A short helix in the C-terminal region of LolA is important for the specific membrane localization of lipoproteins

The structures of a lipoprotein carrier, LolA, and a lipoprotein receptor, LolB, are similar except for an extra C-terminal loop containing a 3(10) helix and beta-strand 12 in LolA. Lipoprotein release was significantly reduced when beta-12 was deleted. Deletion of the 3(10) helix also inhibited the lipoprotein release. Furthermore, lipoproteins were non-specifically localized to membranes when LolA lacked the 3(10) helix. Thus, the membrane localization of lipoproteins with the LolA derivative lacking the 3(10) helix was independent of LolB whereas LolB was essential for the outer membrane localization of lipoproteins with the wild-type LolA.     

An octaheme c-type cytochrome from Shewanella oneidensis can reduce nitrite and hydroxylamine

A c-type cytochrome from Shewanella oneidensis MR-1, containing eight hemes, has been previously designated as an octaheme tetrathionate reductase (OTR). The structure of OTR revealed that the active site contains an unusual lysine-ligated heme, despite the presence of a CXXCH motif in the sequence that would predict histidine ligation. This lysine ligation has been previously observed only in the pentaheme nitrite reductases, suggesting that OTR may have a possible role in nitrite reduction. We have now shown that OTR is an efficient nitrite and hydroxylamine reductase and that ammonium ion is the product. These results indicate that OTR may have a role in the biological nitrogen cycle.     

Cytochrome P450 monooxygenase from Clostridium acetobutylicum: a new alpha-fatty acid hydroxylase

Cytochrome P450 monooxygenase from the anaerobic microorganism Clostridium acetobutylicum (CYP152A2) has been produced in Escherichia coli. CYP152A2 was shown to bind a broad range of saturated and unsaturated fatty acids and corresponding methyl esters and demonstrated a high peroxygenase activity of up to 200min(-1) with myristic acid. Although a high concentration of hydrogen peroxide of 200microM was necessary for high activities of the enzyme, it led to a fast enzyme inactivation within 2-4min. This might reflect the natural function of CYP152A2 as a rapid hydrogen peroxide scavenging enzyme. In two different reconstituted systems with NADPH, CYP152A2 was able to convert 10 times more substrate, if provided with flavodoxin and flavodoxin reductase from E. coli and even 30-40 times more substrate with the CYP102A1-reductase from Bacillus megaterium. According to the clear preference for hydroxylation at alpha-position, CYP152A2 can be referred to as fatty acid alpha-hydroxylase.     

Identification of the residue responsible for catalysing regeneration of activity in the inactive glutamate dehydrogenase mutant D165N

Previously a mutant of clostridial glutamate dehydrogenase with the catalytic Asp-165 replaced by Asn was shown to regain activity through spontaneous, specific deamidation of this residue. A double mutant D165N/K125A has now been constructed, in which the catalytic Lys is also replaced. This was successfully over-expressed and according to several criteria appears to be correctly folded. The double mutant was incubated for 35 days under conditions where D165N reactivates. LC-MS analysis of tryptic digests of timed samples showed no significant deamidation. This confirms that the reactivation of D165N is a consequence of the catalytic chemistry of the enzyme's active site.     

Studying subunit-subunit interactions in a bacterial ABC transporterby in vitro assembly

The thermostable arginine ABC transporter of Geobacillus stearothermophilus consists of a solute binding protein, ArtJ; a transmembrane subunit, ArtM; and a nucleotide-binding subunit, ArtP. An ArtM/His₆-ArtP complex was functionally assembled from separately purified subunits as demonstrated by assaying stimulation of its ATPase activity by arginine-loaded ArtJ in proteoliposomes. Studying in vitro assembly with variants carrying mutations in the conserved Q loop and/or the EAA loop of ArtP and ArtM, respectively, confirmed the predicted roles of both motifs in intersubunit signaling and physical interaction, respectively. In vitro assembly is considered a useful tool for investigating assembly defects of ABC transporters caused by mutations.     

