Multiwavelength Anomalous Dispersion Method Research Articles

Structural Studies Reveal the Functional Modularity of the Scc2-Scc4 Cohesin Loader.

The remarkable accuracy of eukaryotic cell division is partly maintained by the cohesin complex acting as a molecular glue to prevent premature sister chromatid separation. The loading of cohesin onto chromosomes is catalyzed by the Scc2-Scc4 loader complex. Here, we report the crystal structure of Scc4 bound to the N terminus of Scc2 and show that Scc4 is a tetratricopeptide repeat (TPR) superhelix. The Scc2 N terminus adopts an extended conformation and is entrapped by the core of the Scc4 superhelix. Electron microscopy (EM) analysis reveals that the Scc2-Scc4 loader complex comprises three domains: a head, body, and hook. Deletion studies unambiguously assign the Scc2N-Scc4 as the globular head domain, whereas in vitro cohesin loading assays show that the central body and the hook domains are sufficient to catalyze cohesin loading onto circular DNA, but not chromatinized DNA in vivo, suggesting a possible role for Scc4 as a chromatin adaptor.

Crystal structure of the fibre head domain of the Atadenovirus Snake Adenovirus 1.

Adenoviruses are non-enveloped icosahedral viruses with trimeric fibre proteins protruding from their vertices. There are five known genera, from which only Mastadenoviruses have been widely studied. Apart from studying adenovirus as a biological model system and with a view to prevent or combat viral infection, there is a major interest in using adenovirus for vaccination, cancer therapy and gene therapy purposes. Adenoviruses from the Atadenovirus genus have been isolated from squamate reptile hosts, ruminants and birds and have a characteristic gene organization and capsid morphology. The carboxy-terminal virus-distal fibre head domains are likely responsible for primary receptor recognition. We determined the high-resolution crystal structure of the Snake Adenovirus 1 (SnAdV-1) fibre head using the multi-wavelength anomalous dispersion (MAD) method. Despite the absence of significant sequence homology, this Atadenovirus fibre head has the same beta-sandwich propeller topology as other adenovirus fibre heads. However, it is about half the size, mainly due to much shorter loops connecting the beta-strands. The detailed structure of the SnAdV-1 fibre head and other animal adenovirus fibre heads, together with the future identification of their natural receptors, may lead to the development of new strategies to target adenovirus vectors to cells of interest.

Purification, crystallization and preliminary X-ray diffraction studies of UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) from Synechococcus sp. PCC 7942.

A UDP-glucose:tetrahydrobiopterin α-glucosyltransferase (BGluT) enzyme was discovered in the cyanobacterium Synechococcus sp. PCC 7942 which transfers a glucose moiety from UDP-glucose to tetrahydrobiopterin (BH4). BGluT protein was overexpressed with selenomethionine labelling for structure determination by the multi-wavelength anomalous dispersion method. The BGluT protein was purified by nickel-affinity and size-exclusion chromatography. It was then crystallized by the hanging-drop vapour-diffusion method using a well solution consisting of 0.1 M bis-tris pH 5.5, 19%(w/v) polyethylene glycol 3350 with 4%(w/v) D(+)-galactose as an additive. X-ray diffraction data were collected to 1.99 Å resolution using a synchrotron-radiation source. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 171.35, b = 77.99, c = 53.77 Å, β = 90.27°.

Crystal structure of PfbA, a surface adhesin of Streptococcus pneumoniae, provides hints into its interaction with fibronectin

PfbA is a surface adhesin and invasin of Streptococcus pneumoniae that binds to human fibronectin and plasminogen of the host extracellular matrix. It is a virulence factor for its pathogenesis. The crystal structure of recombinant PfbA150–607 from S. pneumoniae strain R6, was determined using multiwavelength anomalous dispersion (MAD) method and refined to 1.90Å resolution. The structure of rPfbA150–607 revealed that residues Thr150 to Lys570 form a rigid parallel beta helix, followed by a short disordered region (571–607) that consists of beta hairpins. The structural organization of the beta helix resembles that of polysaccharide-modifying enzymes. The structural and sequence features essential for fibronectin-binding observed in the well characterized fibronectin-binding proteins such as FnBPA of Staphylococcus aureus, SfbI of Streptococcus pyogenes and BBK32 of Borrelia burgdorferi has been found in rPfbA150–607. Based on this, it is predicted that the disordered region following the beta helix could be the fibronectin-binding region in PfbA. PfbA150–607 contains relatively high number of surface exposed lysines and these residues are probably involved in binding plasmin(ogen) as observed in other plasminogen-binding proteins.

