The crystal structure of DusC from E. coli was determined at 2.1 Å resolution.
Dihydrouridine (D) is one of the most widely conserved tRNA modifications. Dihydrouridine synthase (Dus) is responsible for introducing D modifications into RNA by the reduction of uridine. Recently, a unique substrate-recognition mechanism using a small adapter molecule has been proposed for Thermus thermophilus Dus (TthDusC). To acquire insight regarding its substrate-recognition mechanism, the crystal structure of DusC from Escherichia coli (EcoDusC) was determined at 2.1 Å resolution. EcoDusC was shown to be composed of two domains: an N-terminal catalytic domain and a C-terminal tRNA-binding domain. An L-shaped electron density surrounded by highly conserved residues was found in the active site, as observed for TthDus. Structure comparison with TthDus indicated that the N-terminal region has a similar structure, whereas the C-terminal domain has marked differences in its relative orientation to the N-terminal domain as well as in its own structure. These observations suggested that Dus proteins adopt a common substrate-recognition mechanism using an adapter molecule, whereas the manner of tRNA binding is diverse.
dihydrouridine; dihydrouridine synthase; tRNA modification
The crystal structure of Na-GST-3 from the human hookworm parasite N. americanus is reported.
Necator americanus is the major cause of human hookworm infection, which is a global cause of anemia in the developing world. Ongoing efforts to control hookworm infection include the identification of candidate vaccine antigens as well as potential therapeutic targets from the infective L3 larval stages and adult stages of the parasite. One promising family of proteins are the adult-stage-secreted cytosolic glutathione S-transferases (GSTs). Nematode GSTs facilitate the inactivation and degradation of a variety of electrophilic substrates (drugs) via the nucleophilic addition of reduced glutathione. Parasite GSTs also play significant roles in multi-drug resistance and the modulation of host immune defense mechanisms. Here, the structure of Na-GST-3, one of three GSTs secreted by adult-stage N. americanus, is reported. Unlike most GST structures, the Na-GST-3 crystal contains a monomer in the asymmetric unit. However, the monomer forms a prototypical GST dimer across the crystallographic twofold. A glutathione from the fermentation process is bound to the monomer. The overall binding cavity of Na-GST-3 is reminiscent of that of other N. americanus GSTs and is larger and capable of binding a wider array of ligands than GSTs from organisms that have other major detoxifying mechanisms. Furthermore, despite having low sequence identity to the host GST, Na-GST-3 has a greater tertiary-structure similarity to human sigma-class GST than was observed for the other N. americanus GSTs.
hookworms; GST; Necator americanus; vaccines; inhibition
The crystal structure of 1,5-anhydro-d-fructose reductase from S. meliloti has been determined to 1.93 Å resolution. It is compared with that of 1,5-anhydro-d-fructose reductase from S. morelense and the significance of the observed open conformation of the active site is discussed.
1,5-Anhydro-d-fructose (1,5-AF) is an interesting building block for enantioselective and stereoselective organic synthesis. Enzymes acting on this compound are potential targets for structure-based protein/enzyme design to extend the repertoire of catalytic modifications of this and related building blocks. Recombinant 1,5-anhydro-d-fructose reductase (AFR) from Sinorhizobium meliloti 1021 was produced in Escherichia coli, purified using a fused 6×His affinity tag and crystallized in complex with the cofactor NADP(H) using the hanging-drop technique. Its structure was determined to 1.93 Å resolution using molecular replacement. The structure displays an empty substrate-binding site and can be interpreted as an open conformation reflecting the enzyme state shortly after the release of product, presumably with bound oxidized cofactor NADP+. Docking simulations indicated that amino-acid residues Lys94, His151, Trp162, Arg163, Asp176 and His180 are involved in substrate binding, catalysis or product release. The side chain of Lys94 seems to have the ability to function as a molecular switch. The crystal structure helps to characterize the interface relevant for dimer formation as observed in solution. The crystal structure is compared with the structure of the homologue from S. morelense, which was solved in a closed conformation and for which dimer formation in solution could not be verified but seems to be likely based on the presented studies of S. meliloti AFR.
carbohydrate metabolism; redox enzymes; biotechnology
Endo-β-1,4-galactanase from E. nidulans crystallized in the presence of 0.2 M zinc acetate contains 15 zinc ions.
