analysis and molecular model of the third domain of Japanese quail ovomucoid,a Kazal type inhibitor

The third domain of Japanese quail ovomucoid, a Kazal type inhibitor, has been crystallized and its crystal structure determined at 2.5 Å resolution using multiple isomorphous replacement techniques. The asymmetric unit contains four molecules. In the crystal the molecules are arranged in two slightly different octamers with approximate D4 symmetry. The molecules are held together mainly by interactions of the N-terminal residues, which form a novel secondary structural element, a β-channel.The molecule is globular with approximate dimensions 35 Å × 27 Å × 19 Å. The secondary structural elements are a double-stranded anti-parallel β-sheet of residues Pro22 to Gly32 and an α-helix from Asn33 to Ser44. The reactive site Lys18-Asp19 is located in an exposed loop. It is close to Asn33 at the N terminus of the helical segment. The polypeptide chain folding of ovomucoid bears some resemblance to other inhibitors in the existence of an anti-parallel double strand following the reactive site loop.     

The Crystal Structure of Glutamine-binding Protein fromEscherichia coli

The crystal structure of the glutamine-binding protein (GlnBP) fromEscherichia coliin a ligand-free “open” conformational state has been determined by isomorphous replacement methods and refined to anR-value of 21.4% at 2.3 Å resolution. There are two molecules in the asymmetric unit, related by pseudo 4-fold screw symmetry. The refined model consists of 3587 non-hydrogen atoms from 440 residues (two monomers), and 159 water molecules. The structure has root-mean-square deviations of 0.013 Å from “deal” bond lengths and 1.5° from “ideal” bond angles.The GlnBP molecule has overall dimensions of approximately 60 Å × 40 Å × 35 Å and is made up of two domains (termed large and small), which exhibit a similar supersecondary structure, linked by two antiparallel β-strands. The small domain contains three α-helices and four parallel and one antiparallel β-strands. The large domain is similar to the small domain but contains two additional α-helices and three more short antiparallel β-strands. A comparison of the secondary structural motifs of GlnBP with those of other periplasmic binding proteins is discussed.A model of the “closed form” GlnBP-Gln complex has been proposed based on the crystal structures of the histidine-binding protein-His complex and “open form” GlnBP. This model has been successfully used as a search model in the crystal structure determination of the “closed form” GlnBP-Gln complex by molecular replacement methods. The model agrees remarkably well with the crystal structure of the Gln-GlnBP complex with root-mean-square deviation of 1.29 Å. Our study shows that, at least in our case, it is possible to predict one conformational state of a periplasmic binding protein from another conformational state of the protein. The glutamine-binding pockets of the model and the crystal structure are compared and the modeling technique is described.     

A crystallographic model for azurin a 3 A resolution.

The structure of the blue copper protein azurin (M_r 14,000) from Pseudomonas aeruginosa has been determined from a 3.0 Å resolution electron density map computed with phases based on a uranyl derivative to 3 Å resolution and a platinum derivative to 3.7 Å. Interpretation of the somewhat noisy map was based on comparison of the density of the four molecules in the asymmetric unit with their averaged density. The polypeptide chain folds into an eight-strand β barrel with an additional flap containing a short helix. The copper atom is bound at one end and on the inside of the barrel, probably to a cysteine, a methionine, and two histidine residues.     

Sequencing a protein by x-ray crystallography. II. Refinement of yeast hexokinase B co-ordinates and sequence at 2.1 A resolution.

Although the amino acid sequence of yeast hexokinase B has not been determined by chemical means, crystallographic refinement of the hexokinase monomer was carried out at 2.1 Å resolution to improve both the atomic co-ordinates and the amino acid sequence, which had been obtained from a 2.5 Å electron density map. The atomic co-ordinates were adjusted by real-space refinement into a multiple isomorphous replacement map, followed by automated difference Fourier refinement, and restrained parameter structure factor least-squares refinement. The amino acid sequence was altered periodically after visual inspection of (F_o ? F_c) difference electron density maps. Evidence of the improvement in the amino acid sequence was provided by the better agreement between the X-ray and chemically derived amino acid compositions, and most importantly by the ability to locate two short peptides which had been chemically sequenced. While only 6 out of the 18 residues in these two peptides agree with the sequence of the original model, 12 residues agree with the sequence of the refined model and the others differ by only an atom or two. The refined model contains 3293 of of the 3596 non-hydrogen atoms expected from the amino acid composition and 152 bound water molecules. The crystallographic R factor at 2.1 Å is 0.25.We show that there are several advantages to refining the structure of even a protein of unknown sequence. (1) Improved phases can be obtained to the resolution limit of the diffraction pattern starting with a model derived from a 2.5 Å map. (2) The accuracy of the amino acid sequence derived by X-ray methods alone can be substantially improved. (3) Functionally important residues can be identified before chemical sequence information is available. (4) The improved X-ray sequence should greatly reduce the effort required to obtain a chemical sequence; since peptides as short as eight or nine residues can be located in the refined X-ray sequence, peptides do not need to be overlapped by chemical means.     

