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BBA - Proteins and Proteomics (v.1804, #9)
Computational approaches for protein function prediction: A combined strategy from multiple sequence alignment to molecular docking-based virtual screening by Ciro Leonardo Pierri; Giovanni Parisi; Vito Porcelli (pp. 1695-1712).
The functional characterization of proteins represents a daily challenge for biochemical, medical and computational sciences. Although finally proved on the bench, the function of a protein can be successfully predicted by computational approaches that drive the further experimental assays. Current methods for comparative modeling allow the construction of accurate 3D models for proteins of unknown structure, provided that a crystal structure of a homologous protein is available. Binding regions can be proposed by using binding site predictors, data inferred from homologous crystal structures, and data provided from a careful interpretation of the multiple sequence alignment of the investigated protein and its homologs. Once the location of a binding site has been proposed, chemical ligands that have a high likelihood of binding can be identified by using ligand docking and structure-based virtual screening of chemical libraries. Most docking algorithms allow building a list sorted by energy of the lowest energy docking configuration for each ligand of the library. In this review the state-of-the-art of computational approaches in 3D protein comparative modeling and in the study of protein–ligand interactions is provided. Furthermore a possible combined/concerted multistep strategy for protein function prediction, based on multiple sequence alignment, comparative modeling, binding region prediction, and structure-based virtual screening of chemical libraries, is described by using suitable examples. As practical examples, Abl-kinase molecular modeling studies, HPV-E6 protein multiple sequence alignment analysis, and some other model docking-based characterization reports are briefly described to highlight the importance of computational approaches in protein function prediction.
Keywords: Abbreviations; NMR; Nuclear magnetic resonance; HPV; Human papilloma virus; MSA; Multiple sequence alignments; BLAST; Basic Local Alignment Search Tools; PSI-BLAST; Position Specific Iterative Basic Local Alignment Search Tools; NCBI; National Center for Biotechnology Information; EMBL-EBI; European Molecular Biology Laboratory-European Bioinformatics Institutes; DDBJ; DNA Data Bank of Japan; nr; non-redundant; T-Coffee; Tree-based Consistency Objective Function for Alignment Evaluation; E; -value; The expectation value; PAM; Point Accepted Mutation; BLOSUM; Blocks Substitution Matrix; PHAT; Predicted Hydrophobic and Transmembrane; GUI; Graphical user interface; PDZ; PSD95/Dlg/ZO-1; AHS; Amidohydrolases; SAH; S-Adenosylhomocysteine; RMSD; Root mean square deviation; PGK; Phosphoglycerate kinase; SCOP; Structural classification of proteinsProtein function prediction; Protein–ligand interaction; Multiple sequence alignment; Comparative modeling; Binding site prediction; Docking-based virtual screening of chemical library; Abl-kinase; HPV-E6
Dystrophin: More than just the sum of its parts by Elisabeth Le Rumeur /; Steve J. Winder; Jean-François Hubert (pp. 1713-1722).
Dystrophin is one of a number of large cytoskeleton associated proteins that connect between various cytoskeletal elements and often are tethered to the membrane through other transmembrane protein complexes. These cytolinker proteins often provide structure and support to the cells where they are expressed, and mutations in genes encoding these proteins frequently gives rise to disease. Dystrophin is no exception in any of these respects, providing connections between a transmembrane complex known as the dystrophin–glycoprotein complex and the underlying cytoskeleton. The most established connection and possibly the most important is that to F-actin, but more recently evidence has been forthcoming of connections to membrane phospholipids, intermediate filaments and microtubules. Moreover it is becoming increasingly clear that the multiple spectrin-like repeats in the centre of the molecule, that had hitherto been thought to be largely redundant, harbour binding activities that have a significant impact on dystrophin functionality. This functionality is particularly apparent when assessed by the ability to rescue the dystrophic phenotype in mdx mice. This review will focus on the relatively neglected but functionally vital coiled-coil region of dystrophin, highlighting the structural relationships and interactions of the coiled-coil region and providing new insights into the functional role of this region.
Keywords: Dystrophin; Spectrin-like repeats; Coiled-coil; Muscular dystrophy; Actin; nNOS; Cytoskeleton; Membrane phospholipids; Membrane binding; Microtubules; Intermediate filaments
Oligomerization properties of FurA from the cyanobacterium Anabaena sp. PCC 7120: Direct visualization by in situ atomic force microscopy under different redox conditions by Anabel Lostao; María Luisa Peleato; Gomez-Moreno Carlos Gómez-Moreno; María F. Fillat (pp. 1723-1729).
Fur proteins are global prokaryotic transcriptional regulators. Functional studies of FurA from the cyanobacterium Anabaena sp. PCC 7120 evidenced the influence of the redox environment in the activity of the regulator and its ability to aggregate through disulphide bridges. Atomic force microscopy allows single-molecule imaging and monitorization of the status of FurA under different redox conditions mimicking a physiological environment. The estimated FurA average diameter was of 4nm. In the absence of reducing agents, the purified FurA is mainly associated as trimers, being 40° the prevalent angle α conformed by protein monomers. Reducing conditions induces trimer rearrangement to protein monomers and a major fraction of FurA dimers. Disruption of the dimeric assemblies and appearance of higher order aggregates, namely trimers and tetramers are induced by oxidation with diamide or hydrogen peroxide. The homogeneity of the angles exhibited by the trimeric particles, as well as the occurrence of dimers in the presence of DTT, suggests the participation of relatively specific hydrophobic interactions maintaining the dimer. Direct visualization of the regulator under liquid phase at molecular resolution unravels the importance of non-polar interactions in FurA dynamics and shows that in Anabaena disulphide bridges are not essential for the dimerization of FurA.
Keywords: Transcriptional regulation; Cyanobacteria; Ferric uptake regulator; Atomic force microscopy; Redox status; Oligomerization
Probing the primary structural determinants of streptokinase inter-domain linkers by site-specific substitution and deletion mutagenesis by Suman Yadav; Girish Sahni (pp. 1730-1737).