A novel Emericella nidulans endo-β-1,4-galactanase (EnGAL) demonstrates a strong capacity to generate high levels of very potent prebiotic oligosaccharides from potato pulp, a by-product of the agricultural potato-starch industry. EnGAL belongs to glycoside hydrolase family 53 and shows high (72.5%) sequence identity to an endo-β-1,4-galactanase from Aspergillus aculeatus. Diffraction data extending to 2.0 Å resolution were collected from a crystal of EnGAL grown from conditions containing 0.2 M zinc acetate. The crystal structure showed a high similarity between EnGAL and other endo-β-1,4-galactanases belonging to GH53. It also revealed 15 zinc ions bound to the protein, one of which is located in the active site, where it is coordinated by residues Glu136 and Glu246 which comprise the catalytic machinery. The majority of the zinc ions are located on the surface of the enzyme, in some cases with side chains from two different molecules as ligands, thus explaining why the presence of zinc ions was essential for crystallization.
galactanases; glycosyl hydrolases; family GH53; zinc binding; crystallization
The preliminary crystallographic analysis of neuraminidase N2 from influenza virus A/Myanmar/M187/2007 is reported. The crystals belonged to space group P212121, with four monomers per asymmetric unit.
Influenza virus is a major viral respiratory pathogen that causes yearly epidemics in temperate climates. The H3N2 subtype is one of the major causative agents of severe epidemics and plays a critical role in vaccine development. The neuraminidase (NA) inhibitors oseltamivir and zanamivir are two commercially available NA-targeted competitive antiviral drugs. However, their effectiveness has been compromised by the rapid emergence of resistance. Q136K is a novel mutation in NA which confers resistance to zanamivir. In this study, a Q136K mutant N2 protein was expressed in a baculovirus system and crystals were obtained. The crystal of N2 belonged to space group P212121, with unit-cell parameters a = 109.5, b = 112.8, c = 165.2 Å. Data were collected to 2.4 Å resolution. Four monomers were found in the asymmetric unit. The Matthews coefficient and solvent content were calculated to be 3.0 Å3 Da−1 and 59.0%, respectively.
influenza A virus; neuraminidases; drug resistance
Selenophosphate synthetases from L. major and T. brucei were crystallized for the first time using limited proteolysis and microbatch techniques and suitable crystals for X-ray diffraction experiments were obtained.
Selenophosphate synthetase (SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the activation of selenide with adenosine 5′-triphosphate (ATP) to generate selenophosphate, the essential selenium donor for selenocysteine synthesis. Recombinant full-length Leishmania major SPS (LmSPS2) was recalcitrant to crystallization. Therefore, a limited proteolysis technique was used and a stable N-terminal truncated construct (ΔN-LmSPS2) yielded suitable crystals. The Trypanosoma brucei SPS orthologue (TbSPS2) was crystallized by the microbatch method using paraffin oil. X-ray diffraction data were collected to resolutions of 1.9 Å for ΔN-LmSPS2 and 3.4 Å for TbSPS2.
selenocysteine; selenophosphate synthetase; Trypanosoma brucei; Leishmania major
The staphylococcal α-haemolysin H35A mutant has been cloned, expressed and crystallized. The crystals belonged to space group P61, with unit-cell parameters a = b = 151.3, c = 145.0 Å.
Staphylococcal α-haemolysin is a β-barrel pore-forming toxin expressed by Staphylococcus aureus. α-Haemolysin is secreted as a water-soluble monomeric protein which binds to target membranes and forms membrane-inserted heptameric pores. Although the crystal structures of the heptameric pore and monomer bound to an antibody have been determined, that of monomeric α-haemolysin without binder has yet to be elucidated. Previous mutation studies showed that mutants of His35 retain the monomeric structure but are unable to assemble into heptamers. Here, α-haemolysin H35A mutants were expressed, purified and crystallized. Diffraction data were collected to 2.90 Å resolution. The crystals belonged to space group P61, with unit-cell parameters a = b = 151.3, c = 145.0 Å. Molecular replacement found four molecules in an asymmetric unit. The relative orientation among molecules was distinct from that of the pore, indicating that the crystal contained monomeric α-haemolysin.
staphylococcal α-haemolysin; monomer; pore-forming toxins
P. cuspidatum bifunctional chalcone synthase/benzalacetone synthase was crystallized in the presence of the product and diffraction data were collected to 2.0 Å resolution.
