A relaxin‐like gonad‐stimulating peptide identified from the starfish Astropecten scoparius

A relaxin‐like gonad‐stimulating peptide (RGP) in starfish was the first identified invertebrate gonadotropin responsible for final gamete maturation. An RGP ortholog was newly identified from Astropecten scoparius of the order Paxillosida. The A. scoparius RGP (AscRGP) precursor is encoded by a 354 base pair open reading frame and is a 118 amino acid (aa) protein consisting of a signal peptide (26 aa), B‐chain (21 aa), C‐peptide (47 aa), and A‐chain (24 aa). There are three putative processing sites (Lys‐Arg) between the B‐chain and C‐peptide, between the C‐peptide and A‐chain, and within the C‐peptide. This structural organization revealed that the mature AscRGP is composed of A‐ and B‐chains with two interchain disulfide bonds and one intrachain disulfide bond. The C‐terminal residues of the B‐chain are Gln‐Gly‐Arg, which is a potential substrate for formation of an amidated C‐terminal Gln residue. Non‐amidated (AscRGP‐GR) and amidated (AscRGP‐NH₂) peptides were chemically synthesized and their effect on gamete shedding activity was examined using A. scoparius ovaries. Both AscRGP‐GR and AscRGP‐NH₂ induced oocyte maturation and ovulation in similar dose‐dependent manners. This is the first report on a C‐terminally amidated functional RGP. Collectively, these results suggest that AscRGP‐GR and AscRGP‐NH₂ act as a natural gonadotropic hormone in A. scoparius.     

Structural and biochemical characterization of a metagenome‐derived esterase with a long N‐terminal extension

The genes encoding six novel esterolytic/lipolytic enzymes, termed LC‐Est1～6, were isolated from a fosmid library of a leaf‐branch compost metagenome by functional screening using tributyrin agar plates. These enzymes greatly vary in size and amino acid sequence. The highest identity between the amino acid sequence of each enzyme and that available from the database varies from 44 to 73%. Of these metagenome‐derived enzymes, LC‐Est1 is characterized by the presence of a long N‐terminal extension (LNTE, residues 26–283) between a putative signal peptide (residues 1–25) and a C‐terminal esterase domain (residues 284–510). A putative esterase from Candidatus Solibacter usitatus (CSu‐Est) is the only protein, which shows the significant amino acid sequence identity (46%) to the entire region of LC‐Est1. To examine whether LC‐Est1 exhibits activity and its LNTE is important for activity and stability of the esterase domain, LC‐Est1 (residues 26–510), LC‐Est1C (residues 284–510), and LC‐Est1C* (residues 304–510) were overproduced in E. coli, purified, and characterized. LC‐Est1C* was only used for structural analysis. The crystal structure of LC‐Est1C* highly resembles that of the catalytic domain of Thermotoga maritima esterase, suggesting that LNTE is not required for folding of the esterase domain. The enzymatic activity of LC‐Est1C was lower than that of LC‐Est1 by 60%, although its substrate specificity was similar to that of LC‐Est1. LC‐Est1C was less stable than LC‐Est1 by 3.3°C. These results suggest that LNTE of LC‐Est1 rather exists as an independent domain but is required for maximal activity and stability of the esterase domain.     

Engineering a recyclable elastin‐like polypeptide capturing scaffold for non‐chromatographic protein purification

Previously, we reported a non‐chromatographic protein purification method exploiting the highly specific interaction between the dockerin and cohesin domains from Clostridium thermocellum and the reversible aggregation property of elastin‐like polypeptide (ELP) to provide fast and cost‐effective protein purification. However, the bound dockerin‐intein tag cannot be completely dissociated from the ELP‐cohesin capturing scaffold due to the high binding affinity, resulting in a single‐use approach. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium‐coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA‐mediated dissociation of the bound dockerin‐intein tag from the ELP‐cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non‐chromatographic based affinity method provides an attractive approach for efficient and cost‐effective protein purification. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:968–971, 2013     

Structural characterization of the heme‐based oxygen sensor,AfGcHK, its interactions with the cognate response regulator,and their combined mechanism of action in a bacterial two‐component signaling system

The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109‐5 forms a two‐component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N‐terminal sensor domain causes the C‐terminal kinase domain of AfGcHK to autophosphorylate at His183 and then transfer this phosphate to Asp52 or Asp169 of the RR protein. Analytical ultracentrifugation revealed that AfGcHK and the RR protein form a complex with 2:1 stoichiometry. Hydrogen‐deuterium exchange coupled to mass spectrometry (HDX‐MS) suggested that the most flexible part of the whole AfGcHK protein is a loop that connects the two domains and that the heme distal side of AfGcHK, which is responsible for oxygen binding, is the only flexible part of the sensor domain. HDX‐MS studies on the AfGcHK:RR complex also showed that the N‐side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the β‐strand B2 area of the RR protein's Rec1 domain, and that the C‐side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and β‐strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Proteins 2016; 84:1375–1389. © 2016 Wiley Periodicals, Inc.     