The bacterial protein streptokinase (SK) contains three independently folded domains (α, β and γ), interconnected by two flexible linkers with noticeable sequence homology. To investigate their primary structure requirements, the linkers were swapped amongst themselves i.e. linker 1 (between α and β domains) was swapped with linker 2 (between β and γ domains) and vice versa. The resultant construct exhibited very low activity essentially due to an enhanced proteolytic susceptibility. However, a SK mutant with two linker 1 sequences, which was proteolytically as stable as WT-rSK retained about 10% of the plasminogen activator activity of rSK When the native sequence of each linker was substituted with 9 consecutive glycine sequences, in case of the linker 1 substitution mutant substantial activity was seen to survive, whereas the linker 2 mutant lost nearly all its activity. The optimal length of linkers was then studied through deletion mutagenesis experiments, which showed that deletion beyond three residues in either of the linkers resulted in virtually complete loss of activator activity. The effect of length of the linkers was then also examined by insertion of extraneous pentapeptide sequences having a propensity for adopting either an extended conformation or a relatively rigid conformation. The insertion of poly-Pro sequences into native linker 2 sequence caused up to 10-fold reduction in activity, whereas its effect in linker 1 was relatively minor. Interestingly, most of the linker mutants could form stable 1:1 complexes with human plasminogen. Taken together, these observations suggest that (i) the functioning of the inter-domain linkers of SK requires a critical minimal length, (ii) linker 1 is relatively more tolerant to insertions and sequence alterations, and appears to function primarily as a covalent connector between the α and β domains, and (iii) the native linker 2 sequence is virtually indispensable for the activity of SK probably because of structural and/or flexibility requirements in SK action during catalysis.
Keywords: Abbreviations; EDC; N′-ethylcarbodiimide; HPG; human plasminogen; HPN; human plasmin; IBs; inclusion bodies; IPTG; isopropyl-1-thio-β-; d; -galactopyranoside; K; d; equilibrium rate constant; k; off; rate of dissociation; k; on; rate of association; μPG; microplasminogen; NHS; N-hydroxysuccinimide; NPGB; p-nitrophenyl p-guanidinobenzoate; nSK; SK derived from; S. equisimilis; strain H46A; SK; streptokinase; SOE-PCR; splicing overlap extension-polymerase chain reaction; SPR; surface plasmon resonance; STI; soybean trypsin inhibitor; WT-SK/rSK; SK of; S. equisimilis; expressed in; E. coliEnzyme–substrate interaction; Linker; Inter-domain interaction; Plasminogen activator; Streptokinase
The C-terminal domain is sufficient for host-binding activity of the Mu phage tail-spike protein by Hidetaka Suzuki; Seiko Yamada; Yoshiharu Toyama; Shigeki Takeda (pp. 1738-1742).
The Mu phage virion contains tail-spike proteins beneath the baseplate, which it uses to adsorb to the outer membrane of Escherichia coli during the infection process. The tail spikes are composed of gene product 45 (gp45), which contains 197 amino acid residues. In this study, we purified and characterized both the full-length and the C-terminal domains of recombinant gp45 to identify the functional and structural domains. Limited proteolysis resulted in a Ser64-Gln197 sequence, which was composed of a stable C-terminal domain. Analytical ultracentrifugation of the recombinant C-terminal domain (gp45-C) indicated that the molecular weight of gp45-C was about 58kDa and formed a trimeric protomer in solution. Coprecipitation experiments and a quartz crystal microbalance (QCM) demonstrated that gp45-C irreversibly binds to the E. coli membrane. These results indicate that gp45 shows behaviors similar to tail-spike proteins of other phages; however, gp45 did not show significant sequence homology with the other phage tail-spike structures that have been identified.
Keywords: Abbreviations; EDTA; ethylenediaminetetraacetic acid; gp45-C; the recombinant C-terminal domain (Ser64-Gln197) of gp45 fused with a histidine tag; PBS; phosphate-buffered saline; PCR; polymerase chain reaction; PMSF; phenylmethanesulfonyl fluoride; QCM; a quartz crystal microbalance; SAM; self-assembling monolayer; SDS-PAGE; sodium dodecyl sulfate–polyacrylamide gel electrophoresis; Tris; tris(hydroxymethyl)aminomethaneAnalytical ultracentrifugation; Bacteriophage; Domain; Limited proteolysis; Tail spike
Reversible binding of zinc in Plasmodium falciparum Sir2: Structure and activity of the apoenzyme by Subhra Prakash Chakrabarty; Hemalatha Balaram (pp. 1743-1750).
Reversible zinc chelation via thiol groups of cysteines leading to modulation of activity in redox regulated proteins forms a basis for switching on–off of various biochemical processes. Silent information regulator 2 (Sir2), a NAD+ dependent deacetylase, contains a non-catalytic zinc ion coordinated by thiol groups of cysteines. Using Plasmodium falciparum Sir2 (PfSir2), we have examined the effect of zinc removal on the structure and activity of this enzyme. Our studies show that the enzyme with high affinity for zinc exhibits partial collapse of structure upon removal of the metal ion. Zinc reconstitution of apo PfSir2 led to recovery of both structure and activity highlighting the reversibility of the process.
Keywords: Sir2; Plasmodium falciparum; Zinc affinity; Deacetylase activity; Structural stability; Zinc reconstitution
Biochemical characterization of two thymidylate synthases in Corynebacterium glutamicum NCHU 87078 by Shu-Chen Kan; Jai-Shin Liu; Hui-Yu Hu; Chia-Ming Chang; Wei-De Lin; Wen-Ching Wang; Wen-Hwei Hsu (pp. 1751-1759).
The genome of Corynebacterium glutamicum NCHU87078 contains two putative thymidylate synthase genes, designated CgthyA and CgthyX. These two genes were expressed in Escherichia coli NovaBlue and the expressed His6-tagged enzymes were purified by nickel-chelate chromatography. The purified CgThyA had a specific activity of 414mU mg−1 protein, whereas thymidylate synthase activity for CgThyX could not be detected in a functional complementation assay using a 10-day incubation period. Gel filtration chromatography and chemical cross-linking experiments showed that CgThyX may exist as a dimer in solution, unlike a typical ThyX protein with homotetrameric structure for catalytic activity. Spectroscopic analysis indicated that purified CgThyX lacked the cofactor FAD. The 2.3Å resolution crystal structure of CgThyX-FAD demonstrated a loose tetramer, in which FAD is chelated between the subunits via a manner distinct from that of other flavin-dependent thymidylate synthases. Structure-based mutational studies have identified a non-conserved segment (residues 70–73) of CgThyX protein with crucial role in binding to FAD. Taken together, our biochemical and structural analyses highlight unique features of the C. glutamicum ThyX that distinguish this enzyme from ThyX proteins from other organisms. Our results also suggest that thymidylate synthesis in C. glutamicum requires ThyA but not ThyX.
