Search

The parameters used to evaluate biomacromolecular crystal quality [Rmerge, I/sigma(I), maximum resolution and mosaicity] strongly depend on the experimental diffraction conditions. In this paper, the distinctive features of the relative Wilson plot method are described and it is shown that the overall B factor obtained from this plot is more appropriate for the characterization of protein crystals. The relative Wilson plot has been applied to the characterization of crystals of the B-DNA decamer d(CCATTAATGG) and crystals of the proteins DsrD (dissimilatory sulfite reductase D) and hen egg-white lysozyme (HEWL), which were studied by neutron diffraction. It was found that the crystal quality of the B-DNA decamer and DsrD depended significantly on the regions of the crystallization phase diagram from which the samples were taken. However, in the case of HEWL crystal quality appears to be independent of the region of the crystallization phase diagram., (Copyright 2004 International Union of Crystallography)

The crystal structure of a self-complementary RNA duplex r(GGCGBrUGCGCU)2 with terminal G·U and internal tandem G·U base pairs has been determined at 2.1 Å resolution. The crystals belong to the tetragonal space group P43, with unit-cell parameters a = b = 37.69, c = 96.28 Å and two duplexes in the asymmetric unit. The two strands of each duplex are related by a pseudodyad axis. The structure was refined to final Rwork and Rfree values of 20.9 and 25.3%, respectively. The duplexes stack in an end-to-end manner, forming infinite columns along the c axis. This is the first structural study of an RNA duplex containing G·U pairs at the termini. The stacking overlaps of the terminal G·U base pairs with their adjacent Watson–Crick base pairs are larger than those of Watson–Crick base pairs of the 5′-YR-3′/3′-RY-5′ type. The terminal G·U base pairs of neighbouring duplexes are also stacked with each other. An alternating underwound–overwound pattern of the twist angles is seen at each step along the duplex. This observation is typical for internal tandem G·U pairs, while the terminal G·U base pairs exhibit high twist angles with the adjacent Watson–Crick pairs. The 3′-­side of U of the internal G·U base pair, which is unstacked, appears to be stabilized by π-cation interaction with an Mg2+ ion. [ABSTRACT FROM AUTHOR]

EMS16 is a member of the snake venom-derived C-type lectin family of proteins (CLPs) found in the venom of Echis multisquamatus. It binds to glycoprotein Ia/IIa (integrin α2β1), a major collagen receptor of platelets, acting as a potent antagonist of platelet aggregation and cell migration. Amino acid sequencing and cDNA cloning of EMS16 have revealed that it is composed of an A chain of 134 amino acid residues and a B chain of 128 residues. Crystals of EMS16 belong to space group P212121, with unit-cell parameters a = 46.57, b = 59.93, and c = 115.74 Å, and diffract to a resolution of 1.9 Å. Phase determination is underway by means of molecular replacement with the structure of blood coagulation factor IX-binding protein (IX-bp) from habu snake venom (PDB code 1bj3) as the search model. [ABSTRACT FROM AUTHOR]

The crystal structure of a thermostable a-amylase from Bacillus stearothermophilus (BSTA) has been determined at 2.0 A resolution. The main-chain fold is almost identical to that of the known crystal structure of Bacillus licheniformis α-amylase (BLA). BLA is known to be more stable than BSTA. A structural comparison between the crystal structures of BSTA and BLA showed significant differences that may account for the difference in their thermostabilities, as follows, (i) The two-residue insertion in BSTA, De181- Gly182, pushes away the spatially contacting region including Asp207, which corresponds to Ca2+-coordinating Asp204 in BLA. As a result, Asp207 cannot coordinate the Ca2+. (ii) BSTA contains nine fewer hydrogen bonds than BLA, which costs about 12 kcal/mol. This tendency is prominent in the (β/α)8-barrel, where 10 fewer hydrogen bonds were observed in BSTA. BLA forms a denser hydrogen bond network in the interhelical region, which may stabilize a-helices in the barrel, (iii) A few small voids observed in the α-helical region of the (β/α)8-barrel in BSTA decrease inter-helical compactness and hydrophobic interactions, (iv) The solvent-accessible surface area of charged residues in BLA is about two times larger than that in BSTA. [ABSTRACT FROM AUTHOR]