Identification of novel halotolerant bacillopeptidase F-like proteinases from a moderately halophilic bacterium, Virgibacillus sp. SK37

Aims: Virgibacillus sp. SK37 isolated from Thai fish sauce produced numerous NaCl‐activated subtilisin‐like proteinases. Our objectives were to purify, characterize and identify these extracellular proteinases. Methods and Results: Three major subtilisin‐like enzymes including 19, 34 and 44 kDa were partially purified and showed maximum activity at pH 8, 55–60°C, 25–30% NaCl and 70–100 mmol l^?1 CaCl₂. Enzymes showed stability at 0–30% NaCl and <20 mmol l^?1 CaCl₂ and were completely inhibited by phenylmethanesulphonyl fluoride but not by ethylenediaminetetraacetic acid. The isoelectric points of 19‐, 34‐ and 44‐kDa proteinases were at 3·6, 5·2 and 3·8, respectively, based on 2D electrophoresis. Peptide mass fingerprint and de novo peptide homology analysis of tryptic peptides using MALDI‐TOF and LC–MS/MS, respectively, suggested that all three enzymes were novel and homologous to bacillopeptidase F. Conclusions: The three major proteinases are a member of bacillopeptidase F‐like enzymes exhibiting thermophilic and halotolerant characteristics with high stability at 30% NaCl. Significance and Impact of the Study: This is the first report on bacillopeptidase F‐like proteinases in genus Virgibacillus with a distinct halotolerant feature. They showed potential to be a processing aid for food and biotechnological applications, particularly in high salt condition.     

Crystal structure of the N‐terminal methyltransferase‐like domain of anamorsin

Anamorsin is a recently identified molecule that inhibits apoptosis during hematopoiesis. It contains an N‐terminal methyltransferase‐like domain and a C‐terminal Fe‐S cluster motif. Not much is known about the function of the protein. To better understand the function of anamorsin, we have solved the crystal structure of the N‐terminal domain at 1.8 Å resolution. Although the overall structure resembles a typical S‐adenosylmethionine (SAM) dependent methyltransferase fold, it lacks one α‐helix and one β‐strand. As a result, the N‐terminal domain as well as the full‐length anamorsin did not show S‐adenosyl‐l ‐methionine (AdoMet) dependent methyltransferase activity. Structural comparisons with known AdoMet dependent methyltransferases reveals subtle differences in the SAM binding pocket that preclude the N‐terminal domain from binding to AdoMet. The N‐terminal methyltransferase‐like domain of anamorsin probably functions as a structural scaffold to inhibit methyl transfers by out‐competing other AdoMet dependant methyltransferases or acts as bait for protein–protein interactions.Proteins 2014; 82:1066–1071. © 2013 Wiley Periodicals, Inc.     

Molecular cloning and expression of a C‐type lectin‐like protein from orange‐spotted grouper Epinephelus coioides

A C‐type lectin‐like protein (Ec‐CTLP) was cloned from the grouper Epinephelus coioides. The full‐length cDNA of Ec‐CTLP was composed of 905 bp with a 522 bp open reading frame that encodes a 174‐residue protein. The putative amino acid sequence of Ec‐CTLP contains a signal peptide of 19 residues at the N‐terminus and a CLECT domain from Cys43 to Arg169 and a conserved imperfect WND (Trp‐Asn‐Asp) motif. The homologous identity of deduced amino acid sequences is from 32 to 42% with other fishes. The expression of Ec‐CTLP was differently upregulated in E. coioides spleen (germline stem) cells after being challenged at 16 and 4° C. Intracellular localization revealed that Ec‐CTLP was distributed only in the cytoplasm. Recombinant Ec‐CTLP (rEc‐CTLP) was expressed in Escherichia coli BL21 (DE3) and purified for mouse Mus musculus anti‐Ec‐CTLP serum preparation. The rEc‐CTLP fusion protein does not possess haemagglutinating activity, but improves survival from frozen bacteria. The survival of bacteria (including gram‐negative E. coli and gram‐positive Staphylococcus aureus) was positively correlated with the concentration of the rEc‐CTLP. These findings can provide clues to help understand the probable C‐type lectin in marine fish innate immunity.     