The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs) generate the backbones of a variety of plant secondary metabolites. An active bifunctional chalcone synthase/benzalacetone synthase (CHS/BAS) from Polygonum cuspidatum was overexpressed in Escherichia coli as a C-terminally polyhistidine-tagged fusion protein, purified to homogeneity and crystallized using polyethylene glycol 4000 as a precipitant. The production of well shaped crystals of the complex between PcPKS1 and benzalacetone was dependent on the presence of sorbitol and barium chloride as additives. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 80.23, b = 81.01, c = 122.89 Å, and diffracted X-rays to at least 2.0 Å resolution.
chalcone synthase/benzalacetone synthase; Polygonum cuspidatum
Cyanuric acid hydrolase has been crystallized with the bound inhibitor barbituric acid and diffraction data were collected to 2.7 Å resolution.
Cyanuric acid is synthesized industrially and forms during the microbial metabolism of s-triazine herbicides. Cyanuric acid is metabolized by some microorganisms via cyanuric acid hydrolase (CAH), which opens the s-triazine ring as a prelude to further metabolism. CAH is a member of the rare cyanuric acid hydrolase/barbiturase family. Here, the crystallization and preliminary X-ray diffraction analysis of CAH from Azorhizobium caulinodans are reported. CAH was cocrystallized with barbituric acid, a close analog of cyanuric acid that is a tight-binding competitive inhibitor. Crystals suitable for X-ray diffraction experiments were grown in conditions containing PEG 8K or magnesium sulfate as precipitants. An X-ray diffraction data set was collected from CAH–barbituric acid crystals to 2.7 Å resolution. The crystals were found to belong to space group I4122, with unit-cell parameters a = b = 237.9, c = 105.3 Å, α = β = γ = 90°.
cyanuric acid; barbituric acid; triazine; cyanuric acid hydrolase; Azorhizobium caulinodans
Spermidine acetyltransferase (SAT) from E. coli catalyses the transfer of acetyl groups from acetyl-CoA to spermidine. SAT was crystallized and the crystals contained four molecules in the asymmetric unit.
The spermidine acetyltransferase (SAT) from Escherichia coli catalyses the transfer of acetyl groups from acetyl-CoA to spermidine. SAT has been expressed and purified from E. coli. SAT was crystallized by the sitting-drop vapour-diffusion method to obtain a more detailed insight into the molecular mechanism. Preliminary X-ray diffraction studies revealed that the crystals diffracted to 2.5 Å resolution and belonged to the cubic space group P23, with unit-cell parameters a = b = c = 148.7 Å. They contained four molecules per asymmetric unit.
spermidine acetyltransferase; Escherichia coli
Nitroalkane oxidase from P. aeruginosa was purified and crystallized. A complete data set was collected to 1.9 Å resolution.
Nitroalkane oxidase (NAO) is a flavin-dependent enzyme which catalyses the oxidation of nitroalkanes to the corresponding aldehydes or ketones, nitrite and hydrogen peroxide. In order to better understand the structure and function of this enzyme, NAO from Pseudomonas aeruginosa was purified and crystallized as a native and a selenomethionine-substituted (SeMet) enzyme. Both crystals diffracted to a resolution of 1.9 Å and belonged to the primitive orthorhombic space group P21, with unit-cell parameters a = 70.06, b = 55.43, c = 87.74 Å, β = 96.56° for native NAO and a = 69.89, b = 54.83, c = 88.20 Å, β = 95.79° for SeMet NAO. Assuming the presence of two molecules in the asymmetric unit in both crystals, the Matthews coefficients (V
M) for native and SeMet NAO were calculated to be 2.30 and 2.48 Å3 Da−1, with estimated solvent contents of 46.50 and 50.37%, respectively.
nitroalkane oxidase; NAO; Pseudomonas aeruginosa; nitro compounds
The bacterial cell-division protein FtsA from methicillin-resistant S. aureus was crystallized and X-ray diffraction data were collected to a resolution of 2.3 Å.