Keywords: Abbreviations; Thy; thymidylate synthase; CH; 2; H; 4; folate; methylenetetrahydrofolate; H; 2; folate; dihydrofolate; H; 4; folate; tetrahydrofolate; CEL; comparative expression level; DSP; dithiobis-succinimydyl propionate Corynebacterium glutamicum; Thymidylate synthase A; Thymidylate synthase X; FAD binding; Site-based mutation
Two distinct regions of calponin share common binding sites on actin resulting in different modes of calponin–actin interaction by Imen Ferjani; Abdellatif Fattoum; Mohamed Manai; Yves Benyamin; Claude Roustan; Sutherland K. Maciver (pp. 1760-1767).
Calponins are a small family of proteins that alter the interaction between actin and myosin II and mediate signal transduction. These proteins bind F-actin in a complex manner that depends on a variety of parameters such as stoichiometry and ionic strength. Calponin binds G-actin and F-actin, bundling the latter primarily through two distinct and adjacent binding sites (ABS1 and ABS2). Calponin binds other proteins that bind F-actin and considerable disagreements exist as to how calponin is located on the filament, especially in the presence of other proteins. A study (Galkin, V.E., Orlova, A., Fattoum, A., Walsh, M.P. and Egelman, E.H. (2006) J. Mol. Biol. 359, 478–485.), using EM single-particle reconstruction has shown that there may be four modes of interaction, but how these occur is not yet known. We report that two distinct regions of calponin are capable of binding some of the same sites on actin (such as 18–28 and 360–372 in subdomain 1). This accounts for the finding that calponin binds the filament with different apparent geometries. We suggest that the four modes of filament binding account for differences in stoichiometry and that these, in turn, arise from differential binding of the two calponin regions to actin. It is likely that the modes of binding are reciprocally influenced by other actin-binding proteins since members of the α-actinin group also adopt different actin-binding positions and bind actin principally through a domain that is similar to calponin's ABS1.
Keywords: Abbreviations; CH; calponin homology domain; CLIK; calponin-like repeat; acrylodan; 6-acryloyl-2-dimethylaminonaphthalene; AEDANS; N; ,-iodoacetyl-; N; ′-[sulpho-1-naphthyl]-ethylenediamine; FITC; fluorescein 5-isothiocyanateActin cytoskeleton; Calponin; Microfilaments; CH domain; Spectrin; α-Actinin
A solution NMR investigation into the murine amelogenin splice-variant LRAP (Leucine-Rich Amelogenin Protein) by Garry W. Buchko; Barbara J. Tarasevich; Jacky Roberts; Malcolm L. Snead; Wendy J. Shaw (pp. 1768-1774).
Amelogenins are the dominant proteins present in ameloblasts during the early stages of enamel biomineralization, making up >90% of the matrix protein. Along with the full-length protein there are several splice-variant isoforms of amelogenin present including LRAP (Leucine-Rich Amelogenin Protein), a protein that consists of the first 33 and the last 26 residues of full-length amelogenin. Using solution-state NMR spectroscopy we have assigned the1H-15N HSQC spectrum of murine LRAP (rp(H)LRAP) in 2% acetic acid at pH 3.0 by making extensive use of previous chemical shift assignments for full-length murine amelogenin (rp(H)M180). This correlation was possible because LRAP, like the full-length protein, is intrinsically disordered under these solution conditions. The major difference between the1H-15N HSQC spectra of rp(H)M180 and rp(H)LRAP was an additional set of amide resonances for each of the seven non-proline residues between S12* and Y12 near the N-terminus of rp(H)LRAP indicating that the N-terminal region of LRAP exists in two different conformations. Analysis of the proline carbon chemical shifts suggests that the molecular basis for the two states is not a cis-trans isomerization of one or more of the proline residues in the N-terminal region. Starting from 2% acetic acid, where rp(H)LRAP was monomeric in solution, NaCl addition effected residue specific changes in molecular dynamics manifested by the reduction in intensity and disappearance of1H-15N HSQC cross peaks. As observed for the full-length protein, these perturbations may signal early events governing supramolecular self-assembly of rp(H)LRAP into nanospheres. However, the different patterns of1H-15N HSQC cross peak perturbation between rp(H)LRAP and rp(H)M180 in high salt suggest that the termini may behave differently in their respective nanospheres, and perhaps, these differences contribute to the cell signaling properties attributable to LRAP but not to the full-length protein.
Keywords: LRAP; Intrinsic disorder; Nanosphere self-assembly; Amelogenesis; Enamel; NMR spectroscopy; Dynamic light scattering; Biomineralization; Cell signaling
Conversion of the metal-specific activity of Escherichia coli Mn-SOD by site-directed mutagenesis of Gly165Thr by Masaki Osawa; Fumiyuki Yamakura; Masashi Mihara; Yuichiro Okubo; Kazuhiro Yamada; B. Yukihiro Hiraoka (pp. 1775-1779).
Glycine 165, which is located near the active site metal, is mostly conserved in aligned amino acid sequences of manganese-containing superoxide dismutase (Mn-SOD) proteins, but is substituted to threonine in most iron-containing SODs (Fe-SODs). Because threonine 165 is located between Trp128 and Trp130, and Trp128 is one of the metal-surrounding aromatic amino acids, the conversion of this amino acid may affect the metal-specific activity of Escherichia coli Mn-SOD. In order to clarify this possibility, we prepared a mutant of E. coli Mn-SOD with the replacement of Gly165 by Thr. The ratio of the specific activities of Mn- to Fe-reconstituted enzyme increased from 0.006 in the wild-type to 0.044 in the mutant SOD; therefore, the metal-specific SOD was converted to a metal-tolerant SOD. The visible absorption spectra of the Fe- and Mn-reconstituted mutant SODs indicated the loss of Mn-SOD character. It was concluded that Gly at position 165 plays a catalytic role in maintaining the integrity of the metal specificity of Mn-SOD.
Keywords: Abbreviations; SOD; superoxide dismutase; Mn-SOD; manganese-containing superoxide dismutase; Fe-SOD; iron-containing superoxide dismutase; MBP; maltose-binding protein; SDS; sodium dodecyl sulfate; PAGE; polyacrylamide gel electrophoresismetal-specific activity; metal-tolerant activity; superoxide dismutase; metalloenzyme
Expression, purification and preliminary biochemical studies of the N-terminal domain of leucine-rich repeat kinase 2 by Bin Lu; Yujia Zhai; Chan Wu; Xiaoyun Pang; Zhiheng Xu; Fei Sun (pp. 1780-1784).