Cyclic nucleotide-gated (CNG) ion channels play pivotal roles in sensory transduction of retinal and olfactory neurons. The elapid snake toxins pseudechetoxin (PsTx) and pseudecin (Pdc) are the only known protein blockers of CNG channels. These toxins are structurally classified as cysteinerich secretory proteins and exhibit structural features that are quite distinct from those of other known small peptidic channel blockers. This article describes the crystallization and preliminary X-ray diffraction analyses of these toxins. Crystals of PsTx belonged to space group P212121, with unit-cell parameters a = 60.30, b = 61.59, c = 251.69 Å, and diffraction data were collected to 2.25 Å resolution. Crystals of Pdc also belonged to space group P212121, with similar unit-cell parameters a = 60.71, b = 61.67, c = 251.22 Å, and diffraction data were collected to 1.90 Å resolution. [ABSTRACT FROM AUTHOR]

Piscivostatin (PVS) and acostatin are members of the disintegrin family of platelet-aggregation inhibitors found in snake venoms and are dimeric disintegrins which play different roles to the monomeric disintegrins. The crystals of PVS belonged to the P212121 space group, with unit-cell parameters a = 34.3, b = 54.4, c = 115.9 Å, and diffracted to 2.0 Å resolution. The crystals of acostatin belonged to the P21 space group, with unit-cell parameters a = 55.4, b = 69.3, c = 63.5 Å, β = 111.7°, and diffracted to 2.8 Å resolution. [ABSTRACT FROM AUTHOR]

The cysteine-rich secretory proteins (CRISPs) are widely distributed in mammals, reptiles, amphibians and secernenteas, and are involved in a variety of biological reactions. Here we report the crystal structure of triflin, a snake venom derived blocker of high K+-induced artery contraction, at 2.4Å resolution. Triflin consists of two domains. The first 163 residues form a large globular body with an α–β–α sandwich core, which resembles pathogenesis-related proteins of group-1 (PR-1). Two glutamic acid-associated histidine residues are located in an elongated cleft. A Cd2+ resides in this binding site, and forms a five-coordination sphere. The subsequent cysteine-rich domain adopts a rod-like shape, which is stabilized by five disulfide bridges. Hydrophobic residues, which may obstruct the target ion-channel, are exposed to the solvent. A concave surface, which is surrounded by these two domains, is also expected to play a significant role in the binding to the target receptor, leading to ion channel blockage. The C-terminal cysteine-rich region has a similar tertiary structure to voltage-gated potassium channel blocker toxins, such as BgK and ShK. These findings will contribute toward understanding the functions of the widely distributed CRISP family proteins. [Copyright &y& Elsevier]

Snake venoms contain a number of heterodimeric C-type lectin-like proteins (CLPs) that interact specifically with components of the haemostatic system. EMS16 from the venom of Echis multisquamatus binds to the collagen receptor, integrin α2β1, also known as glycoprotein (GP) Ia/IIa, and specifically inhibits collagen binding. Here we report the crystal structure of EMS16 in complex with recombinant integrin α2-I domain that plays a central role in collagen binding. The structure of the complex at 1.9 Å resolution reveals that the collagen-binding site of the α2-I domain is covered completely by the bound EMS16. This blockage by EMS16 appears to spatially inhibit collagen binding to the α2-I domain. The bound α2-I domain adopts a closed conformation, which is seen in the absence of ligand, suggesting that EMS16 stabilizes a closed conformation corresponding to the less active structure of the α2-I domain. EMS16 does not directly bind to the manganese ion and residues of the metal ion-dependent adhesion site (MIDAS) of the α2-I domain, suggesting that EMS16 may have the potential to bind specifically to the α2-I domain in a metal ion-independent fashion. [Copyright &y& Elsevier]