Structure of 2-keto-3-deoxy-6-phosphogluconate aldolase at 2.8 Å resolution

The structure of 2-keto-3-deoxy-6-phosphogluconate aldolase has been extended to 2.8 Å resolution from 3.5 Å resolution by multiple isomorphous replacement methods using three heavy-atom derivatives and anomalous Bijvoet differences to 6 Å resolution (〈m〉 = 0.72). The replacement phases were improved and refined by electron density modification procedures coupled with inverse transform phase angle calculations. A Kendrew model of the molecule was built, which contained all 225 residues of a recently determined amino acid sequence, whereas only 173 were accounted for at 3.5 Å resolution. The missing residues were found to be part of the interior of the molecule and not simply an appendage. The molecule folds to form an eight-strand α/β-barrel structure strikingly similar to triosephosphate isomerase, the A-domain of pyruvate kinase and Taka amylase. With a knowledge of the sequence, the nature of the interfaces of the two kinds of crystallographic trimers have been examined, from which it was concluded that the choice of trimers selected in the 3.5 Å resolution work was probably correct for trimers in solution. The active site region has been established from the position of the Schiff base forming Lys144 but it has not been possible to confirm it conclusively in independent derivative experiments. An apparent anomaly exists in the location of Glu56 (about 25 Å from Lys144). The latter has been reported to assist in catalysis.     

Crystal structure of p-hydroxybenzoate hydroxylase.

The structure of the enzyme p-hydroxybenzoate hydroxylase (EC 1.14.13.2) in a complex with its substrate has been determined at a resolution of 2.5 Å. The molecular weight is 43,000 and the dimensions of one molecule are approximately 70 Å × 50 Å × 45 Å. The crystal structure contains dimers of these molecules. Approximately 16% of the residues occur in β-sheets and 26% in α-heliees. The molecule can be divided into three domains. The active site, near the isoalloxazine ring, is formed by side-chains of the three domains. The N-5 edge of the isoalloxazine ring points to p-hydroxybenzoate, which is bound in a deep cleft.     

Refined 2.5 Å X-ray crystal structure of the complex formed by porcine kallikrein A and the bovine pancreatic trypsin inhibitor: Crystallization,patterson search,structure determination,refinement, structure and comparison with its components and with the bovine trypsin-pancreatic trypsin inhibitor complex

The complex formed by porcine pancreatic kallikrein A with the bovine pancreatic trypsin inhibitor (PTI) has been crystallized at pH 4 in tetragonal crystals of space group P4₁2₁2 with one molecule per asymmetric unit. Its crystal structure has been solved applying Patterson search methods and using a model derived from the bovine trypsin-PTI complex (Huber et al., 1974) and the structure of porcine pancreatic kallikrein A (Bode et al., 1983). The kallikrein-PTI model has been crystallographically refined to an R-value of 0·23 including X-ray data to 2·5 Å.The root-mean-square deviation, including all main-chain atoms, is 0·45 Å and 0·65 Å for the PTI and for the kallikrein component, respectively, compared with the refined models of the free components. The largest differences are observed in external loops of the kallikrein molecule surrounding the binding site, particularly in the C-terminal part of the intermediate helix around His172. Overall, PTI binding to kallikrein is similar to that of the trypsin complex. In particular, the conformation of the groups at the active site is identical within experimental error (in spite of the different pH values of the two structures). Ser195 OG is about 2·5 Å away from the susceptible inhibitor bond Lys15 C and forms an optimal 2·5 Å hydrogen bond with His57 NE.The PTI residues Thr11 to Ile18 and Val34 to Arg39 are in direct contact with kallikrein residues and form nine intermolecular hydrogen bonds. The reactive site Lys15 protrudes into the specificity pocket of kallikrein as in the trypsin complex, but its distal ammonium group is positioned differently to accommodate the side-chain of Ser226. Ser226 OG mediates the ionic interaction between the ammonium group and the carboxylate group of Asp189. Model-building studies indicate that an arginine side-chain could be accommodated in this pocket. The PTI disulfide bridge 14–38 forces the kallikrein residue Tyr99 to swing out of its normal position. Model-building experiments show that large hydrophobic residues such as phenylalanine can be accommodated at this (S2) site in a wedge-shaped hydrophobic cavity, which is formed by the indole ring of Trp215 and by the phenolic side-chain of Tyr99, and which opens towards the bound inhibitor/substrate chain. Arg17 in PTI forms a favorable hydrogen bond and van der Waals' contacts with kallikrein residues, whereas the additional hydrogen bond formed in the trypsin-PTI complex between Tvr39 OEH and Ile19 N is not possible The kallikrein binding site offers a qualitative explanation of the unusual binding and cleavage at the N-terminal Met-Lys site of kininogen. Model-building experiments suggest that the generally restricted capacity of kallikrein to bind protein inhibitors with more extended binding segments might be explained by steric hindrance with some extruding external loops surrounding the kallikrein binding site (Bode et al., 1983).     

