The crystal structure of the H107R variant of the extracellular domain of mouse NKR-P1A was determined using X-ray diffraction from a merohedrally twinned crystal.
The structure of the H107R variant of the extracellular domain of the mouse natural killer cell receptor NKR-P1A has been determined by X-ray diffraction at 2.3 Å resolution from a merohedrally twinned crystal. Unlike the structure of the wild-type receptor in space group I4122 with a single chain per asymmetric unit, the crystals of the variant belonged to space group I41 with a dimer in the asymmetric unit. Different degrees of merohedral twinning were detected in five data sets collected from different crystals. The mutation does not have a significant impact on the overall structure, but led to the binding of an additional phosphate ion at the interface of the molecules.
NKR-P1A; merohedral twinning; mutation
The crystallographic structures of the subunit B mutants F427W and F508W of the Pyrococcus horikoshii OT3 of the A1AO ATP synthase reveal that the exact volume of the adenine ribose binding pocket is essential for ATP-/ADP-binding.
A reporter tryptophan residue was individually introduced by site-directed mutagenesis into the adenine-binding pocket of the catalytic subunit A (F427W and F508W mutants) of the motor protein A1AO ATP synthase from Pyrococcus horikoshii OT3. The crystal structures of the F427W and F508W mutant proteins were determined to 2.5 and 2.6 Å resolution, respectively. The tryptophan substitution caused the fluorescence signal to increase by 28% (F427W) and 33% (F508W), with a shift from 333 nm in the wild-type protein to 339 nm in the mutant proteins. Tryptophan emission spectra showed binding of Mg-ATP to the F427W mutant with a K
d of 8.5 µM. In contrast, no significant binding of nucleotide could be observed for the F508W mutant. A closer inspection of the crystal structure of the F427W mutant showed that the adenine-binding pocket had widened by 0.7 Å (to 8.70 Å) in comparison to the wild-type subunit A (8.07 Å) owing to tryptophan substitution, as a result of which it was able to bind ATP. In contrast, the adenine-binding pocket had narrowed in the F508W mutant. The two mutants presented demonstrate that the exact volume of the adenine ribose binding pocket is essential for nucleotide binding and even minor narrowing makes it unfit for nucleotide binding. In addition, structural and fluorescence data confirmed the viability of the fluorescently active mutant F427W, which had ideal tryptophan spectra for future structure-based time-resolved dynamic measurements of the catalytic subunit A of the ATP-synthesizing enzyme A-ATP synthase.
A1AO ATP synthase; tryptophan; Pyrococcus horikoshii OT3
Cerium was used to enhance the anomalous signal in hen egg-white lysozyme crystals and led to successful in-house SAD phasing.
The anomalous signal of cerium(III) ions present in a derivative of hen egg-white lysozyme (HEWL) crystals obtained by the addition of 0.025 M cerium chloride to the crystallization medium was used for phasing. X-ray intensity data were collected to 2 Å resolution using an in-house Cu Kα radiation data-collection facility. Phasing of a single-wavelength data set purely based on its f′′ led to a clearly interpretable electron-density map. Automated substructure solution by AutoSol in PHENIX resulted in four highest peaks corresponding to cerium(III) ions with data limited to 3 Å resolution, and about 90% of the residues were built automatically by AutoBuild in PHENIX. Cerium(III) ions bound on the surface of the enzyme are found to interact mainly with the main-chain and side-chain carbonyl groups of Asn, Glu, Tyr and Asp and with water molecules. Ce3+ ions were used as potential anomalous scatterers for the in-house single-wavelength anomalous scattering technique, and this is proposed as a tool for macromolecular phasing and for the study of the interactions of trivalent metal ions with proteins and other macromolecules.
cerium; anomalous scattering; automated SAD phasing; hen egg-white lysozyme; cocrystallization; redundancy
A modification of previously published protocols for the crystallization of homotetrameric R67 DHFR was used to crystallize a mixed wild-type and variant tandem dimer of R67 DHFR. Surprisingly, a fully wild-type crystal structure was obtained, apparently as a consequence of selective proteolytic degradation of the variant.