Mapping disease‐related missense mutations in the immunoglobulin‐like fold domain of lamin A/C reveals novel genotype–phenotype associations for laminopathies

Mutations in A‐type nuclear lamins cause laminopathies. However, genotype–phenotype correlations using the 340 missense mutations within the LMNA gene are unclear: partially due to the limited availability of three‐dimensional structure. The immunoglobulin (Ig)‐like fold domain has been solved, and using bioinformatics tools (including Polyphen‐2, Fold X, Parameter OPtimized Surfaces, and PocketPicker) we characterized 56 missense mutations for position, surface exposure, change in charge and effect on Ig‐like fold stability. We find that 21 of the 27 mutations associated with a skeletal muscle phenotype are distributed throughout the Ig‐like fold, are nonsurface exposed and predicted to disrupt overall stability of the Ig‐like fold domain. Intriguingly, the remaining 6 mutations clustered, had higher surface exposure, and did not affect stability. The majority of 9 lipodystrophy or 10 premature aging syndrome mutations also did not disrupt Ig‐like fold domain stability and were surface exposed and clustered in distinct regions that overlap predicted binding pockets. Although buried, the 10 cardiac mutations had no other consistent properties. Finally, most lipodystrophy and premature aging mutations resulted in a ‐1 net charge change, whereas skeletal muscle mutations caused no consistent net charge changes. Since premature aging, lipodystrophy and the subset of 6 skeletal muscle mutations cluster tightly in distinct, charged regions, they likely affect lamin A/C –protein/DNA/RNA interactions: providing a consistent genotype–phenotype relationship for mutations in this domain. Thus, this subgroup of skeletal muscle laminopathies that we term the ‘Skeletal muscle cluster’, may have a distinct pathological mechanism. These novel associations refine the ability to predict clinical features caused by certain LMNA missense mutations. Proteins 2014; 82:904–915. © 2013 Wiley Periodicals, Inc.     

Novel Ca2+‐independent carbohydrate recognition of the C‐type lectins,SPL‐1 and SPL‐2, from the bivalve Saxidomus purpuratus

Novel Ca²⁺‐independent C‐type lectins, SPL‐1 and SPL‐2, were purified from the bivalve Saxidomus purpuratus. They are composed of dimers with either identical (SPL‐2 composed of two B‐chains) or distinct (SPL‐1 composed of A‐ and B‐chains) polypeptide chains, and show affinity for N‐acetylglucosamine (GlcNAc)‐ and N‐acetylgalactosamine (GalNAc)‐containing carbohydrates, but not for glucose or galactose. A database search for sequence similarity suggested that they belong to the C‐type lectin family. X‐ray crystallographic analysis revealed definite structural similarities between their subunits and the carbohydrate‐recognition domain (CRD) of the C‐type lectin family. Nevertheless, these lectins (especially SPL‐2) showed Ca²⁺‐independent binding affinity for GlcNAc and GalNAc. The crystal structure of SPL‐2/GalNAc complex revealed that bound GalNAc was mainly recognized via its acetamido group through stacking interactions with Tyr and His residues and hydrogen bonds with Asp and Asn residues, while widely known carbohydrate‐recognition motifs among the C‐type CRD (the QPD [Gln‐Pro‐Asp] and EPN [Glu‐Pro‐Asn] sequences) are not involved in the binding of the carbohydrate. Carbohydrate‐binding specificities of individual A‐ and B‐chains were examined by glycan array analysis using recombinant lectins produced from Escherichia coli cells, where both subunits preferably bound oligosaccharides having terminal GlcNAc or GalNAc with α‐glycosidic linkages with slightly different specificities.     