Structure of rabbit skeletal myosin: Analysis of the amino acid sequences of two fragments from the rod region

The amino acid sequences of fragments from light meromyosin and heavy meromyosin subfragment-2 have been analysed and structural features noted. As with other α-fibrous protein sequences, there is a regular disposition of apolar residues in positions a and d of the heptapeptide-type repeat characteristic of the coiled-coil conformation. The common occurrence of acidic and basic residues in the e and g positions, respectively, give rise to a maximum number of interchain ionic interactions when the two parallel chains of myosin are in axial register. Although the quasi-repeating heptapeptides in the sequences both have two points of discontinuity (unlike that in most other α-fibrous proteins), secondary structure prediction methods indicate that the fragments will be 90 to 100% α-helical. Fast Fourier transform techniques have revealed a significant periodicity of about 27.4 ± 0.3 residues (~41 Å) in the linear disposition of the acidic residues and the basic residues in both of the fragments. This period is compatible with similarly directed myosin molecules in the thick filament being axially staggered with respect to one another by an odd multiple of 143 Å. Preliminary evidence is also presented to show that the sequence of the rod region of myosin may have a 28 residue gene duplication repeat.     

Three-dimensional image reconstruction of straight flagella from a mutant Salmonella typhimurium.

The structure of the straight flagella from a mutant Salmonella typhimurium was studied by electron microscopy using digital image processing, including three-dimensional reconstruction, to an effective resolution of about 14 Å.Three-dimensional studies suggest that there are two sets of intersubunit bonds, i.e. intraprotofilament bonds along the (n = 11, l = 1) helix at a radius of about 55 Å and interprotofilament bonds along the (n = ?5, l = 7) helix at radii of about 10 to 15 Å and 50 Å, and along the (n = 6, l = 8) helix at a radius of about 45 Å and along the (n = 1, l = 15) helix at a radius of about 20 Å. There are four high density regions in a morphological subunit. These regions are situated at radii of about 15 Å, 40 Å, 70 Å and 80 Å. Variation was seen in the position of the high density regions at radii of about 15 Å and 40 Å among the ten models that were reconstituted individually. The regions at radii of 40 Å and 70 Å are the highest in density. The radial distance between these two regions is consistent with the 32 Å feature of a cylindrically averaged Patterson function calculated using equatorial data from X-ray diffraction pattern (Champness, 1968,1971).At the outer radii of the flagellum the shape of the morphological subunit roughly corresponded to that of the “chevron” described by O'Brien &; Bennett (1972), but there was no corresponding structure at the inner radii; the appearance of chevrons in that region might arise from the superposition of the two sides of the helical lattice.The biological significance of the “beaded” submolecular structure of flagellin and the presence of two sets of intersubunit bonds at the different radii is discussed with reference to the waveform and polymorphic behaviour of flagellar filaments.     

The double-helical molecular structure of crystalline b-amylose

The crystal structure of the B-polymorph of amylose appears to be based on double-stranded helices. The individual strands are in a right-handed six-fold helical conformation repeating in 20.8 Å and are wound parallel around each other. The steric disposition of O-6 is gt. The double helices pack in a hexagonal unit-cell (a  b  18.50 Å, c (fiber repeat)  10.40 Å, γ  120°), with two helices (12 d-glucose residues) per cell. The helices are packed antiparallel and leave an open channel within a hexagonal array that is filled with water molecules. The reliability of the structure analysis is indicated by R  0.22. The structure of B-amylose is consistent with the diffraction diagrams of B-starches and accounts for the physical properties of such starches.     