Trimethoprim is an antibiotic that targets bacterial dihydrofolate reductase (DHFR). A plasmid-encoded DHFR known as R67 DHFR provides resistance to trimethoprim in bacteria. To better understand the mechanism of this homotetrameric enzyme, a tandem dimer construct was created that linked two monomeric R67 DHFR subunits together and mutated the sequence of residues 66–69 of the first subunit from VQIY to INSF. Using a modified crystallization protocol for this enzyme that included in situ proteolysis using chymotrypsin, the tandem dimer was crystallized and the structure was solved at 1.4 Å resolution. Surprisingly, only wild-type protomers were incorporated into the crystal. Further experiments demonstrated that the variant protomer was selectively degraded by chymotrypsin, although no canonical chymotrypsin cleavage site had been introduced by these mutations.
dihydrofolate reductase; antibiotic resistance; in situ proteolysis; protein degradation; type II DHFR
A high-resolution X-ray crystallographic study of the effects of solvent deuteration on the crystallization of proteinase K shows negligibly small degradations of the crystals owing to solvent deuteration and small structural differences between nondeuterated and deuterated crystals of proteinase K.
Deuteration of macromolecules is an important technique in neutron protein crystallography. Solvent deuteration of protein crystals is carried out by replacing water (H2O) with heavy water (D2O) prior to neutron diffraction experiments in order to diminish background noise. The effects of solvent deuteration on the crystallization of proteinase K (PK) with polyethylene glycol as a precipitant were investigated using high-resolution X-ray crystallography. In previous studies, eight NO3
− anions were included in the PK crystal unit cell grown in NaNO3 solution. In this study, however, the PK crystal structure did not contain NO3
− anions; consequently, distortions of amino acids arising from the presence of NO3
− anions were avoided in the present crystal structures. High-resolution (1.1 Å) X-ray diffraction studies showed that the degradation of PK crystals induced by solvent deuteration was so small that this degradation would be negligible for the purpose of neutron protein crystallography experiments at medium resolution. Comparison of the nonhydrogen structures of nondeuterated and deuterated crystal structures demonstrated very small structural differences. Moreover, a positive correlation between the root-mean-squared differences and B factors indicated that no systematic difference existed.
deuteration; polyethylene glycol; proteinase K
The assembly of bacterial outer membrane proteins is catalyzed by the BAM complex, which is made up of five proteins (BamA–E). Structural and bioinformatic analysis of the C-terminal domain of E. coli BamC (BamCC) suggests that a negatively charged conserved surface on BamCC may be an important protein–protein interaction site.
In Gram-negative bacteria, the BAM complex catalyzes the essential process of assembling outer membrane proteins. The BAM complex in Escherichia coli consists of five proteins: one β-barrel membrane protein, BamA, and four lipoproteins, BamB, BamC, BamD and BamE. Here, the crystal structure of the C-terminal domain of E. coli BamC (BamCC: Ala224–Ser343) refined to 1.5 Å resolution in space group H3 is reported. BamCC consists of a six-stranded antiparallel β-sheet, three α-helices and one 310-helix. Sequence and surface analysis reveals that most of the conserved residues within BamCC are localized to form a continuous negatively charged groove that is involved in a major crystalline lattice contact in which a helix from a neighbouring BamCC binds against this surface. This interaction is topologically and architecturally similar to those seen in the substrate-binding grooves of other proteins with BamC-like folds. Taken together, these results suggest that an identified surface on the C-terminal domain of BamC may serve as an important protein-binding surface for interaction with other BAM-complex components or substrates.
NlpB; BamC; outer membrane biogenesis; BAM complex; outer membrane proteins; lipoproteins; β-barrel proteins
Protein crystals typically deteriorate upon soaking in solutions very different from those used to grow them (the ‘mother liquor’). This is particularly true of solutions containing lower salt concentrations and organic solvents (i.e. DMSO, ethanol). Optimization of the relative humidity environment of the crystals can compensate for these effects and allow recovery of diffraction quality.
