The crystal structure of penicillopepsin at 6Åresolution

A 6Åresolution electron density map of crystals of penicillopepsin, an acid protease from Penicillium janthinellum, has been computed from multiple isomorphous replacement phases determined from two heavy metal derivatives, K₂PtCl₆ and UO₂Cl₂. The mean figure of merit of the map is 0.939. The boundaries of the molecules, of which there are four per unit cell, are readily discernible. The molecule is highly asymmetric with approximate dimensions 60Å× 40Å× 30Å. The molecule consists of two distinct lobes separated by a deep cleft, which is probably the extended substrate binding site.     

Refined crystal structure of the potato inhibitor complex of carboxypeptidase A at 2.5 Å resolution

The exopeptidase carboxypeptidase A forms a tight complex with a 39 residue inhibitor protein from potatoes. We have determined the crystal structure of this complex, and refined the atomic model to a crystallographic R-factor of 0.196 at 2.5 Å resolution. The structure of the inhibitor protein is organized around a core of disulfide bridges. No α-helices or β-sheets are present in this protein, although there is one turn of 3₁₀ helix. The four carboxy-terminal residues of the inhibitor protein bind in the active site groove of carboxypeptidase A, defining binding subsites S′₁, S₁, S₂ and S₃ on the enzyme. The carboxy-terminal glycine of the inhibitor is cleaved from the remainder of the inhibitor in the complex, and remains trapped in the back of the active site pocket. Interactions between the inhibitor and residues Tyr248 and Arg71 of carboxypeptidase A resemble possible features of binding stages for substrates both prior and subsequent to peptide bond hydrolysis. Not all of these interactions would be available to different types of ester substrates, however, which may be in part responsible for the observed kinetic differences in hydrolysis between peptides and various classes of esters. With the exception of residues involved in the binding of the inhibitor protein (such as Tyr248), the structure of carboxypeptidase A as determined in the inhibitor complex is quite similar to the structure of the unliganded enzyme (Lipscomb et al., 1968), which was solved from an unrelated crystal form.     

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.     

The crystal structure of d(GTACGTAC) at 2.25 A resolution: are the A-DNA's always unwound approximately 10 degrees at the C-G steps?

The structure of the self-complementary octamer d(GTACGTAC) has been analyzed by a single crystal X-ray diffraction method at 2.25 A resolution. The crystallographic R factor was 0.184 for all 1233 reflections at this resolution. In spite of the alternating purine-pyrimidine sequence, d(GTACGTAC) adopts the A-form conformation rather than the left-handed Z-form. The average helix twist and the mean rise per base pair are 32.1 degrees and 3.18 A, respectively. The d(GTACGTAC) helix is characterized by a wide open major groove and small base-pair tilt (9.7 degrees). The partial unwinding of the helix is observed only at the central pyrimidine-purine C-G step, but not at the other pyrimidine-purine T-A steps. Based on this study and six other X-ray studies, we propose a hypothesis that the A-DNA's are always unwound approximately 10 degrees at the C-G steps. Significant differences in base-pair stacking modes are seen between the purine-pyrimidine step and the pyrimidine-purine step. All deoxyribose rings adopt the C3'-endo conformation. All backbone torsion angles fall into the range expected for the A-DNA form, except for the nucleotide G5, whose alpha and gamma torsion angles adopt the trans, trans conformation instead of the common gauche-, gauche+ conformation.     