Crystal structure of the collagen model peptide (Pro‐Pro‐Gly)4‐Hyp‐Asp‐Gly‐(Pro‐Pro‐Gly)4 at 1.0 Å resolution

The single‐crystal structure of the collagen‐like peptide (Pro‐Pro‐Gly)₄‐Hyp‐Asp‐Gly‐(Pro‐Pro‐Gly)₄, was analyzed at 1.02 Å resolution. The overall average helical twist (θ = 49.6°) suggests that this peptide adopts a 7/2 triple‐helical structure and that its conformation is very similar to that of (Gly‐Pro‐Hyp)₉, which has the typical repeating sequence in collagen. High‐resolution studies on other collagen‐like peptides have shown that imino acid‐rich sequences preferentially adopt a 7/2 triple‐helical structure (θ = 51.4°), whereas imino acid‐lean sequences adopt relaxed conformations (θ < 51.4°). The guest Gly‐Hyp‐Asp sequence in the present peptide, however, has a large helical twist (θ = 61.1°), whereas that of the host Pro‐Pro‐Gly sequence is small (θ = 46.7°), indicating that the relationship between the helical conformation and the amino acid sequence of such peptides is complex. In the present structure, a strong intermolecular hydrogen bond between two Asp residues on the A and B strands might induce the large helical twist of the guest sequence; this is compensated by a reduced helical twist in the host, so that an overall 7/2‐helical symmetry is maintained. The Asp residue in the C strand might interact electrostatically with the N‐terminus of an adjacent molecule, causing axial displacement, reminiscent of the D‐staggered structure in fibrous collagens. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 436–447, 2013.     

Fast‐responding thermal‐death‐time tubes for the determination of thermal bacteria inactivation

The knowledge of thermal inactivation kinetics, usually expressed in terms of D‐ and z‐values, is of crucial importance for the design of sanitation and sterilization processes. In this study, we designed a simple, fast‐responding, and mechanically stable aluminum tube for inactivation measurements and compared these experiments with the successive‐sampling method at different temperatures. Up to 65°C, we determined a come‐up time of approximately 15 s for the tubes, which is lower than the corresponding values of other devices, presumably because of lower wall thickness, material properties, and a higher surface to volume ratio. D‐values of Escherichia coli calculated from tube inactivation experiments by first‐order kinetics were 370 s (56°C), 126 s (58°C), 53.2 s (60°C), 33.8 s (62°C), and 3.22 s (65°C), and the corresponding values determined with the successive‐sampling flask method were insignificantly different (417, 138, 48.6, and 29.1 s for 56, 58, 60, and 62°C, respectively). These data as well as those measured for Enterobacter cloacae, Pseudomonas putida, Serratia odorifera, and Yersinia rhodei were in close accordance with literature values.     

The minimal α‐crystallin domain of Mj Hsp16.5 is functional at non‐heat‐shock conditions

The small heat shock protein (sHSP) from Methanococcus jannaschii (Mj Hsp16.5) forms a monodisperse 24mer and each of its monomer contains two flexible N‐ and C‐terminals and a rigid α‐crystallin domain with an extruding β‐strand exchange loop. The minimal α‐crystallin domain with a β‐sandwich fold is conserved in sHSP family, while the presence of the β‐strand exchange loop is divergent. The function of the β‐strand exchange loop and the minimal α‐crystallin domain of Mj Hsp16.5 need further study. In the present study, we constructed two fragment‐deletion mutants of Mj Hsp16.5, one with both the N‐ and C‐terminals deleted (ΔNΔC) and the other with a further deletion of the β‐strand exchange loop (ΔNΔLΔC). ΔNΔC existed as a dimer in solution. In contrast, the minimal α‐crystallin domain ΔNΔLΔC became polydisperse in solution and exhibited more efficient chaperone‐like activities to prevent amorphous aggregation of insulin B chain and fibril formation of the amyloidogenic peptide dansyl‐SSTSAA‐W than the mutant ΔNΔC and the wild type did. The hydrophobic probe binding experiments indicated that ΔNΔLΔC exposed much more hydrophobic surface than ΔNΔC. Our study also demonstrated that Mj Hsp16.5 used different mechanisms for protecting different substrates. Though Mj Hsp16.5 formed stable complexes with substrates when preventing thermal aggregation, no complexes were detected when preventing aggregation under non‐heat‐shock conditions. Proteins 2014; 82:1156–1167. © 2013 Wiley Periodicals, Inc.     

Increased <Emphasis Type="SmallCaps">d</Emphasis>-allose production by the R132E mutant of ribose-5-phosphate isomerase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Ribose-5-phosphate isomerase from Clostridium thermocellum converted d-psicose to d-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in d-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for d-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively. The specific activity and catalytic efficiency (k _cat/K _m) of the R132E mutant for d-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was used, the conversion yield of d-psicose to d-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min.     