The use of relative humidity control of protein crystals to overcome some of the shortcomings of soaking ligands (i.e. inhibitors, substrate analogs, weak ligands) into pre-grown apoprotein crystals has been explored. Crystals of PurE (EC 220.127.116.11), an enzyme from the purine-biosynthesis pathway of Bacillus anthracis, were used as a test case. The findings can be summarized as follows: (i) using humidity control, it is possible to improve/optimize the diffraction quality of crystals soaked in solutions of organic solvent (DMSO, ethanol) containing ligands/inhibitors; (ii) optimization of the relative humidity can compensate for the deterioration of the diffraction pattern that is observed upon desalting crystals grown in high salt; (iii) combining desalting protocols with the addition of PEG it is possible to achieve very high concentrations of weak ligands (in the 5–10 mM range) in soaking solutions and (iv) fine control of the relative humidity of crystals soaked in these solutions can compensate for the deterioration of crystal diffraction and restore ‘high-resolution’ diffraction for structure-based and fragment-based drug design. It is suggested that these experimental protocols may be useful in other protein systems and may be applicable in academic or private research to increase the probability of obtaining structures of protein–ligand complexes at high resolution.
humidity control; fragment-based drug design; structure-based drug design; PurE; EC 18.104.22.168
ATP-citrate lyase catalyzes the production of acetyl-CoA from citrate, CoA and ATP. Crystals were grown of the amino-terminal portion of the human enzyme in the presence of tartrate, ATP and magnesium ions. The crystal structure shows the inhibitor tartrate and the product ADP–Mg2+ bound to the protein.
Human ATP-citrate lyase (EC 22.214.171.124) is the cytoplasmic enzyme that catalyzes the production of acetyl-CoA from citrate, CoA and ATP. The amino-terminal portion of the enzyme, containing residues 1–817, was crystallized in the presence of tartrate, ATP and magnesium ions. The crystals diffracted to 2.3 Å resolution. The structure shows ADP–Mg2+ bound to the domain that possesses the ATP-grasp fold. The structure demonstrates that this crystal form could be used to investigate the structures of complexes with inhibitors of ATP-citrate lyase that bind at either the citrate- or ATP-binding site.
ATP-citrate lyase; ATP-grasp domain
The crystal structure of recombinant UbiX has been determined to 1.5 Å resolution.
The ubiX gene (PA4019) of Pseudomonas aeruginosa has been annotated as encoding a putative 3-octaprenyl-4-hydroxybenzoate decarboxylase from the ubiquinone-biosynthesis pathway. Based on a transposon mutagenesis screen, this gene was also implicated as being essential for the survival of this organism. The crystal structure of recombinant UbiX determined to 1.5 Å resolution showed that the protein belongs to the superfamily of homo-oligomeric flavine-containing cysteine decarboxylases. The enzyme assembles into a dodecamer with 23 point symmetry. The subunit displays a typical Rossmann fold and contains one FMN molecule bound at the interface between two subunits.
Pseudomonas aeruginosa; PA4019; aromatic acid decarboxylases
The crystal structure of an extremely thermostable multicopper oxidase from a hyperthermophile was determined.
The crystal structure of an extremely thermostable multicopper oxidase (McoP) from the hyperthermophilic archaeon Pyrobaculum aerophilum was determined at a resolution of 2.0 Å. The overall fold was comprised of three cupredoxin-like domains and the main-chain coordinates of the enzyme were similar to those of multicopper oxidases from Escherichia coli (CueO) and Bacillus subtilis (CotA). However, there were clear topological differences around domain 3 between McoP and the other two enzymes: a methionine-rich helix in CueO and a protruding helix in CotA were not present in McoP. Instead, a large loop (PL-1) covered the T1 copper centre of McoP and a short α-helix in domain 3 extended near the N-terminal end of PL-1. In addition, the sizes of several surface loops in McoP were markedly smaller than the corresponding loops in CueO and CotA. Structural comparison revealed that the presence of extensive hydrophobic interactions and a smaller cavity volume are likely to be the main factors contributing to the hyperthermostability of McoP.
hyperthermophiles; Pyrobaculum aerophilum; multicopper oxidases; archaea
The crystal structure of Drosphila melanogaster Rab6 bound to the non-hydrolysable GTP analogue GMPPNP has been determined at a resolution of 1.4 Å.