Structural analysis of the human galectin-9 N-terminal carbohydrate recognition domain reveals unexpected properties that differ from the mouse orthologue

Galectins are a family of β-galactoside-binding lectins that contain a conserved carbohydrate recognition domain (CRD). They exhibit high affinities for small β-galactosides as well as variable binding specificities for complex glycoconjugates. Structural and biochemical analyses of the mechanism governing specific carbohydrate recognition provide a useful template to elucidate the function of these proteins. Here we report the crystal structures of the human galectin-9 N-terminal CRD (NCRD) in the presence of lactose and Forssman pentasaccharide. Mouse galectin-9 NCRD, the structure of which was previously solved by our group, forms a non-canonical dimer in both the crystal state and in solution. Human galectin-9 NCRD, however, exists as a monomer in crystals, despite a high sequence identity to the mouse homologue. Comparative frontal affinity chromatography analysis of the mouse and human galectin-9 NCRDs revealed different carbohydrate binding specificities, with disparate affinities for complex glycoconjugates. Human galectin-9 NCRD exhibited a high affinity for Forssman pentasaccharide; the association constant for mouse galectin-9 NCRD was 100-fold less than that observed for the human protein. The combination of structural data with mutational studies demonstrated that non-conserved amino acid residues on the concave surface were important for determination of target specificities. The human galectin-9 NCRD exhibited greater inhibition of cell proliferation than the mouse NCRD. We discuss the biochemical and structural differences between highly homologous proteins from different species.     

Biochemical characterization of the HydE and HydG iron-only hydrogenase maturation enzymes from Thermatoga maritima

Fe-only hydrogenases contain a di-iron active site complex, in which the two Fe atoms have carbon monoxide and cyanide ligands and are linked together by a putative di(thiomethyl)amine molecule. We have cloned, purified and characterized the HydE and HydG proteins, thought to be involved in the biosynthesis of this peculiar metal site, from the thermophilic organism Thermotoga maritima. The HydE protein anaerobically reconstituted with iron and sulfide binds two [4Fe-4S] clusters, as characterized by UV and EPR spectroscopy. The HydG protein binds one [4Fe-4S] cluster, and probably an additional one. Both enzymes are able to reductively cleave S-adenosylmethionine (SAM) when reduced by dithionite, confirming that they are Radical-SAM enzymes.     

The structure of the carbohydrate backbone of the LPS from Shewanella putrefaciens CN32

The lipopolysaccharide (LPS) from a natural rough strain of Shewanella putrefaciens CN32 was analyzed using NMR and mass spectroscopy and chemical methods, and the following structure of its carbohydrate backbone is proposed: beta-Galf-(1-->3)-beta-Gal-(1-->4)-beta-Glc-(1-->4)-alpha-DDHep2PEtN-(1-->5)-alpha-Kdo4P-(1-->6)-beta-GlcN4P-(1-->6)-alpha-GlcN1P     

Intertwined dimeric structure for the SH3 domain of the c-Src tyrosine kinase induced by polyethylene glycol binding

Here we report the first crystal structure of the SH3 domain of the cellular Src tyrosine kinase (c-Src-SH3) domain on its own. In the crystal two molecules of c-Src-SH3 exchange their -RT loops generating an intertwined dimer, in which the two SH3 units, preserving the binding site configuration, are oriented to allow simultaneous binding of two ligand molecules. The dimerization of c-Src-SH3 is induced, both in the crystal and in solution, by the binding of a PEG molecule at the dimer interface, indicating that this type of conformations are energetically close to the native structure. These results have important implications respect to in vivo oligomerization and amyloid aggregation.     

Viroporin activity of murine hepatitis virus E protein

The viroporin activity of the E protein from murine hepatitis virus (MHV), a member of the coronaviruses, was analyzed. Viroporins are a growing family of viral proteins able to enhance membrane permeability, promoting virus budding. Initially, the MHV E gene was inducibly expressed in Escherichia coli cells, leading to the arrest of bacterial growth, cell lysis and permeabilization to different compounds. Thus, exit of labeled nucleotides from E. coli cells to the cytoplasm was apparent upon expression of MHV E. In addition, enhanced entry of the antibiotic hygromycin B occurred at levels comparable to those observed with the viroporin 6K from Sindbis virus. Mammalian cells are also readily permeabilized by the expression of MHV E protein. Finally, brefeldin A powerfully blocks the viroporin activity of the E protein in BHK cells, suggesting that an intact vesicular system is necessary for this coronavirus to permeabilize mammalian cells.     

