scholarly journals Interrogation of 3D-swapped structure and functional attributes of quintessential Sortase A from Streptococcus pneumoniae

2020 ◽  
Vol 477 (24) ◽  
pp. 4711-4728
Author(s):  
Tora Biswas ◽  
Anurag Misra ◽  
Sreetama Das ◽  
Prity Yadav ◽  
Suryanarayanarao Ramakumar ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Streptococcus Pneumoniae ◽  
Biochemical Characterization ◽  
Surface Proteins ◽  
Size Exclusion ◽  
Sedimentation Equilibrium ◽  
Exclusion Chromatography ◽  
Distance Mapping ◽  
Regulatory Functions ◽  
Pilus Assembly

The anchoring of the surface proteins to the cell wall in gram-positive bacteria involves a peptide ligation reaction catalyzed by transpeptidase sortase. Most bacterial genomes encode multiple sortases with dedicated functions. Streptococcus pneumoniae (Sp) carries four sortases; a housekeeping sortase (SrtA), and three pilin specific sortases (SrtC1, C2, C3) dedicated to the biosynthesis of covalent pilus. Interestingly, SrtA, meant for performing housekeeping roles, is also implicated in pilus assembly of Sp. The allegiance of SpSrtA to the pathogenic pilus assembly makes it an ideal target for clinical inhibitor development. In this paper, we describe biochemical characterization, crystal structure and peptide substrate preference of SpSrtA. Transpeptidation reaction with a variety of substrates revealed that the enzyme preferred elongated LPXTG sequences and transferred them equally well to both Ala- and Gly-terminated peptides. Curiously, the crystal structure of both wild type and an active site (Cys to Ala) mutant of SpSrtA displayed inter-twined 3D-swapped dimers in which each protomer generated a classic eight-stranded beta-barrel ‘sortase fold'. Size-exclusion chromatography and sedimentation equilibrium measurements revealed the predominant presence of a dimer in equilibrium with its monomer. The crystal structure-based Cys–Cys distance mapping with defined chemical cross-linkers established the existence of 3D-swapped structure in solution. The swapping in SpSrtA, unprecedented for sortase family, may be physiologically relevant and meant to perform regulatory functions.

Molecular cloning and biochemical characterization of a NAD-dependent sorbitol dehydrogenase from cold-adapted Pseudomonas mandelii

2021 ◽  
Author(s):  
Quynh DangThu ◽  
Thu-Thuy Nguyen ◽  
Sei-Heon Jang ◽  
ChangWoo Lee
Keyword(s):  
Biochemical Characterization ◽  
Size Exclusion ◽  
Sorbitol Dehydrogenase ◽  
High Catalytic Activity ◽  
Active Site Residues ◽  
Tertiary Structures ◽  
Cold Adapted ◽  
Pseudomonas Mandelii ◽  
Catalytic Active Site ◽  
Exclusion Chromatography

Abstract Sugar alcohols (polyols) have important roles as nutrients, anti-freezing agents, and scavengers of free radicals in cold-adapted bacteria, but the characteristics of polyol dehydrogenases in cold-adapted bacteria remain largely unknown. In this study, based on the observation that a cold-adapted bacterium Pseudomonas mandelii JR-1 predominantly utilized D-sorbitol as its carbon source, among the four polyols examined (D-galactitol, D-mannitol, D-sorbitol, or D-xylitol), we cloned and characterized a sorbitol dehydrogenase (SDH, EC 1.1.1.14) belonging to the short-chain dehydrogenase/reductase family from this bacterium (the SDH hereafter referred to as PmSDH). PmSDH contained Asn111, Ser140, Tyr153, and Lys157 as catalytic active site residues and existed as a ∼67 kDa dimer in size-exclusion chromatography. PmSDH converted D-sorbitol to D-fructose using NAD+ as a coenzyme and, vice versa, D-fructose to D-sorbitol using NADH as a coenzyme. PmSDH maintained its conformational flexibility, secondary and tertiary structures, and thermal stability at 4–25°C. At 40°C, PmSDH was rapidly denatured. These results indicate that PmSDH, which has a flexible structure and a high catalytic activity at colder temperatures, is well-suited to sorbitol utilization in the cold-adapted bacterium P. mandelii JR-1.