Structure of the Rho-specific guanine nucleotide-exchange factor Xpln.

Xpln is a guanine nucleotide-exchange factor (GEF) for Rho GTPases. A Dbl homology (DH) domain followed by a pleckstrin homology (PH) domain is a widely adopted GEF-domain architecture. The Xpln structure solely comprises these two domains. Xpln activates RhoA and RhoB, but not RhoC, although their GTPase sequences are highly conserved. The molecular mechanism of the selectivity of Xpln for Rho GTPases is still unclear. In this study, the crystal structure of the tandemly arranged DH-PH domains of mouse Xpln, with a single molecule in the asymmetric unit, was determined at 1.79 Å resolution by the multiwavelength anomalous dispersion method. The DH-PH domains of Xpln share high structural similarity with those from neuroepithelial cell-transforming gene 1 protein, PDZ-RhoGEF, leukaemia-associated RhoGEF and intersectins 1 and 2. The crystal structure indicated that the α4-α5 loop in the DH domain is flexible and that the DH and PH domains interact with each other intramolecularly, thus suggesting that PH-domain rearrangement occurs upon RhoA binding.

The structure ofL-amino-acid ligase fromBacillus licheniformis

L-Amino-acid ligases (LALs) are enzymes which catalyze the formation of dipeptides by linking two L-amino acids. Although many dipeptides are known and expected to have medical and nutritional benefits, their practical use has been limited owing to their low availability and high expense. LALs are potentially desirable tools for the efficient production of dipeptides; however, the molecular basis of substrate recognition by LAL has not yet been sufficiently elucidated for the design of ideal LALs for the desired dipeptides. This report presents the crystal structure of the LAL BL00235 derived from Bacillus licheniformis NBRC 12200 determined at 1.9 Å resolution using the multi-wavelength anomalous dispersion method. The overall structure of BL00235 is fairly similar to that of YwfE, the only LAL with a known structure, but the structure around the catalytic site contains some significant differences. Detailed structural comparison of BL00235 with YwfE sheds some light on the molecular basis of the substrate specificities.

Crystal Structure of Cucumisin, a Subtilisin-Like Endoprotease from Cucumis melo L.

Cucumisin is a plant serine protease, isolated as an extracellular glycoprotein from the melon fruit Cucumis melo L. var. Prince. Cucumisin is composed of multiple domain modules, including catalytic, protease-associated, and fibronectin-III-like domains. The crystal structure of cucumisin was determined by the multiwavelength anomalous dispersion method and refined at 2.75Å resolution. A structural homology search indicated that the catalytic domain of cucumisin shares structural similarity with subtilisin and subtilisin-like fold enzymes. According to the Z-score, the highest structural similarity is with tomato subtilase 3 (SBT3), with an rmsd of 3.5Å for the entire region. The dimer formation mediated by the protease-associated domain in SBT3 is a distinctive structural characteristic of cucumisin. On the other hand, analytical ultracentrifugation indicated that cucumisin is mainly monomeric in solution. Although the locations of the amino acid residues composing the catalytic triad are well conserved between cucumisin and SBT3, a disulfide bond is uniquely located near the active site of cucumisin. The steric circumstances of the active site with this disulfide bond are distinct from those of SBT3, and it contributes to the substrate preference of cucumisin, especially at the P2 position. Among the plant serine proteases, the thermostability of cucumisin is higher than that of its structural homologue SBT3, as determined by their melting points. A structural comparison between cucumisin and SBT3 revealed that cucumisin possesses less surface area and shortened loop regions. Consequently, the higher thermostability of cucumisin is achieved by its more compact structure.