Leucine-rich repeat kinase 2 gene is a key factor for Parkinson's disease and encodes for a large protein kinase LRRK2 (280kDa) with multiple domains, including the different repeat sequences at the N-terminus such as ankyrin domain. Here, we successfully expressed and purified two kinds of LRRK2's N-terminal fragments N1 (aa12–320) and N2 (aa12–860). The purified N2 protein was identified by mass spectrometry and N1's molecular weight was determined to be 33.23kDa. Gel filtration revealed that N1 exhibits as monomer, dimer and tetramer and N2 as oligomer in solution. N1's multiple oligomeric states were further proved by native-page and cross-linking gel experiments. Circular dichroism spectrum indicated that N1 and N2 contain both α helixes and β sheets. The polymerization character of LRRK2 N-terminal region would be speculated to relate with its biological function.
Keywords: Abbreviations; IPTG; isopropyl-b-; d; -thiogalactopyranoside; GST; glutathione S-transferase; BSA; bovine serum albumin; PMSF; phenylmethylsulfonyl fluoride; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresisLeucine-rich repeat kinase 2; N-terminal domain; Ankyrin domain; Expression and purification; Oligomerization
Unique biochemical properties of the protein tyrosine phosphatase activity of PTEN—Demonstration of different active site structural requirements for phosphopeptide and phospholipid phosphatase activities of PTEN by Joel Yeong-Chit Chia; Joanna E. Gajewski; Yi Xiao; Hong-Jian Zhu; Heung-Chin Cheng (pp. 1785-1795).
Missense PTEN mutations of the active site residues Asp-92, Cys-124 and Gly-129 contribute to Cowden syndrome. How their mutations affect phospholipid phosphatase activity and tumor suppressor function of PTEN has been defined. In this study, we investigated how their mutations affect the kinetics and catalytic mechanism of PTEN phosphoprotein phosphatase activity. Our data suggest that PTEN catalysis of phosphoprotein dephosphorylation follows a two-step mechanism with Cys-124 transiently phosphorylated to form the phosphoenzyme intermediate. In spite of this, we were unable to trap the genuine phosphoenzyme intermediate; instead, we unexpectedly discovered a novel phosphotransfer reaction in which the phosphate group is transferred from a tyrosyl phosphopeptide to PTEN to form a unique phosphorylated protein. Even though the finding is novel, the phosphotransfer reaction is likely an in vitro non-enzymatic reaction. Kinetic analysis revealed that mutation of Asp-92 has negligible impacts on phosphopeptide phosphatase activity of PTEN, suggesting that Asp-92 does not participate in the phosphopeptide dephosphorylation reaction. The results also imply that allosteric regulators facilitating the recruitment of Asp-92 to participate in catalysis will increase the activity of PTEN in dephosphorylating phosphoprotein and phosphopeptide substrates. Furthermore, kinetic analysis revealed that the G129E mutation has different effects on phospholipid and phosphoprotein phosphatase activities. Taken together, the data show that while the two phosphatase activities of PTEN follow a similar catalytic mechanism, they have notable differences in the requirements of the active site structure.
Keywords: PTEN; Protein tyrosine phosphatase; Catalytic mechanism; Phosphotransfer reaction; Kinetic analysis; Enzymology
A biophysical map of the dystrophin rod by Ahmed Mirza; Mirnalini Sagathevan; Neha Sahni; Lien Choi; Nick Menhart (pp. 1796-1809).
We have conducted a biophysical scan of the rod region of dystrophin, targeting all 24 single spectrin type repeat, STR, motifs and 23 2-STR tandem motifs. Of these 47 targets, we were able to express and purify 39 and have characterized them with regard to various stability metrics: thermodynamic stability as assessed by thermal and solvent denaturation, as well as resistance to proteolysis. We find that while all measured parameters varied greatly throughout the rod, there was no general stabilization of the 2-STR motifs over single STR motifs. However, stabilization by thermodynamic interaction was seen in six regions: strongly in D16:17 and D21:22 and to a lesser extent in D2:3, D4:5, D6:7 and D20:21. This indicates that these STRs interact structurally. In the rest of the rod, no cooperativity was seen and STRs appear to be thermodynamically independent. Stability also varied widely along the rod, with some motifs that are barely stable, beginning to unfold at physiological temperatures; these are largely found in the central rod region from D7 to D15. Regions of high stability were found in the interacting motifs, as well as a general trend toward increasing stability at the C-terminus of the rod. Interestingly, the rod region nNOS binding site occurs at such an interacting, very stable site, D16:17. Overall this describes a highly heterogeneous rod region.
Keywords: Dystrophin; Spectrin type repeat; Domain; Stability
Analysis of the interaction mode between hyperthermophilic archaeal group II chaperonin and prefoldin using a platform of chaperonin oligomers of various subunit arrangements by Muhamad Sahlan; Taro Kanzaki; Tamotsu Zako; Mizuo Maeda; Masafumi Yohda (pp. 1810-1816).
Prefoldin is a co-chaperone that captures an unfolded protein substrate and transfers it to the group II chaperonin for completion of protein folding. Group II chaperonin of a hyperthermophilic archaeon, Thermococcus strain KS-1, interacts and cooperates with archaeal prefoldins. Although the interaction sites within chaperonin and prefoldin have been analyzed, the binding mode between jellyfish-like hexameric prefoldin and the double octameric ring group II chaperonin remains unclear. As prefoldin binds the chaperonin β subunit more strongly than the α subunit, we analyzed the binding mode between prefoldin and chaperonin in the context of Thermococcus group II chaperonin complexes of various subunit compositions and arrangements. The oligomers exhibited various affinities for prefoldins according to the number and order of subunits. Binding affinity increased with the number of Cpnβ subunits. Interestingly, chaperonin complexes containing two β subunits adjacently exhibited stronger affinities than other chaperonin complexes containing the same number of β subunits. The result suggests that all four β tentacles of prefoldin interact with the helical protrusions of CPN in the PFD–CPN complex as the previously proposed model that two adjacent PFD β subunits seem to interact with two CPN adjacent subunits.
Keywords: Abbreviations; T; . KS-1; hyperthermophilic archaeon; Thermococcus; sp. strain KS-1; PFD; prefoldin; PhPFD; Pyrococcus horikoshii; prefoldin; CPN; group II chaperonin; SPR; surface plasmon resonance; PAGE; polyacrylamide gel electrophoresis; c; Cpn; connected group II chaperonin mutantsPrefoldin; Chaperonin; Binding mode; Molecular chaperone; Surface plasmon resonance
Kinetic and structural characterization of caspase-3 and caspase-8 inhibition by a novel class of irreversible inhibitors by Zhigang Wang; William Watt; Nathan A. Brooks; Melissa S. Harris; Jan Urban; Douglas Boatman; Michael McMillan; Michael Kahn; Robert L. Heinrikson; Barry C. Finzel; Arthur J. Wittwer; James Blinn; Satwik Kamtekar; Alfredo G. Tomasselli (pp. 1817-1831).