EWI-2 modulates lymphocyte integrin α4β1 functions

Blood ◽  
2004 ◽  
Vol 103 (8) ◽  
pp. 3013-3019 ◽  
Author(s):  
Tatiana V. Kolesnikova ◽  
Christopher S. Stipp ◽  
Ravi M. Rao ◽  
William S. Lane ◽  
Francis W. Luscinskas ◽  
...  
Keyword(s):  
Cell Surface ◽  
Cell Adhesion Molecule ◽  
Apparent Size ◽  
Surface Proteins ◽  
Size Exclusion ◽  
Leukemia Cells ◽  
Wild Type ◽  
Vascular Cell Adhesion ◽  
Exclusion Chromatography ◽  
Cell Adhesion Molecule 1

Abstract The most prominent cell-surface integrin α4β1 partner, a 70-kDa protein, was isolated from MOLT-4 T leukemia cells, using anti–α4β1 integrin antibody-coated beads. By mass spectrometry, this protein was identified as EWI-2, a previously described cell-surface partner for tetraspanin proteins CD9 and CD81. Wild-type EWI-2 overexpression had no effect on MOLT-4 cell tethering and adhesion strengthening on the α4β1 ligand, vascular cell adhesion molecule-1 (VCAM-1), in shear flow assays. However, EWI-2 markedly impaired spreading and ruffling on VCAM-1. In contrast, a mutant EWI-2 molecule, with a different cytoplasmic tail, neither impaired cell spreading nor associated with α4β1 and CD81. The endogenous wild-type EWI-2–CD81–α4β1 complex was fully soluble, and highly specific as seen by the absence of other MOLT-4 cell-surface proteins. Also, it was relatively small in size (0.5 × 106 Da to 4 × 106 Da), as estimated by size exclusion chromatography. Overexpression of EWI-2 in MOLT-4 cells caused reorganization of cell-surface CD81, increased the extent of CD81-CD81, CD81-α4β1, and α4β1-α4β1 associations, and increased the apparent size of CD81-α4β1 complexes. We suggest that EWI-2–dependent reorganization of α4β1-CD81 complexes on the cell surface is responsible for EWI-2 effects on integrin-dependent morphology and motility functions. (Blood. 2004;103: 3013-3019)

scholarly journals Crystal structure of bacteriophage T4 Spackle as determined by native SAD phasing

2020 ◽  
Vol 76 (9) ◽  
pp. 899-904
Author(s):  
Ke Shi ◽  
Fredy Kurniawan ◽  
Surajit Banerjee ◽  
Nicholas H. Moeller ◽  
Hideki Aihara
Keyword(s):  
Crystal Structure ◽  
Bacteriophage T4 ◽  
Early Gene ◽  
Size Exclusion ◽  
Amino Terminal ◽  
Intramolecular Disulfide Bond ◽  
Exclusion Chromatography ◽  
T4 Bacteriophage ◽  
Sad Phasing ◽  
T4 Phage

The crystal structure of a bacteriophage T4 early gene product, Spackle, was determined by native sulfur single-wavelength anomalous diffraction (SAD) phasing using synchrotron radiation and was refined to 1.52 Å resolution. The structure shows that Spackle consists of a bundle of five α-helices, forming a relatively flat disc-like overall shape. Although Spackle forms a dimer in the crystal, size-exclusion chromatography with multi-angle light scattering shows that it is monomeric in solution. Mass spectrometry confirms that purified mature Spackle lacks the amino-terminal signal peptide and contains an intramolecular disulfide bond, consistent with its proposed role in the periplasm of T4 phage-infected Escherichia coli cells. The surface electrostatic potential of Spackle shows a strikingly bipolar charge distribution, suggesting a possible mode of membrane association and inhibition of the tail lysozyme activity in T4 bacteriophage superinfection exclusion.

scholarly journals Molecular cloning and biochemical characterization of rabbit factor XI

2002 ◽  
Vol 367 (1) ◽  
pp. 49-56 ◽  
Author(s):  
Dipali SINHA ◽  
Mariola MARCINKIEWICZ ◽  
David GAILANI ◽  
Peter N. WALSH
Keyword(s):  
Human Factor ◽  
Biochemical Characterization ◽  
Protein A ◽  
Size Exclusion ◽  
Factor Xi ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Intracellular Process ◽  
And Function

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.

scholarly journals Purification and partial biochemical characterization of normal human interleukin 1.

1984 ◽  
Vol 160 (3) ◽  
pp. 772-787 ◽  
Author(s):  
J A Schmidt
Keyword(s):  
High Performance ◽  
Biochemical Characterization ◽  
Interleukin 1 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Thymocyte Proliferation ◽  
Exclusion Chromatography ◽  
Normal Human

A protocol for the rapid, efficient purification of the major charged species of human interleukin 1 (IL-1) has been developed using high performance anion exchange and size exclusion chromatography. The isolated material is pure as determined by sodium dodecyl sulfate (SDS) gradient polyacrylamide gel electrophoresis (PAGE) and analytical isoelectric focusing (IEF). The molecular weight of the purified material is 15,000 and the isoelectric point (pI) is 6.8, values that are in good agreement with those previously reported for human IL-1. 10(-10) M concentrations of the purified material give half-maximal stimulation in the thymocyte proliferation assay. Amounts of IL-1 sufficient for receptor studies and detailed biochemical analysis can now be produced on a regular basis.