Domain-Swapped Dimeric Structure of a Stable and Functional De Novo Four-Helix Bundle Protein, WA20

To probe the potential for activity in unevolved amino acid sequence space, we created a third generation combinatorial library of de novo four-helix bundle proteins. The "artificial superfamily" of helical bundles was designed using binary patterning of polar and nonpolar residues, and expressed in Escherichia coli from a library of synthetic genes. WA20, picked from the library, is one of the most stable proteins in the superfamily, and has rudimentary activities such as esterase and lipase. Here we report the crystal structure of WA20, determined by the multiwavelength anomalous dispersion method. Unexpectedly, the WA20 crystal structure is not a monomeric four-helix bundle, but a dimeric four-helix bundle. Each monomer comprises two long α-helices that intertwist with the helices of the other monomer. The two monomers together form a 3D domain-swapped four-helix bundle dimer. In addition, there are two hydrophobic pockets, which may potentially provide substrate binding sites. Small-angle X-ray scattering shows that the molecular weight of WA20 is ~25 kDa and the shape is rod-like (the maximum length, D(max) = ~8 nm), indicating that WA20 forms a dimeric four-helix bundle in solution. These results demonstrate that our de novo protein library contains not only simple monomeric proteins, but also stable and functional multimeric proteins.

Single-wavelength phasing strategy for quasi-racemic protein crystal diffraction data

Racemic protein crystallography offers two key features: an increased probability of crystallization and the potential advantage of phasing centric diffraction data. In this study, a phasing strategy is developed for the scenario in which a crystal is grown from a mixture in which anomalous scattering atoms have been incorporated into only one enantiomeric form of the protein molecule in an otherwise racemic mixture. The structure of a protein crystallized in such a quasi-racemic form has been determined in previous work [Pentelute et al. (2008), J. Am. Chem. Soc. 130, 9695-9701] using the multiwavelength anomalous dispersion (MAD) method. Here, it is shown that although the phases from such a crystal are not strictly centric, their approximate centricity provides a powerful way to break the phase ambiguity that ordinarily arises when using the single-wavelength anomalous dispersion (SAD) method. It is shown that good phases and electron-density maps can be obtained from a quasi-racemic protein crystal based on single-wavelength data. A prerequisite problem of how to establish the origin of the anomalous scattering substructure relative to the center of pseudo-inversion is also addressed.

Purification, crystallization and preliminary X-ray crystallographic analysis of the C-terminal cytoplasmic domain of FlhB from Salmonella typhimurium.

FlhB is a key protein in the regulation of protein export by the bacterial flagellar secretion system. It is composed of two domains: an N-terminal transmembrane domain and a C-terminal cytoplasmic domain (FlhBc). FlhBc from Salmonella typhimurium has been successfully crystallized using the vapour-diffusion method. The crystals diffracted to 2.45 Å resolution and belonged to space group P4(2)2(1)2, with unit-cell parameters a=b=49.06, c=142.94 Å. A selenomethionine-containing variant of FlhBc has also been crystallized in the same space group and was used for initial phase calculation by the multiwavelength anomalous dispersion (MAD) method.

Structure of Peptide-Induced Transmembrane Pore Determined by Anomalous X-Ray Diffraction

We determined the structure of the melittin-induced transmembrane pore by X-ray diffraction. The multibilayer sample on substrate was prepared in full hydration. The peptide-to-lipid ratio, P/L, of the melittin-lipid mixtures were in the condition where pores were present, as established previously by neutron in-plane scattering in correlation with oriented circular dichroism. At low hydration levels, the interbilayer distance shortened and caused the membrane pores to become long-ranged correlated and form a periodically ordered lattice of rhombohedral symmetry. Here we used the multiwavelength anomalous dispersion (MAD) method to solve the phase problem for a rhombohedral phase of a phospholipid with brominated chains and performed multiwavelength anomalous diffraction at the bromine K edge. The X-ray light source in BL23A beam line of NSRRC and home-made humidity-temperature controlled chamber will be applied in the measurements. We found the melittin-induced pores were at least partially framed by a lipid monolayer. Evidence suggests that the pore structure is of the toroid type different from the barrel-stave type induced by alamethicin.

Crystal Structure of HugZ, a Novel Heme Oxygenase from Helicobacter pylori

The crystal structure of a heme oxygenase (HO) HugZ from Helicobacter pylori complexed with heme has been solved and refined at 1.8 Å resolution. HugZ is part of the iron acquisition mechanism of H. pylori, a major pathogen of human gastroenteric diseases. It is required for the adaptive colonization of H. pylori in hosts. Here, we report that HugZ is distinct from all other characterized HOs. It exists as a dimer in solution and in crystals, and the dimer adopts a split-barrel fold that is often found in FMN-binding proteins but has not been observed in hemoproteins. The heme is located at the intermonomer interface and is bound by both monomers. The heme iron is coordinated by the side chain of His(245) and an azide molecule when it is present in crystallization conditions. Experiments show that Arg(166), which is involved in azide binding, is essential for HugZ enzymatic activity, whereas His(245), surprisingly, is not, implying that HugZ has an enzymatic mechanism distinct from other HOs. The placement of the azide corroborates the observed γ-meso specificity for the heme degradation reaction, in contrast to most known HOs that have α-meso specificity. We demonstrate through sequence and structural comparisons that HugZ belongs to a new heme-binding protein family with a split-barrel fold. Members of this family are widespread in pathogenic bacteria and may play important roles in the iron acquisition of these bacteria.