Rab6 is a small GTPase that belongs to the p21 Ras superfamily. It is involved in vesicle trafficking between the Golgi apparatus and endosomes/ER in eukaryotes. The GDP-bound inactive protein undergoes conformational changes when the nucleotide is exchanged to GTP, allowing Rab6 to interact with a variety of different effector proteins. To further understand how these changes affect downstream protein binding, the crystal structure of Rab6 from Drosophila melanogaster has been solved to 1.4 Å resolution, the highest resolution for a Rab6 structure to date. The crystals belonged to space group C2, with unit-cell parameters a = 116.5, b = 42.71, c = 86.86 Å, α = 90, β = 133.12, γ = 90°. The model was refined to an R factor of 14.5% and an R
free of 17.3%.
Rab6; GTP binding; GMPPNP
Apo and GDP-bound crystal structures of an essential GTPase, YsxC, from T. maritima were determined to maximal resolutions of 2.3 and 1.9 Å, respectively. Switch I in GDP–YsxC can adopt both an ‘open’ and ‘closed’ conformation, suggesting a mechanism for diffusion of GDP out of the nucleotide-binding pocket.
YsxC belongs to the YihA family of TRAFAC class GTPases. The protein is involved in the biogenesis of ribosomes and is essential for the survival of a wide range of bacteria. Here, crystal structures of YsxC from Thermotoga maritima and its complex with GDP were determined at maximal resolutions of 2.3 and 1.9 Å, respectively. Major structural differences are observed in the switch I region, which is disordered in the apo form but exists in both an ‘open’ and a ‘closed’ conformation in the GDP-bound state. A comparison with the structure of the GMPPNP–YsxC complex from Bacillus subtilis provides insights into the mechanism of conformational change in the switch I and II regions upon hydrolysis of GTP.
GTPases; ribosome assembly; conformational switches; GTP binding
The 2.0 Å resolution crystal structure of tetrameric (αβ)2 R-state human adult aquomethemoglobin reveals axial water coordination at both the α and β heme sites.
The crystal structure of tetrameric (αβ)2 R-state human adult aquomethemoglobin is reported at 2.0 Å resolution. The asymmetric unit contained one αβ subunit pair. The R-state crystal belonged to space group P41212, with unit-cell parameters a = b = 53.6, c = 192.8 Å. An Fe-bound water molecule was modeled into the heme distal pockets of each of the α and β subunits. In the α subunit, a highly ordered liganded water was modeled with an Fe—O(water) distance of 2.2 Å and appears to be protected against escape from the distal pocket by the conformation of the heme propionate groups, which point upwards towards the distal His58 residue aided by a hydrogen-bonding network involving the solvent. In the β subunit, the liganded water exhibited greater motion and was modeled with a longer Fe—O(water) distance of 2.5 Å; in this subunit both propionate groups point downwards away from the distal His63 residue, presumably allowing greater motion of the liganded water in and out of the distal pocket.
human hemoglobin; aquomethemoglobin; ferric; R state
The structure of a recombinant form of the sweet-tasting protein thaumatin I was determined at 1.1 Å resolution and refined to an R
work of 9.1% and an R
free of 11.7%. Comparisons with plant thaumatin revealed the electron density of recombinant thaumatin I to be significantly improved, especially around Asn46 and Ser63.
Thaumatin, an intensely sweet-tasting plant protein, elicits a sweet taste at a concentration of 50 nM. The crystal structure of a recombinant form of thaumatin I produced in the yeast Pichia pastoris has been determined to a resolution of 1.1 Å. The model was refined with anisotropic B parameters and riding H atoms. A comparison of the diffraction data and refinement statistics for recombinant thaumatin I with those for plant thaumatin I revealed no significant differences in the diffraction data. The R values for recombinant thaumatin I and plant thaumatin I (F
o > 4σ) were 9.11% and 9.91%, respectively, indicating the final model to be of good quality. Notably, the electron-density maps around Asn46 and Ser63, which differ between thaumatin variants, were significantly improved. Furthermore, a number of H atoms became visible in an OMIT map and could be assigned. The high-quality structure of recombinant thaumatin with H atoms should provide details about sweetness determinants in thaumatin and provide valuable insights into the mechanism of its interaction with taste receptors.
thaumatin; sweet-tasting proteins; Pichia pastoris; H atoms
The crystal structure of C. thermocellum CelK CMB4 confirms unique extended binding pocket features of clostridial CBM4s.