Snake venoms contain a number of hemostatically active C-type lectin-like proteins (CLPs), which affect the blood coagulation system, endothelial cells, and platelets. CLPs have broad similarities in structure and possess distinct biological functions. EMS 16, a CLP from Echis multisquamatus venom, which is a potent and selective inhibitor of the collagen receptor, glycoprotein Ia/IIa (integrin α2β1), has been used in the present study to examine structure-function relationships in venom CLPs by X-ray crystallography. The structure of EMSI6, determined at a resolution of 1.9 Å, revealed a heterodimer involved with domain swapping of the central loop as observed in the structures of other CLPs. A part of the glycan was observed and identified as N-acetyl-D-glucosamine (GlcNAc) in the electron density map at Asn21 of subunit B, an expected glycosylation site. EMS 16 had a unique, positively charged electrostatic potential patch on the concave surface that may qualify as a site for interaction with the I-domain of the glycoprotein Ia/IIa. [ABSTRACT FROM AUTHOR]

Disintegrins are a family of small proteins containing an Arg-Gly-Asp (RGD) sequence motif that binds specifically to integrin receptors. Since the integrin is known to serve as the final common pathway leading to aggregation via formation of platelet–platelet bridges, disintegrins act as fibrinogen receptor antagonists. Here, we report the first crystal structure of a disintegrin, trimestatin, found in snake venom. The structure of trimestatin at 1.7 A˚ resolution reveals that a number of turns and loops form a rigid core stabilized by six disulfide bonds. Electron densities of the RGD sequence are visible clearly at the tip of a hairpin loop, in such a manner that the Arg and Asp side-chains point in opposite directions. A docking model using the crystal structure of integrin αVβ3 suggests that the Arg binds to the propeller domain, and Asp to the βA domain. This model indicates that the C-terminal region is another potential binding site with integrin receptors. In addition to the RGD sequence, the structural evidence of a C-terminal region (Arg66, Trp67 and Asn68) important for disintegrin activity allows understanding of the high affinity and selectiveness of snake venom disintegrin for integrin receptors. The crystal structure of trimestatin should provide a useful framework for designing and developing more effective drugs for controlling platelet aggregation and anti-angiogenesis cancer. [Copyright &y& Elsevier]

Rice dwarf virus (RDV), the causal agent of rice dwarf disease, is a member of the genus Phytoreovirus in the family Reoviridae. RDV is a double-shelled virus with a molecular mass of approximately 70 million Dalton. This virus is widely prevalent and is one of the viruses that cause the most economic damage in many Asian countries. The atomic structure of RDV was determined at 3.5 A˚ resolution by X-ray crystallography. The double-shelled structure consists of two different proteins, the core protein P3 and the outer shell protein P8. The atomic structure shows structural and electrostatic complementarities between both homologous (P3-P3 and P8-P8) and heterologous (P3-P8) interactions, as well as overall conformational changes found in P3-P3 dimer caused by the insertion of amino-terminal loop regions of one of the P3 protein into the other. These interactions suggest how the 900 protein components are built into a higher-ordered virus core structure. [Copyright &y& Elsevier]

Gradient elution chromatography of recombinant apoaequorin carried out in the presence of Ca2+ revealed two isoforms of apoaequorin, reduced and oxidized, whereas in the presence of EDTA 3 isoforms were observed. In a regeneration mixture of apoaequorin, coelenterazine, EDTA, and 2-mercaptoethanol, four isoforms were obtained, of which only one, aequorin, gave light with Ca2+. A method is described for the preparation of highly pure aequorin. The aequorin was stable in solution for approximately 10 days at 4 °C and pH 7.6, and then it gradually lost activity with a half-life of about 20 days until it was almost completely inactive on day 30. [Copyright &y& Elsevier]