Expression, crystallization and preliminary X-ray analysis of an anomeric inverting agarase from Pseudoalteromonas sp. CY24.

AgaB from Pseudoalteromonas sp. CY24 is a novel agarase that hydrolyzes agarose to generate products with inverted anomeric configuration and that has been proposed to have a larger catalytic cleft than other β-agarases. Here, the expression, purification, crystallization and data collection of AgaB in both wild-type and selenomethionine-substituted forms is described. The crystals of wild-type AgaBdiffracted to 1.97 Å resolution and belonged to space group C222(1). The selenomethionine derivative crystallized in space group I222. The phasing problem was solved by the multiwavelength anomalous dispersion (MAD) method. These results will facilitate detailed structural and enzymatic analysis of AgaB.

Crystal Structure of Yeast Rpn14, a Chaperone of the 19 S Regulatory Particle of the Proteasome

The ubiquitin-proteasome pathway is a major proteolytic system in eukaryotic cells and regulates various cellular processes. The 26 S proteasome, the central enzyme of this pathway, consists of a proteolytic core particle and two 19 S regulatory particles (RPs) composed of ATPase (Rpt) and non-ATPase (Rpn) subunits. Growing evidence indicates that proteasome assembly is assisted by a variety of chaperones. In particular, it has been reported recently that Nas2, Nas6, Rpn14, and Hsm3 bind specific Rpt subunits, thereby contributing to the formation of 19 S RP. Rpn14 transiently binds to the C-terminal domain of the Rpt6 subunit (Rpt6-C) during maturation of the ATPase ring of 19 S RP. In this study, we determined the crystal structure of yeast Rpn14 at 2.0 A resolution, which revealed that this chaperone consists of a unique N-terminal domain with unknown function and a C-terminal domain assuming a canonical seven-bladed beta-propeller fold. The Rpt6-binding site on Rpn14 was predicted based on structural comparison with the complex formed between Nas6 and Rpt3-C. The top face of Rpn14 exhibits a highly acidic surface area, whereas the putative interacting surface of Rpt6-C is basic. By inspection of structural data along with genetic and biochemical data, we determined the specific residues of Rpn14 and Rpt6 for complementary charge interactions that are required for 19 S RP assembly.

Calcium Binds to LipL32, a Lipoprotein from Pathogenic Leptospira, and Modulates Fibronectin Binding

Tubulointerstitial nephritis is a cardinal renal manifestation of leptospirosis. LipL32, a major lipoprotein and a virulence factor, locates on the outer membrane of the pathogen Leptospira. It evades immune response by recognizing and adhering to extracellular matrix components of the host cell. The crystal structure of Ca(2+)-bound LipL32 was determined at 2.3 A resolution. LipL32 has a novel polyD sequence of seven aspartates that forms a continuous acidic surface patch for Ca(2+) binding. A significant conformational change was observed for the Ca(2+)-bound form of LipL32. Calcium binding to LipL32 was determined by isothermal titration calorimetry. The binding of fibronectin to LipL32 was observed by Stains-all CD and enzyme-linked immunosorbent assay experiments. The interaction between LipL32 and fibronectin might be associated with Ca(2+) binding. Based on the crystal structure of Ca(2+)-bound LipL32 and the Stains-all results, fibronectin probably binds near the polyD region on LipL32. Ca(2+) binding to LipL32 might be important for Leptospira to interact with the extracellular matrix of the host cell.