Because of their central role in programmed cell death, the caspases are attractive targets for developing new therapeutics against cancer and autoimmunity, myocardial infarction and ischemic damage, and neurodegenerative diseases. We chose to target caspase-3, an executioner caspase, and caspase-8, an initiator caspase, based on the vast amount of information linking their functions to diseases. Through a structure-based drug design approach, a number of novel β-strand peptidomimetic compounds were synthesized. Kinetic studies of caspase-3 and caspase-8 inhibition were carried out with these urazole ring-containing irreversible peptidomimetics and a known irreversible caspase inhibitor, Z-VAD-fmk. Using a stopped-flow fluorescence assay, we were able to determine individual kinetic parameters of caspase-3 and caspase-8 inhibition by these inhibitors. Z-VAD-fmk and the peptidomimetic inhibitors inhibit caspase-3 and caspase-8 via a three-step kinetic mechanism. Inhibition of both caspase-3 and caspase-8 by Z-VAD-fmk and of caspase-3 by the peptidomimetic inhibitors proceeds via two rapid equilibrium steps followed by a relatively fast inactivation step. However, caspase-8 inhibition by the peptidomimetics goes through a rapid equilibrium step, a slow-binding reversible step, and an extremely slow inactivation step. The crystal structures of inhibitor complexes of caspases-3 and -8 validate the design of the inhibitors by illustrating in detail how they mimic peptide substrates. One of the caspase-8 structures also shows binding at a secondary, allosteric site, providing a possible route to the development of noncovalent small molecule modulators of caspase activity.
Keywords: Abbreviations; AMC; 7-amino-4-methyl-coumarin; DMSO; dimethyl sulfoxide; DTT; dithiothreitol; fmk; flouromethyl ketone; MMX; Molecumetics Company, Ltd; RMSD; root mean squared deviationCaspase-3; Caspase-8; Peptidomimetic inhibitors; Irreversible inhibition; Urazole; Crystal structure
An interpretation of positional displacement of the helix12 in nuclear receptors: Preexistent swing-up motion triggered by ligand binding by Chiduru Watanabe; Hirofumi Watanabe; Shigenori Tanaka (pp. 1832-1840).
Positional displacement of helix12 (H12) in the estrogen receptor α, which belongs to the nuclear receptor (NR) superfamily, is studied by the molecular dynamics (MD) simulation and the linear response theory. Tendency of the H12 to swing up upon ligand binding, which is consistent with X-ray structures and earlier MD simulations, is reproduced by the calculation of the conformational fluctuation in apo state and the response to the external perturbation. Our study thus provides an interpretation of the positional change of the H12 such that it is derived by the preexistent swing-up motion where the ligand binding works only as a trigger. Our finding, which illustrates underlying mechanism of the H12 motion, would contribute to finding a way to regulate the transcriptional activity by synthesized ligands because the transcriptional activity of the NR is governed by the position of the H12.
Keywords: Abbreviations; NR; nuclear receptor; H12; helix 12; FMO; fragment molecular orbital; ERα; estrogen receptor α; MD; molecular dynamics; RA; retinoic acid; RAR; retinoic acid receptor; RMSD; root mean square deviation; PC1; first principal component; PC2; second principal component; PC3; third principal component; EST; 17β-estradiol; OHT; 4-hydroxytamoxifenNuclear receptor; Helix 12; Allosteric effect; Protein structural change; Molecular dynamics (MD); Linear response theory
Asymmetric synthesis of ( S)-ethyl-4-chloro-3-hydroxy butanoate using a Saccharomyces cerevisiae reductase: Enantioselectivity and enzyme–substrate docking studies by Jihye Jung; Hyun Joo Park; Ki-Nam Uhm; Dooil Kim; Hyung-Kwoun Kim (pp. 1841-1849).
Ethyl ( S)-4-chloro-3-hydroxy butanoate (ECHB) is a building block for the synthesis of hypercholesterolemia drugs. In this study, various microbial reductases have been cloned and expressed in Escherichia coli. Their reductase activities toward ethyl-4-chloro oxobutanoate (ECOB) have been assayed. Amidst them, Baker's yeast YDL124W, YOR120W, and YOL151W reductases showed high activities. YDL124W produced ( S)-ECHB exclusively, whereas YOR120W and YOL151W made ( R)-form alcohol. The homology models and docking models with ECOB and NADPH elucidated their substrate specificities and enantioselectivities. A glucose dehydrogenase-coupling reaction was used as NADPH recycling system to perform continuously the reduction reaction. Recombinant E. coli cell co-expressing YDL124W and Bacillus subtilis glucose dehydrogenase produced ( S)-ECHB exclusively.
Keywords: Abbreviations; ECOB; ethyl-4-chloro-3-oxo butanoate; ECHB; ethyl-4-chloro-3-hydroxy butanoate; GDH; glucose dehydrogenase; NAD(P)H; nicotinamide adenine dinucleotide (phosphate); DMSO; dimethyl sulfoxide; GC; gas chromatographyReductase; Enantioselectivity; Chiral intermediate; Hyperlipidemia drug; Docking model
Stability curves of laboratory evolved thermostable mutants of a Bacillus subtilis lipase by Md. Zahid Kamal; Shoeb Ahmad; Poornima Yedavalli; Nalam Madhusudhana Rao (pp. 1850-1856).
Shape of the protein stability curves changes to achieve higher melting temperature. Broadly, these changes have been classified as upward shift (increased ∆ Gs), rightward shift (increase in Ts) and flattening of the stability curves (decrease in ∆ Cp). Comparative studies on homologous mesophilic–thermophilic protein pairs highlighted the differential contribution of these three strategies amongst proteins. But unambiguous way of identification of the strategies, which will be preferred for a protein, is still not achieved. We have performed comparative thermodynamic studies using differential scanning calorimeter (DSC) on thermostable variants of a lipase from Bacillus subtilis. These variants are products of 1, 2, 3 and 4 rounds of directed evolution and harbor mutations having definite contribution in thermostability unlike natural thermophilic proteins. We have shown that upward and rightward shift in stability curves are prime strategies in this lipase. Our results along with that from the other study on laboratory evolved xylanase A suggest that optimization of suboptimal thermodynamic parameters is having a dominant influence in selection of thermodynamic strategies for higher thermostability.
Keywords: Lipase; Thermostability; In vitro evolution; Thermodynamics; Calorimetry; Free energy
Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα) by Darja Lavogina; Christian K. Nickl; Erki Enkvist; Gerda Raidaru; Marje Lust; Angela Vaasa; Asko Uri; Wolfgang R. Dostmann (pp. 1857-1868).