Here, a 2.0 Å resolution X-ray structure of Clostridium thermocellum cellulase K family 4 carbohydrate-binding module (CelK CBM4) is reported. The resulting structure was refined to an R factor of 0.212 and an R
free of 0.274. Structural analysis shows that this new structure is very similar to the previously solved structure of C. thermocellum CbhA CBM4. Most importantly, these data support the previously proposed notion of an extended binding pocket using a novel tryptophan-containing loop that may be highly conserved in clostridial CBM4 proteins.
carbohydrate-binding modules; CBM; CBM4; CelK
Here, the crystal structure of mature fHbp determined at 2 Å resolution is presented and is compared with the structure of the same protein in complex with factor H domains 6 and 7 recently solved using X-ray techniques.
fHbp, a highly immunogenic outer membrane protein of Neisseria meningitidis, is responsible for binding to human factor H, a multi-domain protein which is the central regulator of the alternative complement pathway. Here, the crystal structure of mature fHbp determined at 2 Å resolution is presented and is compared with the structure of the same protein in complex with factor H domains 6 and 7 recently solved using X-ray techniques. While the overall protein fold is well conserved, modifications are observed mainly in the loop regions involved in the interaction, reflecting a specific adaptation of fHbp in complexing factor H with high affinity. Such a comparison has to date been impaired by the fact that fHbp models determined by NMR show remarkable differences over the entire structure.
Neisseria meningitidis; factor H; fHbp; complement system; outer membrane proteins; surface antigens
The full-length RNA-chaperone Hfq from Escherichia coli (102 amino acids) has been crystallized. The crystal belongs to the P1 space group. The structure was solved to 2.85 Å resolution, revealing that the disordered C-terminal resides on the proximal face of the hexamer.
The structure of full-length host factor Qβ (Hfq) from Escherichia coli obtained from a crystal belonging to space group P1, with unit-cell parameters a = 61.91, b = 62.15, c = 81.26 Å, α = 78.6, β = 86.2, γ = 59.9°, was solved by molecular replacement to a resolution of 2.85 Å and refined to R
work and R
free values of 20.7% and 25.0%, respectively. Hfq from E. coli has previously been crystallized and the structure has been solved for the N-terminal 72 amino acids, which cover ∼65% of the full-length sequence. Here, the purification, crystallization and structural data of the full 102-amino-acid protein are presented. These data revealed that the presence of the C-terminus changes the crystal packing of E. coli Hfq. The crystal structure is discussed in the context of the recently published solution structure of Hfq from E. coli.
RNA chaperones; Hfq; riboregulation; crystal packing
The crystal structure of flap endonuclease 1 from the hyperthermophilic archaeon D. amylolyticus was determined at 2.00 Å resolution.
Flap endonuclease 1 (FEN1) is a key enzyme in DNA repair and DNA replication. It is a structure-specific nuclease that removes 5′-overhanging flaps and the RNA/DNA primer during maturation of the Okazaki fragment. Homologues of FEN1 exist in a wide range of bacteria, archaea and eukaryotes. In order to further understand the structural basis of the DNA recognition, binding and cleavage mechanism of FEN1, the structure of FEN1 from the hyperthermophilic archaeon Desulfurococcus amylolyticus (DaFEN1) was determined at 2.00 Å resolution. The overall fold of DaFEN1 was similar to those of other archaeal FEN1 proteins; however, the helical clamp and the flexible loop exhibited a putative substrate-binding pocket with a unique conformation.
FEN1; DNA repair; DNA replication
Comparison of the 2.45 Å resolution crystal structure of homotrimeric RipC, a putative citrate lyase β subunit from Y. pestis, with structural homologs reveals conserved RipC residues that are implicated in CoA binding.
Yersinia pestis remains a threat, with outbreaks of plague occurring in rural areas and its emergence as a weapon of bioterrorism; thus, an improved understanding of its various pathogenicity pathways is warranted. The rip (required for intracellular proliferation) virulence operon is required for Y. pestis survival in interferon-γ-treated macrophages and has been implicated in lowering macrophage-produced nitric oxide levels. RipC, one of three gene products from the rip operon, is annotated as a citrate lyase β subunit. Furthermore, the Y. pestis genome lacks genes that encode citrate lyase α and γ subunits, suggesting a unique functional role of RipC in the Y. pestis
rip-mediated survival pathway. Here, the 2.45 Å resolution crystal structure of RipC revealed a homotrimer in which each monomer consists of a (β/α)8 TIM-barrel fold. Furthermore, the trimeric state was confirmed in solution by size-exclusion chromatography. Through sequence and structure comparisons with homologous proteins, it is proposed that RipC is a putative CoA- or CoA-derivative binding protein.