Crystallization of selenomethionyl exo-β-1,3-galactanase from the basidiomycetePhanerochaete chrysosporium

Exo-beta-1,3-galactanase from Phanerochaete chrysosporium (Pc1,3Gal43A) consists of a glycoside hydrolase family 43 catalytic domain and a substrate-binding domain that belongs to carbohydrate-binding module family 35. It catalyzes the hydrolysis of beta-1,3-galactan, which is the backbone of the arabinogalactan proteins; the C-terminal carbohydrate-binding module family 35 domain increases the local concentration of the enzyme around beta-1,3-galactan by its high affinity for the substrate. To enable phase determination using the multiwavelength anomalous dispersion method, selenomethionyl Pc1,3Gal43A was crystallized at 298 K using the hanging-drop vapour-diffusion method. The presence of selenium in the crystals was confirmed from the X-ray absorption spectrum. The crystals belonged to space group P2(1) and diffracted to 1.8 A resolution.

The PHCCEx domain of Tiam1/2 is a novel protein- and membrane-binding module

Tiam1 and Tiam2 (Tiam1/2) are guanine nucleotide-exchange factors that possess the PH–CC–Ex (pleckstrin homology, coiled coil and extra) region that mediates binding to plasma membranes and signalling proteins in the activation of Rac GTPases. Crystal structures of the PH–CC–Ex regions revealed a single globular domain, PHCCEx domain, comprising a conventional PH subdomain associated with an antiparallel coiled coil of CC subdomain and a novel three-helical globular Ex subdomain. The PH subdomain resembles the β-spectrin PH domain, suggesting non-canonical phosphatidylinositol binding. Mutational and binding studies indicated that CC and Ex subdomains form a positively charged surface for protein binding. We identified two unique acidic sequence motifs in Tiam1/2-interacting proteins for binding to PHCCEx domain, Motif-I in CD44 and ephrinB's and the NMDA receptor, and Motif-II in Par3 and JIP2. Our results suggest the molecular basis by which the Tiam1/2 PHCCEx domain facilitates dual binding to membranes and signalling proteins.

Structural basis of typhoid: Salmonella typhi type IVb pilin (PilS) and cystic fibrosis transmembrane conductance regulator interaction

The type IVb pilus of the enteropathogenic bacteria Salmonella typhi is a major adhesion factor during the entry of this pathogen into gastrointestinal epithelial cells. Its target of adhesion is a stretch of 10 residues from the first extracellular domain of cystic fibrosis transmembrane conductance regulator (CFTR). The crystal structure of the N-terminal 25 amino acid deleted S. typhi native PilS protein (DeltaPilS), which makes the pilus, was determined at 1.9 A resolution by the multiwavelength anomalous dispersion method. Also, the structure of the complex of DeltaPilS and a target CFTR peptide, determined at 1.8 A, confirms that residues 113-117 (NKEER) of CFTR are involved in binding with the pilin protein and gives us insight on the amino acids that are essential for binding. Furthermore, we have also explored the role of a conserved disulfide bridge in pilus formation. The subunit structure and assembly architecture are crucial for understanding pilus functions and designing suitable therapeutics against typhoid.

Overexpression, purification, characterization and preliminary crystallographic study of phosphoglycolate phosphatase fromShigella flexneri2a strain 301

Phosphoglycolate phosphatase has a salvage function in the metabolism of the 2-phosphoglycolate formed during bacterial DNA repair. In order to better understand its dimerization behaviour, the influence of metal ions on its activity and its catalytic mechanism at the molecular level, recombinant phosphoglycolate phosphatase from Shigella flexneri was overexpressed, purified, characterized and crystallized by the hanging-drop vapour-diffusion method at 291 K using polyethylene glycol 3500 as a precipitant and zinc acetate as an additive. The crystals belonged to space group R3, with unit-cell parameters a = 88.1, b = 88.1, c = 259.2 A, corresponding to the presence of two molecules in the asymmetric unit. SeMet-labelled protein was also prepared and crystallized for use in phase determination. Initial structure determination using the multiwavelength anomalous dispersion (MAD) method clearly revealed that SfPGPase bears an alpha-helical cap domain that differs from that of a previously reported orthologue.

Crystal Structures of F420-dependent Glucose-6-phosphate Dehydrogenase FGD1 Involved in the Activation of the Anti-tuberculosis Drug Candidate PA-824 Reveal the Basis of Coenzyme and Substrate Binding

The modified flavin coenzyme F(420) is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F(420)-dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F(420) for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F(420) and citrate at resolutions of 1.90 and 1.95 A(,) respectively. The structure reveals a highly specific F(420) binding mode, which is shared with several other F(420)-dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F(420) and is shown to be a competitive inhibitor (IC(50) 43 microm). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F(420) and helps promote a butterfly bend conformation that facilitates F(420) reduction and catalysis.