Type I cGMP-dependent protein kinase (PKGIα) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIα is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIα would lead to advances in the treatment of a variety of cardiovascular diseases. Aim: Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIα to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. Results: Structure–activity profiling of ARCs with PKGIα in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC50 values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1α-induced vasodilation.
Keywords: Abbreviations; Ac; ethanoic acid moiety; Adc; adenosine-4′-dehydroxymethyl-4′-carboxylic acid; Ahx; 6-aminohexanoic acid moiety; AMTH; 5-(2-aminopyrimidin-4-yl)thiophene-2-carboxylic acid; ARC; conjugate of adenosine analogue and oligo-; d; -arginine; BSA; bovine serum albumin; cAMP; cyclic adenosine monophosphate; CCDA; carbocyclic 3′-deoxyadenosine; cGMP; cyclic guanosine monophosphate; CPP; cell-penetrating peptide; DTT; dithiothreitol; FA; fluorescence polarization/anisotropy; FI; fluorescence intensity; Fmoc; N-(fluorenyl-9-methoxycarbonyl) moiety; FRET; Förster resonance energy transfer; Hex; n-hexanoic acid moiety; MALDI-TOF; matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; MLC; 20; myosin regulatory light chain; PBS; phosphate-buffered saline; PKA; cAMP-dependent protein kinase (protein kinase A); PKAc; cAMP-dependent protein kinase catalytic subunit type α; PKGI; cGMP-dependent protein kinase (protein kinase G) isoform I; PKI; thermostable protein kinase inhibitor peptide; PSS; physiological salt solution; PUPI; 1-(9; H; -purin-6-yl)piperidin-4-amine; PUTR; 1-(9; H; -purin-6-yl)-1; H; -1,2,3-triazole-4-carboxylic acid; R-II; cAMP-dependent protein kinase catalytic subunit type II; ROCK-II; Rho kinase type II; (Rp)-8-Br-PET-cGMPS; 8-bromo-β-phenyl-1,N; 2; -ethenoguanosine-3′,5′-cyclic monophosphorothioate, Rp-isomer; (Rp)-8-Br-cGMPS; 8-bromoguanosine-3′,5′-cyclic monophosphorothioate, Rp-isomer; SA profile; structure–activity profile; TAMRA; 5′-carboxy tetramethylrhodamineProtein kinase inhibitor; ARC; cGMP-dependent protein kinase (PKG); cAMP-dependent protein kinase (PKA); Fluorescence anisotropy; Pressure myography
Proteomic analysis of the transitional endoplasmic reticulum in hepatocellular carcinoma: An organelle perspective on cancer by Line Roy; Sylvie LaBoissière; Eman Abdou; Geneviève Thibault; Nathalie Hamel; Maryam Taheri; Daniel Boismenu; Joël Lanoix; Robert E. Kearney; Jacques Paiement (pp. 1869-1881).
The transitional endoplasmic reticulum (tER) is composed of both rough and smooth ER membranes and thus participates in functions attributed to both these two subcellular compartments. In this paper we have compared the protein composition of tER isolated from dissected liver tumor nodules of aflatoxin B1-treated rats with that of tER from control liver. Tandem mass spectrometry (MS), peptide counts and immunoblot validation were used to identify and determine the relative expression level of proteins. Inhibitors of apoptosis (i.e. PGRMC1, tripeptidyl peptidase II), proteins involved in ribosome biogenesis (i.e. nucleophosmin, nucleolin), proteins involved in translation (i.e. eEF-2, and subunits of eIF-3), proteins involved in ubiquitin metabolism (i.e. proteasome subunits, USP10) and proteins involved in membrane traffic (i.e. SEC13-like 1, SEC23B, dynactin 1) were found overexpressed in tumor tER. Transcription factors (i.e. Pur-beta, BTF3) and molecular targets for C-Myc and NF-kappa B were observed overexpressed in tER from tumor nodules. Down-regulated proteins included cytochrome P450 proteins and enzymes involved in fatty acid metabolism and in steroid metabolism. Unexpectedly expression of the protein folding machinery (i.e. calreticulin) and proteins of the MHC class I peptide-loading complex did not change. Proteins of unknown function were detected in association with the tER and the novel proteins showing differential expression are potential new tumor markers. In many cases differential expression of proteins in tumor tER was comparable to that of corresponding genes reported in the Oncomine human database. Thus the molecular profile of tumor tER is different and this may confer survival advantage to tumor cells in cancer.
Keywords: Hepatocellular carcinoma; Quantitative organelle proteomics; Transitional endoplasmic reticulum; Cancer biomarker
Proteomic analysis of primary porcine endothelial cells after infection by classical swine fever virus by Su Li; Hui Qu; Jianwei Hao; Jinfu Sun; Huancheng Guo; Changming Guo; Boxing Sun; Changchun Tu (pp. 1882-1888).
Endothelial cells are the main target of classical swine fever virus during infection, and extensive hemorrhage is the most typical clinical sign of classical swine fever. To investigate the molecular mechanism of hemorrhagic pathogenesis, two-dimensional difference gel electrophoresis with fluorescent dyes (2D-DIGE) was used to analyze the proteomic profile of primary porcine umbilical vein endothelial cells (PUVECs) following CSFV infection. Of 15 protein spots with differential expression, 8 were characterized by MALDI-TOF-MS/MS in infected PUVECs at 48h p.i.: moesin, peroxiredoxin 6, stathmin-1, a protein similar to nascent polypeptide-associated complex alpha subunit isoform 2, phosphoglycerate kinase 1, glucosidase II, transketolase and α-tubulin. These could be sorted into 5 functional groups: glycometabolism, cell proliferation, anti-oxidative stress, inflammatory response and cytoskeleton. Western blot and real-time RT-PCR analysis confirmed the down-regulation of phosphoglycerate kinase 1 (PGK1) and up-regulation of moesin identified by 2D-DIGE. Pathway analysis of these 15 differentially expressed proteins showed that CSFV infection altered the metabolism, cytoskeleton and cell proliferation of PUVECs, and that consequently an inflammatory response was induced.
Keywords: Porcine; Endothelial cells; Classical swine fever virus; Proteomics; 2D-DIGE
High-affinity binding of seminal plasma PSP94 to human immunoglobulin is through the Fab domain by Dhanashree D. Jagtap; Deepak N. Modi; Mukesh Kumar; Bhakti R. Pathak; Smita D. Mahale (pp. 1889-1894).