Molybdenum active centre of DMSO reductase from Rhodobacter capsulatus: crystal structure of the oxidised enzyme at 1.82-Å resolution and the dithionite-reduced enzyme at 2.8-Å resolution

The 1.82-Å X-ray crystal structure of the oxidised (Mo^(VI)) form of the enzyme dimethylsulfoxide reductase (DMSOR) isolated from Rhodobacter capsulatus is presented. The structure has been determined by building a partial model into a multiple isomorphous replacement map and fitting the crystal structure of DMSOR from Rhodobacter sphaeroides to the partial model. The enzyme structure has been refined, at 1.82-Å resolution, to an R factor of 14.8% (R _free?=?18.4%). The molybdenum is coordinated by seven ligands: four dithiolene sulfurs, Oγ of Ser147 and two oxo groups. The four sulfur ligands, at a metal-sulfur distance of 2.4?Å or 2.5?Å, are contributed by the two molybdopterin guanine dinucleotide (MGD) cofactors. The coordination sphere of the molybdenum is different from that in previously reported structures of DMSOR from R. sphaeroides and R. capsulatus. The 2.8-Å structure of DMSOR, reduced by addition of sodium dithionite, is also described and differs from the structure of the oxidised enzyme by the removal of a single oxo ligand from the molybdenum coordination sphere. A structure, at 2.5-Å resolution, has also been obtained from crystals soaked in mother liquor buffered at pH?7.0. No differences are observed in the structure at pH?7 when compared with the native crystal structure at pH?5.5.     

Insights into base selectivity from the 1.8 Å resolution structure of an RB69 DNA polymerase ternary complex

Bacteriophage RB69 DNA polymerase (RB69 pol) has served as a model for investigating how B family polymerases achieve a high level of fidelity during DNA replication. We report here the structure of an RB69 pol ternary complex at 1.8 ? resolution, extending the resolution from our previously reported structure at 2.6 ? [Franklin, M. C., et al. (2001) Cell 105, 657-667]. In the structure presented here, a network of five highly ordered, buried water molecules can be seen to interact with the N3 and O2 atoms in the minor groove of the DNA duplex. This structure reveals how the formation of the closed ternary complex eliminates two ordered water molecules, which are responsible for a kink in helix P in the apo structure. In addition, three pairs of polar-nonpolar interactions have been observed between (i) the Cα hydrogen of G568 and the N3 atom of the dG templating base, (ii) the O5' and C5 atoms of the incoming dCTP, and (iii) the OH group of S565 and the aromatic face of the dG templating base. These interactions are optimized in the dehydrated environment that envelops Watson-Crick nascent base pairs and serve to enhance base selectivity in wild-type RB69 pol.     

The crystal structure of papain: II. A three-dimensional fourier synthesis at 4.5 Å resolution

A three-dimensional crystal structure investigation of papain to a resolution of 4.5 Å was performed by the use of the isomorphous replacement method.11° precession photographs were taken of 23 reciprocal lattice planes of the native compound and three derivatives containing p-chloromercuribenzoate, Na₂PtCl₆ and HgCl₂, respectively. The intensities were measured on an automatic integrating microdensitometer. The layers were scaled together and the intensities, after Lorentz polarization correction, were put on a previously determined absolute scale. The heavy-atom parameters, already roughly known, were refined by least squares methods. In the subsequent phase calculation a mean figure of merit of 0.85 was obtained. The absolute heavy-atom configuration could be found from anomalous dispersion measurements, which made it possible to choose the correct phases for finding the protein structure in absolute configuration from the subsequent “best” Fourier synthesis. The molecules could be separated fairly easily. They are more or less spheroidal with dimensions 36 × 48 × 36 Å. Their centres lie close to the positions previously predicted and all heavy-atom binding sites are at the surface. The structure is very complex and probably only a small number of short pieces of α-helix are present. The course of the peptide chain cannot be followed, owing to the large number of intersections. The -SH group, known to be part of the active centre, has been located. It lies at the surface in a slight depression, which enlarges the area of contact with the substrate.     