RipC; citrate lyase β subunit; Yersinia pestis
The structure of immunoglobulin-like repeat 10 from human filamin A solved at 2.44 Å resolution suggests the potential effects of mutations correlated with otopalatodigital syndrome spectrum disorders.
Filamin A (FlnA) plays a critical role in cytoskeletal organization, cell motility and cellular signaling. FlnA utilizes different binding sites on a series of 24 immunoglobulin-like domains (Ig repeats) to interact with diverse cytosolic proteins and with cytoplasmic portions of membrane proteins. Mutations in a specific domain, Ig10 (FlnA-Ig10), are correlated with two severe forms of the otopalatodigital syndrome spectrum disorders Melnick–Needles syndrome and frontometaphyseal dysplasia. The crystal structure of FlnA-Ig10 determined at 2.44 Å resolution provides insight into the perturbations caused by these mutations.
filamin; immunoglobulin-like domains; Melnick–Needles syndrome; frontometaphyseal dysplasia
Dodecyl-β-d-selenomaltoside in a leukotriene C4 synthase crystal exhibited sufficient anomalous diffraction for multiwavelength anomalous diffraction phasing.
Dodecyl-β-d-selenomaltoside (SeDDM) is a seleno-detergent with a β-glycosidic seleno-ether in place of the ether moiety in dodecyl-β-d-maltoside. Seleno-detergents are candidates for heavy-atom agents in experimental phasing of membrane proteins in protein crystallography. Crystals of a nuclear membrane-embedded enzyme, leukotriene C4 synthase (LTC4S), in complex with SeDDM were prepared and a multiwavelength anomalous diffraction (MAD) experiment was performed. The SeDDM in the LTC4S crystal exhibited sufficient anomalous diffraction for determination of the structure using MAD phasing.
membrane proteins; MAD phasing; seleno-detergents; leukotriene C4 synthase
Recombinant hyperthermophilic β-glucosidase from P. furiosus was crystallized. The crystal structure was solved to a resolution of 2.35 Å.
Three categories of cellulases, endoglucanases, cellobiohydrolases and β-glucosidases, are commonly used in the process of cellulose saccharification. In particular, the activity and characteristics of hyperthermophilic β-glucosidase make it promising in industrial applications of biomass. In this paper, the crystal structure of the hyperthermophilic β-glucosidase from Pyrococcus furiosus (BGLPf) was determined at 2.35 Å resolution in a new crystal form. The structure showed that there is one tetramer in the asymmetric unit and that the dimeric molecule exhibits a structure that is stable towards sodium dodecyl sulfate (SDS). The dimeric molecule migrated in reducing SDS polyacrylamide gel electrophoresis (SDS–PAGE) buffer even after boiling at 368 K. Energy calculations demonstrated that one of the two dimer interfaces acquired the largest solvation free energy. Structural comparison and sequence alignment with mesophilic β-glucosidase A from Clostridium cellulovorans (BGLACc) revealed that the elongation at the C-terminal end forms a hydrophobic patch at the dimer interface that might contribute to hyperthermostability.
hyperthermophilic; cellulases; biomass; Pyrococcus furiosus
B. melitensis is a NIAID Category B microorganism that is responsible for brucellosis and is a potential agent for biological warfare. Here, the solution structure of the 116-residue arsenate reductase-related protein Bm-YffB (BR0369) from this organism is reported.