Prostate secretory protein of 94 amino acids (PSP94) is one of the major proteins present in human seminal plasma. We had earlier reported that PSP94 has the ability to bind to human IgG. The aims of the present study were to further delineate the PSP94–IgG interaction and to understand whether this could have any significance in sperm function. Direct binding of IgG fragments to PSP94 showed maximal binding with F(ab′)2 followed by Fab, while Fc displayed least binding in ELISA. Binding kinetics of PSP94–IgG interaction using surface plasmon resonance (SPR) revealed high-affinity binding of IgG to PSP94 with a dissociation constant ( KD) of 8.8×10−11M. PSP94–IgG interaction was found to be through the Fab domains of IgG. Real-time interaction kinetics revealed association constants for binding of IgG, Fab, and F(ab′)2 towards PSP94 to be of the same order but with altered dissociation constants. IgG and its F(ab′)2 fragment once complexed to PSP94 demonstrated negligible dissociation, while dissociation rate of Fab fragment was 6.6×10−4. In silico molecular modeling of PSP94–IgG complex identified N- and C-terminal β-strands of PSP94 to be the most plausible region involved in IgG interaction. Immunofluorescence studies revealed that IgG bound to human spermatozoa predominantly in the tail region, which could be prevented when IgG was preincubated with PSP94. This study reports for the first time that IgG forms a high-affinity complex with PSP94 through its F(ab′)2 domain and reveals the ability of PSP94 to prevent binding of IgG to spermatozoa.
Keywords: Human seminal plasma inhibin (HSPI); β-Microseminoprotein (β-MSP); Immunoglobulin binding factor (IgBF); Prostate secretory protein 94 (PSP94)
Propofol lowers serum PF4 level and partially corrects hypercoagulopathy in endotoxemic rats by Jing Tang; Yijuan Sun; William Ka Kei Wu; Tianyu Zhong; Yawei Liu; Jinfang Xiao; Tao Tao; Zhenlong Zhao; Miaoning Gu (pp. 1895-1901).
Propofol, an anesthetic drug, has been shown to exhibit antioxidant and anti-inflammatory properties in vitro and in vivo. Hypercoagulopathy is a common clinical feature of sepsis, but the effects of propofol on the coagulation system in septic conditions are unclear. Using the gel-based comparative proteomic approach, together with Western blot analysis, ELISA, antithrombin III activity assay, and blood coagulation test, the effect of propofol on serum proteomic profiles in endotoxemic rats was examined. We identified that serum platelet factor-4 (PF4), an endogenous pro-coagulant, was up-regulated in LPS-challenged rats ( p<0.001). Endotoxemia also resulted in hypercoagulopathy as evidenced by the shortening of thromboplastin time and thrombin time. Administration of propofol attenuated LPS-stimulated PF4 release and partially reversed the effect of LPS on thromboplastin time ( p=0.0012) and thrombin time ( p=0.0072). We demonstrated that propofol reduces serum levels of PF4 and partially corrects the hypercoagulopathy associated with endotoxemia in rats.
Keywords: 2D gel electrophoresis; Lipopolysaccharides; Platelet factor 4; Propofol; Rats
Biophysical characterization of highly active recombinant Gaussia luciferase expressed in Escherichia coli by Tharangani Rathnayaka; Minako Tawa; Shihori Sohya; Masafumi Yohda; Yutaka Kuroda (pp. 1902-1907).
Recently, the smallest bioluminescent protein (MW: 19.9kDa), Gaussia luciferase (GLuc), has been isolated from the marine copepod Gaussia prince p s and has attracted much attention as a reporter protein. However, preparation of large quantities of homogeneous natively folded recombinant GLuc appears to be difficult due to its ten cysteines. Here, we report the biophysical characterization of recombinant GLuc expressed using a novel Escherichia coli expression system based on a cold induced expression vector (pCold). Using this system, a large fraction of the protein was expressed in the soluble fraction. GLuc, purified exclusively from the supernatant using nickel affinity chromatography, yielded a large amount of pure GLuc with a native disulfide bond pattern (Soluble-GLuc). Soluble-GLuc had a strong bioluminescence activity and it retained 65% of its activity after 30min incubation at 95°C. Soluble-GLuc remained fully folded until 40°C, as assessed by circular dichroism; and the thermal denaturation curve was S-shaped, indicating a cooperative transition, with a midpoint temperature of 56°C. These results indicate that both the structure and bioluminescence activity of GLuc remain stable at high temperatures, and they strongly suggest GLuc's potential as a reporter protein.
Keywords: Abbreviations and nomenclature; GLuc; Gaussia; luciferase; Soluble-GLuc; GLuc purified from the soluble fraction using nickel affinity chromatography; Refolded-GLuc; GLuc purified from the inclusion body using nickel affinity chromatography; Recombinant GLuc; Escherichia coli; expressed GLuc (including Refolded- and Soluble-GLuc)Circular dichroism; Reverse phase chromatography; Disulfide bond; Natively folded; Thermal stability
Thermodynamic study of the BRCT domain of BARD1 and its interaction with the -pSER-X-X-Phe- motif-containing BRIP1 peptide by Angelos Thanassoulas; Michail Nomikos; Maria Theodoridou; Drakoulis Yannoukakos; Dimitris Mastellos; George Nounesis (pp. 1908-1916).
The BRCA1-associated RING domain protein 1 (BARD1) is the heterodimeric partner of BRCA1. The BRCA1/BARD1 complex demonstrates ubiquitin ligase activity and has been implicated in genomic stability and tumor suppression. Both proteins possess a structurally conserved C-terminal domain (BRCT). While BRCA1–BRCT has been shown to mediate BRCA1 interactions with phosphoproteins such as BRIP1 by recognizing the pSer-X-X-Phe motif, attempts to demonstrate analogous interactions of its dimeric counterpart BARD1–BRCT, have so far been unsuccessful. In this study, chemical-denaturation experiments of BARD1–BRCT domain suggest that its low thermodynamic stability (Δ G=2.5kcal/mol) at room temperature, may affect some of its biochemical properties, such as its interaction with phosphopeptides. The stability of BARD1–BRCT domain at 10°C, increases to 7.5kcal/mol and isothermal titration calorimetry (ITC) experiments at this lower temperature showed binding to the BRIP1 phosphopeptide via an enthalpy-driven interaction, which appears to be specific to the pSer-X-X-Phe peptide-binding motif. Substitution of either pSer at position 0 with Ser (non-phosphorylated peptide) or Phe with Val at position +3, leads to no-binding ITC results. While these findings are indicative that BRIP1 is a potential BARD1 binding partner, it becomes evident that in vitro binding assays involving the entire BARD1 protein and in vivo experiments are also needed to establish its binding partners and its potential role in tumor suppression pathways.