Insight into the antimicrobial mechanism of action of β2,2-amino acid derivatives from molecular dynamics simulation: Dancing the can-can at the membrane surface

The development of antimicrobial agents that target and selectively disrupt biofilms is a pressing issue since, so far, no antibiotics have been developed that achieve this effectively. Previous experimental work has found a promising set of antibacterial peptides: β^2,2-amino acid derivatives, relatively small molecules with common structural elements composed of a polar head group and two non-polar hydrocarbon arms. In order to develop insight into possible mechanisms of action of these novel antibacterial agents, we have performed an in silico investigation of four leading β^2,2-amino acid derivatives, interacting with models of both bacterial (target) and eukaryotic (host) membranes, using molecular dynamics simulation with a model with all-atom resolution. We found an unexpected result that could shed light on the mechanism of action of these antimicrobial agents: the molecules assume a conformation where one of the hydrophobic arms is directed downward into the membrane core while the other is directed upwards, out of the membrane and exposed above the position of the membrane headgroups; we dubbed this conformation the “can-can pose”. Intriguingly, the can-can pose was most closely linked to the choice of headgroup. Also, the compound previously found to be most effective against biofilms displayed the strongest extent of this behavior and, additionally, this behavior was more pronounced for this compound in the bacterial than in the eukaryotic membrane. We hypothesize that adopting the can-can pose could possibly disrupt the protective peptidoglycan macronet found on the exterior of the bacterial membrane.     

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     

Refined crystal structure of γ-chymotrypsin at 1.9 Å resolution: Comparison with other pancreatic serine proteases

The crystal structure of γ-chymotrypsin, the monomeric form of chymotrypsin, has been determined and refined to a crystallographic R-factor of 0.18 at 1.9 Å resolution. The details of the catalytic triad involving Asp102, His57 and Ser195 agree well with the results found for trypsin (Chambers & Stroud, 1979) and Streptomyces griseus protease A (Sielecki et al., 1979). As in many of the other serine proteases, the Oγ of Ser195 does not appear to be hydrogen-bonded to His57.The three-dimensional structures of γ- and α-chymotrypsin (Birktoft & Blow, 1972) are closely similar. The largest backbone differences occur in the “calcium binding loop” (residues 75 to 78) and in the “autolysis loop” (residues 146, 149 and 150). Ala149 and Asn150 are disordered in γ-chymotrypsin, whereas they are stabilized by intermolecular interactions in α-chymotrypsin. The conformation of Ser218 is also different, presumably the indirect result of the dimeric interactions of α-chymotrypsin. These results are discussed in terms of the slow, pH-dependent interconversion of α- and γ-chymotrypsin.     

NMR structure determination of the tetramerization domain of the Mnt repressor: An asymmetric α-helical assembly in slow exchange

The structure and dynamics of the chymotryptic tetramerization domain of the Mnt repressor of Salmonella bacteriophage P22 have been studied by NMR spectroscopy. Two sets of resonances (A and B) were found, representing the asymmetry within the homotetramer. Triple-resonance techniques were used to obtain unambiguous assignments of the A and B resonances. Intra-monomeric NOEs, which were distinguished from the inter-monomeric NOEs by exploiting ¹³C/¹⁵N-filtered NOE experiments, demonstrated a continuous -helix of approximately seven turns for both the A and B monomers. The asymmetry facilitated the interpretation of inter-subunit NOEs, whereas the antiparallel alignment of the subunits allowed further discrimination of inter-monomeric NOEs. The three-dimensional structure revealed an unusual asymmetric packing of a dimer of two antiparallel right-handed intertwined coiled -helices. The A and B forms exchange on a timescale of seconds by a mechanism that probably involves a relative sliding of the two coiled coils. The amide proton solvent exchange rates demonstrate a stable tetrameric structure. The essential role of Tyr 78 in oligomerization of Mnt, found by previous mutagenesis studies, can be explained by the many hydrophobic and hydrogen bonding interactions that this residue participates in with adjacent monomers.     