Brucella melitensis is the etiological agent responsible for brucellosis. Present in the B. melitensis genome is a 116-residue protein related to arsenate reductases (Bm-YffB; BR0369). Arsenate reductases (ArsC) convert arsenate ion (H2AsO4
−), a compound that is toxic to bacteria, to arsenite ion (AsO2
−), a product that may be efficiently exported out of the cell. Consequently, Bm-YffB is a potential drug target because if arsenate reduction is the protein’s major biological function then disabling the cell’s ability to reduce arsenate would make these cells more sensitive to the deleterious effects of arsenate. Size-exclusion chromatography and NMR spectroscopy indicate that Bm-YffB is a monomer in solution. The solution structure of Bm-YffB (PDB entry 2kok) shows that the protein consists of two domains: a four-stranded mixed β-sheet flanked by two α-helices on one side and an α-helical bundle. The α/β domain is characteristic of the fold of thioredoxin-like proteins and the overall structure is generally similar to those of known arsenate reductases despite the marginal sequence similarity. Chemical shift perturbation studies with 15N-labeled Bm-YffB show that the protein binds reduced glutathione at a site adjacent to a region similar to the HX
3R catalytic sequence motif that is important for arsenic detoxification activity in the classical arsenate-reductase family of proteins. The latter observation supports the hypothesis that the ArsC-YffB family of proteins may function as glutathione-dependent thiol reductases. However, comparison of the structure of Bm-YffB with the structures of proteins from the classical ArsC family suggest that the mechanism and possibly the function of Bm-YffB and other related proteins (ArsC-YffB) may differ from those of the ArsC family of proteins.
arsenate reductases; Brucella melitensis; YffB; brucellosis
One third of the world’s human population is infected with M. tuberculosis, the etiological agent responsible for tuberculosis (TB). Here, the solution structure of the small iron-binding protein from this organism, rubredoxin B (Rv3250c), is reported in the zinc-substituted form.
Owing to the evolution of multi-drug-resistant and extremely drug-resistant Mycobacterium tuberculosis strains, there is an urgent need to develop new antituberculosis strategies to prevent TB epidemics in the industrial world. Among the potential new drug targets are two small nonheme iron-binding proteins, rubredoxin A (Rv3251c) and rubredoxin B (Rv3250c), which are believed to play a role in electron-transfer processes. Here, the solution structure and biophysical properties of one of these two proteins, rubredoxin B (Mt-RubB), determined in the zinc-substituted form are reported. The zinc-substituted protein was prepared by expressing Mt-RubB in minimal medium containing excess zinc acetate. Size-exclusion chromatography and NMR spectroscopy indicated that Mt-RubB was a monomer in solution. The structure (PDB entry 2kn9) was generally similar to those of other rubredoxins, containing a three-stranded antiparallel β-sheet (β2–β1–β3) and a metal tetrahedrally coordinated to the S atoms of four cysteine residues (Cys9, Cys12, Cys42 and Cys45). The first pair of cysteine residues is at the C-terminal end of the first β-strand and the second pair of cysteine residues is towards the C-terminal end of the loop between β2 and β3. The structure shows the metal buried deeply within the protein, an observation that is supported by the inability to remove the metal with excess EDTA at room temperature. Circular dichroism spectroscopy shows that this stability extends to high temperature, with essentially no change being observed in the CD spectrum of Mt-RubB upon heating to 353 K.
rubredoxin B; Mycobacterium tuberculosis; Rv3250c
An introduction and overview of the focus, goals and overall mission of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) is given.
The Seattle Structural Genomics Center for Infectious Disease (SSGCID) is a consortium of researchers at Seattle BioMed, Emerald BioStructures, the University of Washington and Pacific Northwest National Laboratory that was established to apply structural genomics approaches to drug targets from infectious disease organisms. The SSGCID is currently funded over a five-year period by the National Institute of Allergy and Infectious Diseases (NIAID) to determine the three-dimensional structures of 400 proteins from a variety of Category A, B and C pathogens. Target selection engages the infectious disease research and drug-therapy communities to identify drug targets, essential enzymes, virulence factors and vaccine candidates of biomedical relevance to combat infectious diseases. The protein-expression systems, purified proteins, ligand screens and three-dimensional structures produced by SSGCID constitute a valuable resource for drug-discovery research, all of which is made freely available to the greater scientific community. This issue of Acta Crystallographica Section F, entirely devoted to the work of the SSGCID, covers the details of the high-throughput pipeline and presents a series of structures from a broad array of pathogenic organisms. Here, a background is provided on the structural genomics of infectious disease, the essential components of the SSGCID pipeline are discussed and a survey of progress to date is presented.
SSGCID; structural genomics; structure-based drug design; infectious diseases; pathogens; emerging and re-emerging diseases