Keywords: Chemical unfolding; Thermodynamic stability; ITC binding experiments
Heterologous expression and purification of a biologically active legume lectin from Cratylia mollis seeds (CRAMOLL 1) by Varejao Nathalia Varejão; Marcius da Silva Almeida; Nuccia N.T. De Cicco; Geórgia C. Atella; Luana C.B.B. Coelho; M Tereza S. Correia; Debora Foguel (pp. 1917-1924).
CRAMOLL 1 is a mannose/glucose isolectin isolated from Cratylia mollis seeds. This lectin has 82% sequence identity with Con A and essentially the same quaternary structure. As with Con A, CRAMOLL 1 seems to undergo complex post-translational processing which makes it difficult to the use of traditional molecular cloning for heterologous expression. Here we report the expression and purification of functional recombinant CRAMOLL 1 (rCRAMOLL 1) in Escherichia coli. This was accomplished by introducing a chemically synthesized DNA encoding the mature CRAMOLL 1 amino acid sequence into a bacterial expression vector under T7 promoter control. Most of the recombinant lectin was found in insoluble aggregates (inclusion bodies), but we were able to recover reasonable amounts of soluble lectin in the active form by decreasing the protein induction temperature. The recombinant lectin was purified to homogeneity with one-step affinity chromatography. The plant CRAMOLL 1 (pCRAMOLL 1) and rCRAMOLL 1 share several physicochemical properties such as molecular mass, charge density and secondary and tertiary structures. However, pCRAMOLL 1 has a lower thermodynamic stability than rCRAMOLL 1 when probed by acidification, high temperature or high hydrostatic pressure, and this is probably caused by the presence of tetramers composed of fragmented monomers, which are formed in the plant cotyledon but absent from the recombinant protein. rCRAMOLL 1 behaves identically to its plant counterpart with respect to its specificity for monosaccharides, and to its agglutinating activities against rabbit erythrocytes and Trypanosoma cruzi epimastigote cells.
Keywords: Abbreviations; rCRAMOLL 1; recombinant CRAMOLL 1; pCRAMOLL 1; plant CRAMOLL 1 (extracted from; Cratylia mollis; seeds); IPTG; isopropyl β-; d; -thiogalactopyranoside; MALDI-TOF; matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis; ANS; 8-anilinonaphthalene-1-sulfonate; bis-ANS; bis(8-anilinonaphthalene-1-sulfonate); MES; 4-morpholineethanesulfonic acid; PBS; phosphate buffer saline; LDL; low density lipoprotein; LIT; liver infusion tryptoseLegume lectins; Cratylia mollis; seed lectin; Gene synthesis; Heterologous expression; Protein stability
Molecular identification of unsaturated uronate reductase prerequisite for alginate metabolism in Sphingomonas sp. A1 by Ryuichi Takase; Akihito Ochiai; Bunzo Mikami; Wataru Hashimoto; Kousaku Murata (pp. 1925-1936).
In Sphingomonas sp. A1, alginate is degraded by alginate lyases to its constituent monosaccharides, which are nonenzymatically converted to an α-keto acid, namely, 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH). The properties of the DEH-metabolizing enzyme and its gene in strain A1 were characterized. In the presence of alginate, strain A1 cells inducibly produced an NADPH-dependent DEH reductase (A1-R) in their cytoplasm. Molecular cloning of the enzyme gene indicated that A1-R belonged to the short-chain dehydrogenase/reductase superfamily and catalyzed the conversion of DEH to 2-keto-3-deoxy-d-gluconic acid most efficiently at around pH 7.0 and 50°C. Crystal structures of A1-R and its complex with NADP were determined at around 1.6Å resolution by X-ray crystallography. The enzyme consists of three layers (α/β/α), with a coenzyme-binding Rossmann fold. NADP is surrounded by positively charged residues, and Gly-38 and Arg-39 are crucial for NADP binding. Site-directed mutagenesis studies suggest that Ser-150, Tyr-164, and Lys-168 located around the Rossmann fold constitute the catalytic triad. To our knowledge, this is the first report on molecular cloning and structure determination of a bacterial DEH reductase responsible for alginate metabolism.
Keywords: Abbreviations; Strain A1; Sphingomonas; sp. strain A1; DEH; 4-deoxy-; l; -erythro-5-hexoseulose uronic acid; KDG; 2-keto-3-deoxy-; d; -gluconic acid; A1-R; DEH-specific strain A1 reductase; TLC; thin-layer chromatography; KPB; potassium phosphate buffer; LC; liquid chromatography; SDS–PAGE; sodium dodecyl sulfate–polyacrylamide gel electrophoresis; MS; mass spectrometry; ESI; electrospray ionization; NMR; nuclear magnetic resonance; A1-R/NADP; A1-R in complex with NADP; r.m.s.d.; root-mean-square deviation; PDB; Protein Data Bank; MALDI-TOF; matrix-assisted laser desorption/ionization time-of-flight; SDR; short-chain dehydrogenase/reductaseAlginate; Crystal structure; 2-Keto-3-deoxy-; d; -gluconic acid; Short-chain dehydrogenase/reductase; Sphingomonas
Autolysis at the disintegrin domain of patagonfibrase, a metalloproteinase from Philodryas patagoniensis (Patagonia Green Racer; Dipsadidae) venom by María E. Peichoto; Adriana F. Paes Leme; Bianca A. Pauletti; Isabel Correia Batista; Stephen P. Mackessy; Ofelia Acosta; Marcelo L. Santoro (pp. 1937-1942).
Patagonfibrase is a 57.5-kDa hemorrhagic metalloproteinase isolated from the venom of Philodryas patagoniensis (Patagonia Green Racer), a South American rear-fanged snake. Herein we demonstrate that patagonfibrase undergoes autolysis at its pH optimum (7.5) and at 37°C, primarily producing a ∼32.6kDa fragment composed of disintegrin-like and cysteine-rich domains, as identified by mass spectrometry and N-terminal sequencing. The autolysis site for production of this fragment is similar to that observed for metalloproteinases from front-fanged Viperidae snake venoms. In the presence of Ca2+, patagonfibrase was only partially autolysed, giving rise mainly to one fragment of ∼52.2kDa. In addition, calcium markedly enhanced the azocaseinolytic activity of patagonfibrase. Our findings contribute to the understanding of the structural and mechanistic bases of this family of metalloenzymes that are widely distributed among snake venoms, demonstrating that important post-translational modifications such as proteolysis can also contribute to the diversity and complexity of proteins found in rear-fanged snake venoms.
Keywords: Autodegradation; Colubridae; Metalloproteinase