The influence of the N-terminal region proximal to the core domain on the assembly and chaperone activity of αB-crystallin

αB-Crystallin (HSPB5) is a small heat-shock protein that is composed of dimers that then assemble into a polydisperse ensemble of oligomers. Oligomerisation is mediated by heterologous interactions between the C-terminal tail of one dimer and the core “α-crystallin” domain of another and stabilised by interactions made by the N-terminal region. Comparatively little is known about the latter contribution, but previous studies have suggested that residues in the region 54–60 form contacts that stabilise the assembly. We have generated mutations in this region (P58A, S59A, S59K, R56S/S59R and an inversion of residues 54–60) to examine their impact on oligomerisation and chaperone activity in vitro. By using native mass spectrometry, we found that all the αB-crystallin mutants were assembly competent, populating similar oligomeric distributions to wild-type, ranging from 16-mers to 30-mers. However, circular dichroism spectroscopy, intrinsic tryptophan and bis-ANS fluorescence studies demonstrated that the secondary structure differs to wild type, the 54–60 inversion mutation having the greatest impact. All the mutants exhibited a dramatic decrease in exposed hydrophobicity. We also found that the mutants in general were equally active as the wild-type protein in inhibiting the amorphous aggregation of insulin and seeded amyloid fibrillation of α-synuclein in vitro, except for the 54–60 inversion mutant, which was significantly less effective at inhibiting insulin aggregation. Our data indicate that alterations in the part of the N-terminal region proximal to the core domain do not drastically affect the oligomerisation of αB-crystallin, reinforcing the robustness of αB-crystallin in functioning as a molecular chaperone.     

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.     

Three-dimensional structure of deoxycholate-treated purple membrane at 6 Å resolution and molecular averaging of three crystal forms of bacteriorhodopsin

The three-dimensional structure of the deoxycholate-treated form of purple membrane has been determined to a resolution of about 6 Å. Using low temperature electron diffraction data, room temperature electron microscope images and improved methods of data analysis, higher resolution has been reached than was obtained using native membranes of the same size. Statistical analysis of the data shows that the new map is considerably better than earlier maps. The map indicates the probable sites for the lipid molecules that remain in the deoxycholate-treated membranes; some of these sites differ from those suggested by the projection map of Glaeser et al. (1985). Comparison of the bacteriorhodopsin structures now determined independently from three crystal forms shows that the monomer structure is independent of the detailed contacts with lipid molecules. The average of the three structures gives a picture with very little noise showing seven similar rod-like features which are clearly best interpreted as -helices; there is no indication that part of the structure is -sheet as suggested by Jap et al. (1983). Phases from the averaged structure at 6 Å resolution will enable better refinement of the parameters that will be required in the analysis of higher resolution images from tilted specimens needed to extend the projection map at 3.5 Å resolution (Henderson et al. 1986) to produce a three-dimensional atomic resolution map.     

The structure of the FMO protein from Chlorobium tepidum at 2.2 Å resolution

The bacteriochlorophyll protein, or FMO protein, from Chlorobium tepidum, which serves as a light-harvesting complex and directs light energy from the chlorosomes attached to the cell membrane to the reaction center has been crystallized in a new space group. The crystals belong to the cubic space group P4₃32 and the structure has been refined to a resolution 2.2 Å with a R factor of 19.7%. The electron density maps show that the structure is composed of two sheets that surround seven bacteriochlorophylls as previously reported (Li et al. (1997) J Mol Biol 271: 456–471). The availability of the new data allows a more accurate refinement of the pigment–protein complex including identification of bound solvent molecules. Several structural differences probably contribute to the observed spectroscopic differences between the FMO proteins from Cb. tepidum and Prosthecochloris aestuarii, including differences in the planarity of corresponding tetrapyrroles. A citrate molecule is found on the surface of each protein subunit of the trimer from Cb. tepidum. However, the citrate molecule is over 15 Å from any bacteriochlorophyll. The presence of the citrate probably does not contribute to the function of the protein although it does contribute to the crystallization as it interacts with a crystallographically related trimer. Among the 236 water molecules found in the protein are four that appear to play a special role in the properties of bacteriochlorophyll 2, as this tetrapyrrole is coordinated by one of these water molecules and the waters form a hydrogen-bonded network that leads to the surface of the protein.This revised version was published online in October 2005 with corrections to the Cover Date.