The domain structure of talin: Residues 1815-1973 form a five-helix bundle containing a cryptic vinculin-binding site

Talin is a large flexible rod-shaped protein that activates the integrin family of cell adhesion molecules and couples them to cytoskeletal actin. Its rod region consists of a series of helical bundles. Here we show that residues 1815-1973 form a 5-helix bundle, with a topology unique to talin which is optimally suited for formation of a long rod such as talin. This is much more stable than the 4-helix (1843-1973) domain described earlier and as a result its vinculin binding sequence is inaccessible to vinculin at room temperature, with implications for the overall mechanism of the talin-vinculin interaction.

Structured summary

MINT-7722300, MINT-7760951: Talin-1 (uniprotkb:P26039) and Vinculin (uniprotkb:P12003) bind (MI:0407) by molecular sieving (MI:0071)     

Molecular and biochemical characterization of a serine racemase from Arabidopsis thaliana

A cDNA encoding a homolog of mammalian serine racemase, a unique enzyme in eukaryotes, was isolated from Arabidopsis thaliana and expressed in Escherichia coli cells. The gene product, of which the amino acid residues for binding pyridoxal 5'-phosphate (PLP) are conserved in this as well as mammalian serine racemases, catalyzes not only serine racemization but also dehydration of serine to pyruvate. The enzyme is a homodimer and requires PLP and divalent cations, Ca2+, Mg2+, Mn2+, Fe2+, or Ni2+, at alkaline pH for both activities. The racemization process is highly specific toward L-serine, whereas L-alanine, L-arginine, and L-glutamine were poor substrates. The Vmax/Km values for racemase activity of L- and D-serine are 2.0 and 1.4 nmol/mg/min/mM, respectively, and those values for L- and D-serine on dehydratase activity are 13 and 5.3 nmol/mg/min/mM, i.e. consistent with the theory of racemization reaction and the specificity of dehydration toward L-serine. Hybridization analysis showed that the serine racemase gene was expressed in various organs of A. thaliana.     

Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both α and β crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded β-sheets, was composed of a typical β-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.     

lac operon induction in Escherichia coli: Systematic comparison of IPTG and TMG induction and influence of the transacetylase LacA

Most commonly used expression systems in bacteria are based on the Escherichia coli lac promoter. Furthermore, lac operon elements are used today in systems and synthetic biology. In the majority of the cases the gratuitous inducers IPTG or TMG are used. Here we report a systematic comparison of lac promoter induction by TMG and IPTG which focuses on the aspects inducer uptake, population heterogeneity and a potential influence of the transacetylase, LacA. We provide induction curves in E. coli LJ110 and in isogenic lacY and lacA mutant strains and we show that both inducers are substrates of the lactose permease at low inducer concentrations but can also enter cells independently of lactose permease if present at higher concentrations. Using a gfp reporter strain we compared TMG and IPTG induction at single cell level and showed that bimodal induction with IPTG occurred at approximately ten-fold lower concentrations than with TMG. Furthermore, we observed that lac operon induction is influenced by the transacetylase, LacA. By comparing two Plac-gfp reporter strains with and without a lacA deletion we could show that in the lacA⁺ strain the fluorescence level decreased after few hours while the fluorescence further increased in the lacA⁻ strain. The results indicate that through the activity of LacA the IPTG concentration can be reduced below an inducing threshold concentration—an influence that should be considered if low inducer amounts are used.     

Solution structure and dynamics of the N-terminal cytosolic domain of rhomboid intramembrane protease from Pseudomonas aeruginosa: insights into a functional role in intramembrane proteolysis

Rhomboids are ubiquitous integral membrane proteases that release cellular signals from membrane-bound substrates through a general signal transduction mechanism known as regulated intramembrane proteolysis (RIP). We present the NMR structure of the cytosolic N-terminal domain (NRho) of P. aeruginosa Rhomboid. NRho consists of a novel alpha/beta fold and represents the first detailed structural insight into this class of intramembrane proteases. We find evidence that NRho is capable of strong and specific association with detergent micelles that mimic the membrane/water interface. Relaxation measurements on NRho reveal structural fluctuations on the microseconds-milliseconds timescale in regions including and contiguous to those implicated in membrane interaction. This structural plasticity may facilitate the ability of NRho to recognize and associate with membranes. We suggest that NRho plays a role in scissile peptide bond selectivity by optimally positioning the Rhomboid active site relative to the membrane plane.     