FtsA from methicillin-resistant Staphylococcus aureus (MRSA) was cloned, overexpressed and purified. The protein was crystallized using the sitting-drop vapour-diffusion technique. A cocrystal with β-γ-imidoadenosine 5′-phosphate (AMPPNP; a nonhydrolysable ATP analogue) was grown using PEG 3350 as a precipitant at 293 K. X-ray diffraction data were collected to a resolution of 2.3 Å at 100 K. The crystal belonged to the monoclinic space group P21, with unit-cell parameters a = 75.31, b = 102.78, c = 105.90 Å, β = 96.54°. The calculated Matthews coefficient suggested that the asymmetric unit contained three or four monomers.
bacterial divisome; methicillin-resistant Staphylococcus aureus; FtsA
The α subdomain of Lon protease from B. thermoruber WR-249 has been expressed, purified and crystallized. Preliminary X-ray diffraction experiments have been undertaken.
DNA-binding ability has previously been reported as a novel function for the thermostable Lon protease from Brevibacillus thermoruber WR-249 (Bt-Lon), and the α subdomain (amino acids 491–605) of Bt-Lon has been identified as being responsible for DNA binding. However, the physiological role and DNA-recognition mode of Bt-Lon still remain unclear. In this study, the crystallization and preliminary crystallographic analysis of the Bt-Lon α subdomain are presented. Native diffraction data to 2.88 Å resolution were obtained from a vitrified crystal at 100 K on the BL13C1 beamline at the NSRRC (National Synchrotron Radiation Research Center), Taiwan. The crystals belonged to space group P23, with unit-cell parameters a = b = c = 94.28 Å. Solvent-content calculations and molecular-replacement results suggest that there are two molecules of Bt-Lon α subdomain per asymmetric unit.
Lon protease; DNA binding; Brevibacillus thermoruber
The crystallization and preliminary X-ray crystallographic analysis of the MxaJ protein of the mox operon from the marine bacterium M. aminisulfidivorans MPT is reported.
The methanol-oxidizing system (mox) is essential for methylotrophic bacteria to extract energy during the oxidoreduction reaction and consists of a series of electron-transfer proteins encoded by the mox operon. One of the key enzymes is the α2β2 methanol dehydrogenase complex (type I MDH), which converts methanol to formaldehyde during the 2e− transfer through the prosthetic group pyrroloquinoline quinone. MxaJ, a product of mxaJ of the mox operon, is a component of the MDH complex and enhances the methanol-converting activity of the MDH complex. However, the exact functional mechanism of MxaJ in the complex is not clearly known. To investigate the functional role of MxaJ in MDH activity, an attempt was made to determine its crystal structure. Diffraction data were collected from a selenomethionine-substituted crystal to 1.92 Å resolution at the peak wavelength. The crystal belonged to the orthorhombic space group P212121, with unit-cell parameters a = 37.127, b = 63.761, c = 99.246 Å. The asymmetric unit contained one MxaJ molecule with a calculated Matthews coefficient of 2.11 Å3 Da−1 and a solvent content of 41.7%. Three-dimensional structure determination of the MxaJ protein is currently in progress by the single-wavelength anomalous dispersion technique and model building.
methanol-oxidizing system (mox); MxaJ; Methylophaga aminisulfidivorans
The expression and crystallization of the extracellular sensory domain of DraK are reported.
The bacterium Streptomyces coelicolor produces useful antibiotics from its secondary metabolites. DraK is a sensory histidine kinase involved in the differential regulation of antibiotics in S. coelicolor through the DraR/DraK two-component system. Here, the extracellular sensory domain of DraK was overexpressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method. The crystal diffracted to 2.2 Å resolution and belonged to space group C2221, with unit-cell parameters a = 41.91, b = 174.50, c = 145.25 Å, α = β = γ = 90°.
DraK; SCO3062; histidine kinases; two-component systems; Streptomyces coelicolor
The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of a novel AB5 toxin EcxAB are described.
AB5 toxins are key virulence factors found in a range of pathogenic bacteria. AB5 toxins consist of two components: a pentameric B subunit that targets eukaryotic cells by binding to glycans located on the cell surface and a catalytic A subunit that disrupts host cellular function following internalization. To date, the A subunits of AB5 toxins either have RNA-N-glycosidase, ADP-ribosyltransferase or serine protease activity. However, it has been suggested that a novel AB5 toxin produced by clinical isolates of Escherichia coli and Citrobacter freundii has an A subunit with metalloproteinase activity. Here, the expression, purification and crystallization of this novel AB5 toxin from E. coli (EcxAB) and the collection of X-ray data to 1.9 Å resolution are reported.