Chaperone‐like activities of different molecular forms of β‐casein. Importance of polarity of N‐terminal hydrophilic domain

As a member of intrinsically unstructured protein family, β‐casein (β‐CN) contains relatively high amount of prolyl residues, adopts noncompact and flexible structure and exhibits chaperone‐like activity in vitro. Like many chaperones, native β‐CN does not contain cysteinyl residues and exhibits strong tendencies for self‐association. The chaperone‐like activities of three recombinant β‐CNs wild type (WT) β‐CN, C4 β‐CN (with cysteinyl residue in position 4) and C208 β‐CN (with cysteinyl residue in position 208), expressed and purified from E. coli, which, consequently, lack the phosphorylated residues, were examined and compared with that of native β‐CN using insulin and alcohol dehydrogenase as target/substrate proteins. The dimers (β‐CND) of C4‐β‐CN and C208 β‐CN were also studied and their chaperone‐like activities were compared with those of their monomeric forms. Lacking phosphorylation, WT β‐CN, C208 β‐CN, C4 β‐CN and C4 β‐CND exhibited significantly lower chaperone‐like activities than native β‐CN. Dimerization of C208 β‐CN with two distal hydrophilic domains considerably improved its chaperone‐like activity in comparison with its monomeric form. The obtained results demonstrate the significant role played by the polar contributions of phosphorylated residues and N‐terminal hydrophilic domain as important functional elements in enhancing the chaperone‐like activity of native β‐CN. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 623–632, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Protein stabilization with a dipeptide‐mimic triazine‐scaffolded synthetic affinity ligand

Protein stabilization was achieved by a novel approach based on the adsorption and establishment of affinity‐like interactions with a biomimetic triazine‐scaffolded ligand. A synthetic lead compound (ligand 3′/11, K_a ≈ 10⁴ M^?1) was selected from a previously screened solid‐phase library of affinity ligands for studies of adsorption and stabilization of cutinase from Fusarium solani pisi used as a model system. This ligand, directly synthesized in agarose by a well‐established solid‐phase synthesis method, was able to strongly bind cutinase and led to impressive half‐lives of more than 8 h at 70 °C, and of approximately 34 h at 60 °C for bound protein (a 25‐ and 57‐fold increase as compared with the free enzyme, respectively). The ligand density in the solid matrix was found to be a determinant parameter for cutinase stabilization. It is conceivable that the highly stabilizing effect observed results from the binding of more than one ligand residue to the enzyme, creating specific macromolecular configurations that lock structural mobility thus improving molecular stability. Copyright © 2013 John Wiley & Sons, Ltd.     

Linear analogues derived from the first EGF‐like domain of human blood coagulation factor VII: enhanced inhibition of FVIIa/TF complex activity by backbone modification through aspartimide formation

Coagulation factor VII bound to its cofactor tissue factor is the physiological initiator of blood coagulation. The interaction between factor VII and tissue factor involves all four of the structural modules found in factor VII, with the most significant contribution coming from the first EGF‐like domain. In this study, the synthesis and biological activity of several analogues derived from the first EGF‐like domain of FVII comprising the sequence 45–83 are reported on. The six cysteine residues found in the native protein were replaced by Abu. The peptides were isolated from a multicomponent mixture following standard Fmoc solid phase synthesis. Purification and characterisation of the heterogeneous product showed that aspartimide formation was a major side‐reaction, occurring predominantly at the Asp⁴⁶‐Gly⁴⁷ and Asn⁵⁷‐Gly⁵⁸ dipeptides. Although relatively common in peptide synthesis, the extent to which this side‐reaction had taken place was considered surprising. Reported herein are the analytical methods used to isolate and characterise several of the modified products. Also, the inhibitory effect of these peptides on the formation and enzymatic activity of the factor VIIa/tissue factor complex have been compared. Surprisingly, the peptide containing an iso‐Asp residue at position 57 possessed 66‐fold higher inhibitory activity compared with the original target peptide. A possible explanation for this increase in observed activity is presented. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Crystal structure of the ubiquitin‐like small archaeal modifier protein 2 from Haloferax volcanii