The cytochrome ba complex from the thermoacidophilic crenarchaeote Acidianus ambivalens is an analog of bc1 complexes

A novel cytochrome ba complex was isolated from aerobically grown cells of the thermoacidophilic archaeon Acidianus ambivalens. The complex was purified with two subunits, which are encoded by the cbsA and soxN genes. These genes are part of the pentacistronic cbsAB-soxLN-odsN locus. The spectroscopic characterization revealed the presence of three low-spin hemes, two of the b and one of the a_s-type with reduction potentials of + 200, + 400 and + 160 mV, respectively. The SoxN protein is proposed to harbor the heme b of lower reduction potential and the heme a_s, and CbsA the other heme b. The soxL gene encodes a Rieske protein, which was expressed in E. coli; its reduction potential was determined to be + 320 mV. Topology predictions showed that SoxN, CbsB and CbsA should contain 12, 9 and one transmembrane α-helices, respectively, with SoxN having a predicted fold very similar to those of the cytochromes b in bc₁ complexes. The presence of two quinol binding motifs was also predicted in SoxN. Based on these findings, we propose that the A. ambivalens cytochrome ba complex is analogous to the bc₁ complexes of bacteria and mitochondria, however with distinct subunits and heme types.     

Expression and purification of HtpX-like small heat shock integral membrane protease of an unknown organism related to Methylobacillus flagellatus

The M48 conserved family of peptidases contains a single catalytic zinc ion tetrahedrally co-ordinated by two histidines within an HEXXH motif. The proteases of this class are generally toxic to the cell and thus difficult to express and purify. Here, we report the expression and purification of the small HtpX-like heat shock metalloprotease from an unknown organism related to the obligate methylotrophic anaerobic bacterium, Methylobacillus flagellatus. The protease was expressed in the Escherichia coli vector - pT7. Optimization of expression was done to increase the yield and solubility of the expressed protein. Improved refolding procedures from inclusion bodies of pT7 E. coli system were devised to get the protease in an active and stable form. The protease was purified to near homogeneity in its active form from the refolded proteins of the inclusion bodies by a two-step (cation exchange followed by gel filtration) high performance liquid chromatography (HPLC). The purified protease was active on zymography and casein hydrolysis assays. The activity of the protease was found to be optimum at pH 7.4 and at a temperature of 37 degrees C but significant activity was also retained at higher temperatures of 45-50 degrees C. Centrifugal fractionation showed that it is a membrane localized endopeptidase. The methods described here can serve as guidelines to express and purify other homologues of M48 family of proteases for functional and structural studies.     

1-O-Acetyl-beta-D-galactopyranose: a novel substrate for the transglycosylation reaction catalyzed by the beta-galactosidase from Penicillium sp

1-O-Acetyl-beta-D-galactopyranose (AcGal), a new substrate for beta-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters (K(M) and k(cat)) for the hydrolysis of 1-O-acetyl-beta-D-galactopyranose catalyzed by the beta-D-galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k(cat) in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The beta-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the beta-D-galactosidase in the reaction of AcGal and methyl beta-D-galactopyranoside (1) as substrates was investigated by 1H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was beta-D-galactopyranosyl-(1-->6)-1-O-acetyl-beta-D-galactopyranose (with a yield of approximately 70%). In the case of 1 as a substrate, the main transglycosylation product was methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside. Methyl beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranoside was found to be minor product in the latter reaction.     

Beta-galactosidase (Escherichia coli) has a second catalytically important Mg2+ site

It is shown here that Escherichia coli beta-galactosidase has a second Mg2+ binding site that is important for activity. Binding of Mg2+ to the second site caused the k(cat) (with oNPG as the substrate) to increase about 100 s(-1); the Km was not affected. The Kd for binding the second Mg2+ is about 10(-4)M. Since the concentration of free Mg2+ in E. coli is about 1-2 mM, the second site is physiologically significant. Non-polar substitutions (Ala or Leu) for Glu-797, a residue in an active site loop, eliminated the k(cat) increase. This indicates that the second Mg2+ site is near to Glu-797. The Ki values of transition state analogs were decreased by small but statistically significant amounts when the second Mg2+ site was occupied and Arrhenius plots showed that less entropic activation energy is required when the second site is occupied. These inhibitor and temperature results suggest that binding of the second Mg2+ helps to order the active site for stabilization of the transition state.