Evolution of proteins formed by β-sheets: I. Plastocyanin and azurin

Plastocyanin and azurin form a family of small copper-containing proteins, active in the electron transport systems of plants and bacteria, respectively. The crystal structures of two members of this family have been determined: poplar leaf plastocyanin and Pseudomonas aeruginosa azurin. Both proteins contain two β-sheets, packed face-to-face. Using computed superpositions of the structures, we have aligned the sequences, identified homologous positions, and studied how the structures have changed as a result of mutations.The residues in the vicinity of the copper-binding site show minimal amino acid substitution and form almost identical structures. Other portions of these proteins are more variable in sequence and in structure. Buried residues tend to maintain their hydrophobic character, but mutations change their volume. The mean variation in volume of homologous buried residues is 54 ų. The differences in size and shape of these buried residues are accommodated by a 3.8 Å shift in relative position of the packed β-sheets. This shift does not affect the copper binding site, because the residues that form this site are in, or adjacent to, just one of the β-sheets.     

Molecular structure of the alpha-lytic protease from Myxobacter 495 at 2.8 Angstroms resolution.

The α-lytic protease was isolated from an extracellular filtrate of the soil microorganism Myxobacter 495. Trigonal crystals (space group, P3₂21) of this serine enzyme were grown from 1·3 m-Li₂SO₄ at pH 7·2. X-ray reflections from crystals of the native enzyme, comprising the 2·8 Å limiting sphere, were phased by the multiple isomorphous replacement technique. Five heavy-atom derivatives were used and the overall mean figure of merit 〈m?〉 is 0·83. The resulting native electron density map of α-lytic protease has been interpreted in conjunction with the published sequence (Olson et al., 1970) of 198 amino-acid residues.α-Lytic protease has a structural core similar to that of the pancreatic serine proteases (108 α-carbon atom positions are topologically equivalent (within 2·0 Å) to residues of porcine elastase) and its tertiary structure is even more closely related to the two other bacterial serine protease structures previously determined (James et al., 1978; Brayer et al., 1978b; Delbaere et al., 1979a). α-Lytic protease has the following distinctive features in common with the bacterial serine enzymes, Streptomyces griseus proteases A and B: an amino terminus that is exposed to solvent on the enzyme surface, a considerably shortened uranyl loop (residues 65 to 84), a major segment of polypeptide chain from the autolysis loop deleted (residues 144 to 155), a buried guanidinium group of Arg138 in an ion-pair bond with Asp194, and an altered conformation of the methionine loop (residues 168 to 182) relative to the pancreatic enzymes.At the present resolution, the members of the catalytic quartet (Ser214, Asp102, His57 and Ser195) adopt the conformation found in all members of the Gly-Asp-Ser-Gly-Gly serine protease family. The carboxylate of Asp102 is in a highly polar environment, as it is the recipient of four hydrogen bonds. The interaction between the N^ε2 atom of the imidazole ring in His57 and O^γ atom of Ser195 is very weak (3·3 Å) and supports the concept that there is little, if any, enhanced nucleophilicity of the side-chain of Ser195 in the native enzyme.The molecular basis for the observed substrate specificity of α-lytic protease is clear from the distribution of amino acid side-chains in the neighborhood of the active site. An insertion of five residues at position 217, and the conformation of the side-chain of Met192 account for the fact that the specificity pocket can bind only small residues, such as Ala, Ser or Val.     

Escherichia coli lac repressor is elongated with its operator DNA binding domains located at both ends

From small-angle X-ray scattering experiments on solutions of Escherichia coli lac repressor and repressor tryptic core, we conclude that the domains of repressor that bind to operator DNA lie at the ends of an elongated molecule. The addition of the inducer, isopropyl-β-d-thiogalactoside, to either repressor or core does not produce a measurable structural change, since the radius of gyration of repressor is 40.3 ± 1.9 Å without and 42.2 ± 1.7 Å with isopropyl-β-d-thiogalactoside; the core radius of gyration is 35.4 ± 1.1 Å without ligand and 36.3 ± 1.1 Å with isopropyl-β-d-thiogalactoside. In the context of data from single crystals of repressor and core, the measured radii of gyration are shown to be consistent with a core (or repressor) molecule of dimensional anisotropy 1: (1.5 to 2.0): (3.0 to 4.0). The 5 Å difference in radius of gyration between native and core repressor is interpreted to mean that the amino terminal 59 residues (headpieces) lie at the ends of an elongated repressor molecule. This structure implies that the repressor may have DNA binding sites, consisting of two adjacent headpieces, on each end of the molecule and this binds to the DNA with its long axis perpendicular to the DNA.     