AB5 toxins; Escherichia coli; co-expression; proteases
The human TRAF4 TRAF domain was crystallized. The crystals were found to belong to the hexagonal space group P32, with unit-cell parameters a = b = 147.17, c = 202.69 Å. The crystals were obtained at 293 K and diffracted to a resolution of 4.2 Å.
TNF (tumour necrosis factor) receptor-associated factor 4 (TRAF4) is a unique TRAF protein that participates in morphogenetic and developmental function and cell migration. TRAF-family proteins contain a TRAF domain for target interaction. In this study, the short form of the human TRAF4 TRAF domain, corresponding to amino acids 290–462, was overexpressed in Escherichia coli using engineered C-terminal His tags. The short form of the TRAF4 TRAF domain was purified to homogeneity and crystallized at 293 K. Finally, X-ray diffraction data were collected to a resolution of 4.2 Å from a crystal belonging to the hexagonal space group P32, with unit-cell parameters a = b = 147.17, c = 202.69 Å.
TRAF4 TRAF domain
A putative lipase (CpsLip) from C. psychrerythraea strain 34H was expressed, purified and characterized. Crystallization and preliminary X-ray crystallographic analysis of CpsLip were performed.
The putative lipase CpsLip from the psychrophilic bacterium Colwellia psychrerythraea 34H encodes a 34 538 Da, 308-amino-acid protein. In this study, CpsLip (UniProtKB code Q486T5) was expressed as an N-terminal hexahistidine fusion protein in Escherichia coli and purified by affinity and size-exclusion chromatography. The expression and purification of CpsLip enabled characterization of the lipase enzymatic properties of the protein. The optimal activity temperature and pH of the recombinant protein were 298 K and pH 7, respectively. CpsLip maintained over 80% activity in the low-temperature range (278–288 K), thereby suggesting that CpsLip is a cold-active lipase. Substrate-specificity analysis demonstrated that CpsLip exhibits maximum activity towards the C12 acyl group. In addition, sequence-alignment results revealed that CpsLip has a highly conserved catalytic triad in the active site consisting of residues Ser111, Asp135 and His283. Moreover, purified CpsLip was successfully crystallized using the hanging-drop vapour-diffusion method and a complete diffraction data set was collected to 4.0 Å resolution using synchrotron radiation on the BL-5A beamline of the Photon Factory.
cold adaptation; lipases; α/β-hydrolase fold; CpsLip; Colwellia psychrerythraea
Crystals of the β-pore-forming toxin monalysin from the Drosophila pathogen P. entomophila grown from PEG solutions were monoclinic (space group C2) and diffracted to 2.85 Å resolution.
Monalysin was recently described as a novel pore-forming toxin (PFT) secreted by the Drosophila pathogen Pseudomonas entomophila. Recombinant monalysin is multimeric in solution, whereas PFTs are supposed to be monomeric until target membrane association. Monalysin crystals were obtained by the hanging-drop vapour-diffusion method using PEG 8000 as precipitant. Preliminary X-ray diffraction analysis revealed that monalysin crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 162.4, b = 146.2, c = 144.4 Å, β = 122.8°, and diffracted to 2.85 Å resolution using synchrotron radiation. Patterson self-rotation analysis and Matthews coefficient calculation indicate that the asymmetric unit contains nine copies of monalysin. Heavy-atom derivative data were collected and a Ta6Br14 cluster derivative data set confirmed the presence of ninefold noncrystallographic symmetry.
monalysin; Pseudomonas entomophila; pore-forming toxins
An alanine dehydrogenase from B. megaterium WSH-002 was expressed in E. coli and purified. Crystallization and preliminary X-ray crystallographic analysis of the recombinant enzyme were performed.
Alanine dehydrogenase (l-AlaDH) from Bacillus megaterium WSH-002 catalyses the NAD+-dependent interconversion of l-alanine and pyruvate. The enzyme was expressed in Escherichia coli BL21 (DE3) cells and purified with a His6 tag by Ni2+-chelating affinity chromatography for X-ray crystallographic analysis. Crystals were grown in a solution consisting of 0.1 M HEPES pH 8.0, 12%(w/v) polyethylene glycol 8000, 8%(v/v) ethylene glycol at a concentration of 15 mg ml−1 purified protein. The crystal diffracted to 2.35 Å resolution and belonged to the trigonal space group R32, with unit-cell parameters a = b = 125.918, c = 144.698 Å.
alanine dehydrogenase; Bacillus megaterium
Hepatitis B virus-like particles consisting of core protein dimers fused to EGFP were expressed, purified and crystallized. The crystals diffracted to 2.8 Å resolution.