The discovery of ubiquitin‐like small archaeal modifier protein 2 (SAMP2) that forms covalent polymeric chains in Haloferax volcanii has generated tremendous interest in the function and regulation of this protein. At present, it remains unclear whether the Hfx. volcanii modifier protein SAMP1 has such polyubiquitinating‐like activity. Although SAMP1 and SAMP2 use the same conjugation machinery to modify their target proteins, each can impart distinct functional consequences. To better understand the mechanism of SAMP2 conjugation, we have sought to characterize the biophysical and structural properties of the protein from Hfx. volcanii. SAMP2 is only partially structured under mesohalic solution conditions and adopts a well‐folded compact conformation in the presence of 2.5M of NaCl. Its 2.3‐Å‐resolution crystal structure reveals a characteristic α/β central core domain and a unique β‐hinge motif. This motif anchors an unusual C‐terminal extension comprising the diglycine tail as well as two lysine residues that can potentially serve to interlink SAMP2 moieties. Mutational alternation of the structural malleability of this β‐hinge motif essentially abolishes the conjugation activity of SAMP2 in vivo. In addition, NMR structural studies of the putative ubiquitin‐like protein HVO_2177 from Hfx. volcanii show that like SAMP1, HVO_2177 forms a classic β‐grasp fold in a salt‐independent manner. These results provide insights into the structure–function relationship of sampylating proteins of fundamental importance in post‐translational protein modification and environmental cues in Archaea.     

Structure‐based investigation into the functional roles of the extended loop and substrate‐recognition sites in an endo‐β‐1,4‐d‐mannanase from the Antarctic springtail,Cryptopygus antarcticus

Endo‐β‐1,4‐d ‐mannanase from the Antarctic springtail, Cryptopygus antarcticus (CaMan), is a cold‐adapted β‐mannanase that has the lowest optimum temperature (30°C) of all known β‐mannanases. Here, we report the apo‐ and mannopentaose (M5) complex structures of CaMan. Structural comparison of CaMan with other β‐mannanases from the multicellular animals reveals that CaMan has an extended loop that alters topography of the active site. Structural and mutational analyses suggest that this extended loop is linked to the cold‐adapted enzymatic activity. From the CaMan‐M5 complex structure, we defined the mannose‐recognition subsites and observed unreported M5 binding site on the surface of CaMan. Proteins 2014; 82:3217–3223. © 2014 Wiley Periodicals, Inc.     

Solution structure of a bacterial immunoglobulin‐like domain of the outer membrane protein (LigB) from Leptospira

Leptospiral immunoglobulin‐like (Lig) proteins are surface proteins expressed in pathogenic strains of Leptospira. LigB, an outer membrane protein containing tandem repeats of bacterial Ig‐like (Big) domains and a no‐repeat tail, has been identified as a virulence factor involved in adhesion of pathogenic Leptospira interrogans to host cells. A Big domain of LigB, LigBCen2R, was reported previously to bind the GBD domain of fibronectin, suggesting its important role in leptospiral infections. In this study, we determined the solution structure of LigBCen2R by nuclear magnetic resonance (NMR) spectroscopy. LigBCen2R adopts a canonical immunoglobulin‐like fold which is comprised of a beta‐sandwich of ten strands in three sheets. We indicated that LigBCen2R is able to bind to Ca²⁺ with a high affinity by isothermal titration calorimetry assay. NMR perturbation experiment identified a number of residues responsible for Ca²⁺ binding. Structural comparison of it with other Big domains demonstrates that they share a similar fold pattern, but vary in some structural characters. Since Lig proteins play a vital role in the infection to host cells, our study will contribute a structural basis to understand the interactions between Leptospira and host cells. Proteins 2015; 83:195–200. © 2014 Wiley Periodicals, Inc.     

Expression of trypsin-like serine peptidases in pre-imaginal stages of Aedes aegypti (Diptera: Culicidae)

This study reports the biochemical characterization and comparative analyses of highly active serine proteases in the larval and pupal developmental stages of Aedes aegypti (Linnaeus) using substrate‐SDS‐PAGE. Zymographic analysis of larval stadia detected proteolytic activity in 6–8 bands with apparent molecular masses ranging from 20 to 250 kDa, with activity observed from pH 5.5 to 10.0. The pupal stage showed a complex proteolytic activity in at least 11 bands with apparent Mr ranging from 25 to 250 kDa, and pH optimum at 10.0. The proteolytic activities of both larval and pupal stages were strongly inhibited by phenyl‐methyl sulfonyl‐fluoride and N‐α‐Tosyl‐L ‐lysine chloromethyl ketone hydrochloride, indicating that the main proteases expressed by these developmental stages are trypsin‐like serine proteases. The enzymes were active at temperatures ranging from 4 to 85°C, with optimal activity between 37 and 60°C, and low activity at 85°C. Comparative analysis between the proteolytic enzymes expressed by larvae and pupae showed that substantial changes in the expression of active trypsin‐like serine proteases occur during the developmental cycle of A. aegypti. © 2011 Wiley Periodicals, Inc.