Analysis of the primary structure of collagen for the origins of molecular packing

The amino acid sequence in the triplet region of the α1 chain of collagen was analyzed for complementary relationships that would explain the stagger of multiples of 670 Å between the rod-like molecules in the fibril. The analysis was done by moving the sequence of 1011 amino acids past itself and scoring for complementarity between opposing amino acids allowing a range of ±2 to 3 residues. It was found that interactions between amino acids of opposite charge and between large hydrophobic amino acids in the overlapping region between two chains are maximal when the chains are staggered by 0D, 1D, 2D, 3D and 4D, where D = 234 ± 1 residues. The residue repeat derived from this value is 2.86 ± 0.02 Å. The existence of a D separation between interacting residues was shown to be reflected in the actual distribution of large hydrophobic amino acids. Surprisingly, the distribution approximates the pattern ($\text{2D}\text{11}$)₅($\text{D}\text{11}$) repeated over 4.4D intervals. The regularity may arise from structural constraints imposed by super-coiling. The distribution of charged residues is less regular and does not show a well-defined periodicity. However, positively-charged residues tend to be near negatively-charged residues, allowing intramolecular charge neutralization as well as strong intermolecular charge interactions at 0D.     

The crystallographic structure of Panicum Mosaic Virus (PMV)

The structure of Panicum Mosaic Virus (PMV) was determined by X-ray diffraction analysis to 2.9 Å resolution. The crystals were of pseudo symmetry F23; the true crystallographic unit cell was of space group P2₁ with a = 411.7 Å, b = 403.9 Å and c = 412.5 Å, with β = 89.7°. The asymmetric unit was two entire T = 3 virus particles, or 360 protein subunits. The structure was solved by conventional molecular replacement from two distant homologues, Cocksfoot Mottle Virus (CfMV) and Tobacco Necrosis Virus (TNV), of ～20% sequence identity followed by phase extension. The model was initially refined with exact icosahedral constraints and then with icosahedral restraints. The virus has Ca⁺⁺ ions octahedrally coordinated by six aspartic acid residues on quasi threefold axes, which is completely different than for either CfMV or TNV. Amino terminal residues 1–53, 1–49 and 1–21 of the A, B and C subunits, respectively, and the four C-terminal residues (239–242) are not visible in electron density maps. The additional ordered residues of the C chain form a prominent “arm” that intertwines with symmetry equivalent “arms” at icosahedral threefold axes, as was seen in both CfMV and TNV. A 17 nucleotide hairpin segment of genomic RNA is icosahedrally ordered and bound at 60 equivalent sites at quasi twofold A–B subunit interfaces at the interior surface of the capsid. This segment of RNA may serve as a conformational switch for coat protein subunits, as has been proposed for similar RNA segments in other viruses.     

Spatial organization of catalase proteins

The three-dimensional structure of the heme-containing fungal catalase fromPenicillium vitale (m.m. 2,80,000) has been studied by X-ray analysis at 2.0 A resolution. The molecule is tetramer, each subunit contains 670 aminoacid residues identified to construct “X-ray” primary structure. The subunit is built of three compact domains and their connections. The first domain of about 350 residues contains aβ-barrel flanked by helices, the second domain of 70 residues is formed by four helices and the third one is composed of 150 residues and is topologically similar to flavodoxin. The active site including heme is deeply buried near theβ-barrel. A comparison of the structure of catalase fromPenicillium vitale with that of beef liver catalase revealed very close structural homology of the first and the second domain, but the third domain is entirely absent in beef liver catalase. A catalase from thermophillic bacteriaThermus thermophilus (m.m. 2,10,000) has been first isolated, crystallized and studied by X-ray analysis. Crystals are cubic, space group is P2₁3, a = 133.4 Å. The molecule is a hexamer with trigonal symmetry 32. The electron density map at 3 Å resolution made it possible to trace the polypeptide chain. The main structural motif is formed by four near parallel helices. There is no heme inThermus thermophilus catalase, the active site is between the four helices and contains two manganese ions.     