Virus-like particles (VLPs) have many potentially useful applications. The core proteins of human hepatitis B virus self-assemble into icosahedral VLPs. As previously reported, core protein dimers (CPDs), produced by connecting two core proteins via a peptide linker, can also assemble into VLPs. CPDs in which heterologous proteins were connected to the C-terminus (CPD1) were found to rearrange into symmetrical octahedra during crystallization. In this study, a heterologous protein was inserted into the peptide linker of the CPD (CPD2). CPD2 was expressed in Escherichia coli, assembled into VLPs, purified and crystallized. A single crystal diffracted to 2.8 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 218.6 Å. Single-crystal analysis showed that CPD1 and CPD2 rearranged into the same octahedral organization in a crystallization solution.
fusion protein; enhanced green fluorescent protein; hepatitis B virus
B. circulans T-3040 cycloisomaltooligosaccharide glucanotransferase was overexpressed in E. coli in two forms and crystallized by the sitting-drop vapour-diffusion method.
Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase (BcCITase) catalyses an intramolecular transglucosylation reaction and produces cycloisomaltooligosaccharides from dextran. BcCITase was overexpressed in Escherichia coli in two different forms and crystallized by the sitting-drop vapour-diffusion method. The crystal of BcCITase bearing an N-terminal His6 tag diffracted to a resolution of 2.3 Å and belonged to space group P3121, containing a single molecule in the asymmetric unit. The crystal of BcCITase bearing a C-terminal His6 tag diffracted to a resolution of 1.9 Å and belonged to space group P212121, containing two molecules in the asymmetric unit.
Bacillus circulans; cycloisomaltooligosaccharide glucanotransferase; dextran; glycoside hydrolase family 66
All the crystallization communications published in Acta Cryst. F in 2012 were analysed. Details of the analysis are presented, along with some suggestions for making this type of publication more useful.
Crystallization of macromolecules is famously difficult. By knowing what has worked for others, researchers can ease the process, both in the case where the protein has already been crystallized and in the situation where more general guidelines are needed. The 264 crystallization communications published in Acta Crystallographica Section F in 2012 have been reviewed, and from this analysis some information about trends in crystallization has been gleaned. More importantly, it was found that there are several ways in which the utility of these communications could be increased: to make each individual paper a more complete crystallization record; and to provide a means for taking a snapshot of what the current ‘best practices’ are in the field.
crystallization; crystallization communications
The crystal structure of the archaeal peroxiredoxin from P. horikoshii was determined at a resolution of 2.25 Å and compared with that of a homologous protein from A. pernix.
The crystal structure of peroxiredoxin from the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii (PhPrx) was determined at a resolution of 2.25 Å. The overall structure was a ring-type decamer consisting of five homodimers. Citrate, which was included in the crystallization conditions, was bound to the peroxidatic cysteine of the active site, with two O atoms of the carboxyl group mimicking those of the substrate hydrogen peroxide. PhPrx lacked the C-terminal tail that forms a 32-residue extension of the protein in the homologous peroxiredoxin from Aeropyrum pernix (ApPrx).
peroxiredoxins; Pyrococcus horikoshii
The structure of the cellulose-binding module of the C. thermocellum cellulosome-integrating protein A scaffoldin with portions of the C- and N-terminal flanking linker regions has been determined at 1.98 Å resolution.
The cellulosome of the cellulolytic bacterium Clostridium thermocellum has a structural multi-modular protein called CipA (cellulosome-integrating protein A) that includes nine enzyme-binding cohesin modules and a family 3 cellulose-binding module (CBM3a). In the CipA protein, the CBM3a module is located between the second and third cohesin modules and is connected to them via proline/threonine-rich linkers. The structure of CBM3a with portions of the C- and N-terminal flanking linker regions, CBM3a-L, has been determined to a resolution of 1.98 Å. The structure is a β-sandwich with a structural Ca2+ ion. The structure is consistent with the previously determined CipA CBM structure; however, the structured linker regions provide a deeper insight into the overall cellulosome structure and assembly.
family 3 cellulose-binding module; CipA; Clostridium thermocellum; cellulosome