Crystal Structure of Three Consecutive Laminin-type Epidermal Growth Factor-like (LE) Modules of Laminin γ1 Chain Harboring the Nidogen Binding Site

The structure of three consecutive laminin-type EGF-like (LE) modules of mouse laminin γ1 chain, γ1III3-5 (positions 738 to 899), has been determined by multiple isomorphous replacement in a crystal of space groupP6₄22 (a=b=74.57 Å,c=185.11 Å and γ=120°). The crystal structure was refined using restrained crystallographic refinement to an R-factor of 19.72 % for 14,983 independent reflections with intensitiesF_obs> 0 at 2.1 Å resolution, with root mean square deviations of 0.012 Å and 1.690° from ideal bond lengths and bond angles, respectively. The final model consisted of 1179 (non-hydrogen) protein atoms within 162 residues and 119 water molecules. The molecule showed a rod-like structure of about 76 Å length with individual modules twisted relative to each other by about 70°. Each module had the same disulfide bond connections Cys1-Cys3 (loop a), Cys2-Cys4 (loop b), Cys5-Cys6 (loop c) and Cys7-Cys8 (loop d), the first three being identical to epidermal growth factor (EGF). All three LE modules showed little secondary structure which was mainly restricted to loop d, but they differed in several other details of their structure. The interface contacts between the LE modules are based on hydrogen bonds and hydrophobic interactions between the hydrophobic core of loop d of the preceding module and the first cysteine and an exposed residue in loop b of the following module. Module 4 was previously shown to contribute the major nidogen binding site of laminins and site-directed mutagenesis demonstrated a specific binding role for Asp800, Asn802, Val804 and Tyr819 in loops a and c. The side-chains of these four residues are all located on the surface in a linear array and separated by a distance of 17 Å between Tyr819 and Val804. The entire nidogen binding site is stabilizedviamain-chain hydrogen bonds which are in part derived from the link between loops b and c (residues Leu815 and Lys816). The data demonstrate the unique nature of the LE modules and only a remote similarity to EGF. They also indicate that the crucial residues in the binding loops provide direct contacts with nidogen and explain the synergism between loops a and c which is essential for binding.     

Terminase Subunits from the Pseudomonas-Phage E217

Pseudomonas phages are increasingly important biomedicines for phage therapy, but little is known about how these viruses package DNA. This paper explores the terminase subunits from the Myoviridae E217, a Pseudomonas-phage used in an experimental cocktail to eradicate P. aeruginosa in vitro and in animal models. We identified the large (TerL) and small (TerS) terminase subunits in two genes ～58 kbs away from each other in the E217 genome. TerL presents a classical two-domain architecture, consisting of an N-terminal ATPase and C-terminal nuclease domain arranged into a bean-shaped tertiary structure. A 2.05 Å crystal structure of the C-terminal domain revealed an RNase H-like fold with two magnesium ions in the nuclease active site. Mutations in TerL residues involved in magnesium coordination had a dominant-negative effect on phage growth. However, the two ions identified in the active site were too far from each other to promote two-metal-ion catalysis, suggesting a conformational change is required for nuclease activity. We also determined a 3.38 Å cryo-EM reconstruction of E217 TerS that revealed a ring-like decamer, departing from the most common nonameric quaternary structure observed thus far. E217 TerS contains both N-terminal helix-turn-helix motifs enriched in basic residues and a central channel lined with basic residues large enough to accommodate double-stranded DNA. Overexpression of TerS caused a more than a 4-fold reduction of E217 burst size, suggesting a catalytic amount of the protein is required for packaging. Together, these data expand the molecular repertoire of viral terminase subunits to Pseudomonas-phages used for phage therapy.     

Crystallographic study of turkey egg-white lysozyme and its complex with a disaccharide

The crystal structure of turkey egg-white lysozyme, determined by the molecular replacement method at 5 Å resolution (Bott & Sarma, 1976) has now been refined to 2.8 Å resolution and a model has been built to fit the electron density. A comparison of the co-ordinates with those of hen lysozyme indicate a rootmean-square deviation of 1.6 Å for all the main-chain and side-chain atoms. A significant difference is observed in the region of residues 98 to 115 of the structure. The molecules are packed in this crystal form with the entire length of the active cleft positioned in the vicinity of the crystallographic 6-fold axis and is not blocked by neighboring molecules. A difference electron density map calculated between crystals of turkey lysozyme soaked in a disaccharide of N-acetyl glucosamine—N-acetyl muramic acid and the native crystals showed a strong positive peak at subsite C, a weak positive peak at subsite D and two strong peaks that correspond to the subsite E and a new subsite F′. This new site F′ is different from the subsite F predicted for the sixth saccharide from model building in hen lysozyme. The interactions between the saccharides bound at subsites E and F′ and the enzyme molecules are discussed.