scholarly journals Crystal structure of an anti-idiotype variable lymphocyte receptor

2017 ◽  
Vol 73 (12) ◽  
pp. 682-687
Author(s):  
Bernard C. Collins ◽  
Hiro Nakahara ◽  
Sharmistha Acharya ◽  
Max D. Cooper ◽  
Brantley R. Herrin ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structural Similarity ◽  
Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Size Exclusion ◽  
Antigen Receptors ◽  
Diagnostic Assays ◽  
Great Utility ◽  
Exclusion Chromatography ◽  
Leukemic Clone

Variable lymphocyte receptors (VLRs), the leucine-rich repeat (LRR)-based antigen receptors of jawless fish, have great utility in a wide variety of biochemical and biological applications, similar to classical Ig-based antibodies. VLR-based reagents may be particularly useful when traditional antibodies are not available. An anti-idiotype lamprey VLR, VLR39, has previously been identified that recognizes the heavy-chain CDR3 of the B-cell receptor (BCR) of a leukemic clone from a patient with chronic lymphocytic leukemia (CLL). VLR39 was used successfully to track the re-emergence of this clone in the patient following chemotherapy. Here, the crystal structure of VLR39 is presented at 1.5 Å resolution and compared with those of other protein-specific VLRs. VLR39 adopts a curved solenoid fold and exhibits substantial structural similarity to other protein-binding VLRs. VLR39 has a short LRRCT loop that protrudes outwards away from the concave face and is similar to those of its protein-specific VLR counterparts. Analysis of the VLR39–BCR interaction by size-exclusion chromatography and biolayer interferometry using the scFv version of the BCR confirms that VLR39 recognizes the BCR Fv region. Such VLR-based reagents may be useful for identifying and monitoring leukemia in CLL patients and in other clinical diagnostic assays.

scholarly journals Structural and Biochemical Characterization of a Cold-Active PMGL3 Esterase with Unusual Oligomeric Structure

Biomolecules ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 57
Author(s):  
Konstantin M. Boyko ◽  
Mariya V. Kryukova ◽  
Lada E. Petrovskaya ◽  
Elena A. Kryukova ◽  
Alena Y. Nikolaeva ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
3D Structure ◽  
Hormone Sensitive Lipase ◽  
Size Exclusion ◽  
Wild Type ◽  
Prolonged Incubation ◽  
Gene Coding ◽  
Cold Active ◽  
Tetrameric Structure ◽  
Exclusion Chromatography

The gene coding for a novel cold-active esterase PMGL3 was previously obtained from a Siberian permafrost metagenomic DNA library and expressed in Escherichia coli. We elucidated the 3D structure of the enzyme which belongs to the hormone-sensitive lipase (HSL) family. Similar to other bacterial HSLs, PMGL3 shares a canonical α/β hydrolase fold and is presumably a dimer in solution but, in addition to the dimer, it forms a tetrameric structure in a crystal and upon prolonged incubation at 4 °C. Detailed analysis demonstrated that the crystal tetramer of PMGL3 has a unique architecture compared to other known tetramers of the bacterial HSLs. To study the role of the specific residues comprising the tetramerization interface of PMGL3, several mutant variants were constructed. Size exclusion chromatography (SEC) analysis of D7N, E47Q, and K67A mutants demonstrated that they still contained a portion of tetrameric form after heat treatment, although its amount was significantly lower in D7N and K67A compared to the wild type. Moreover, the D7N and K67A mutants demonstrated a 40 and 60% increase in the half-life at 40 °C in comparison with the wild type protein. Km values of these mutants were similar to that of the wt PMGL3. However, the catalytic constants of the E47Q and K67A mutants were reduced by ~40%.

scholarly journals Biochemical Characterization of a Group-5 Soluble Diiron Monooxygenase Hydroxylase and Related Chaperonin-like Component

2020 ◽  
Author(s):  
Chihiro Inoue ◽  
Yoshitaka Abe ◽  
Nobutaka Fujieda
Keyword(s):  
Biochemical Characterization ◽  
Quaternary Structure ◽  
Expression System ◽  
Functional Expression ◽  
Size Exclusion ◽  
Native Page ◽  
Dinuclear Iron ◽  
Exclusion Chromatography ◽  
Group 5

<p>Recently, the functional expression of group-5 hydroxylase component (MimA and MimC) in <i>Escherichia coli </i>along with its related chaperonin-like component (MimG) was reported by Furuya and Kino. In this study, we report the purification via a heterologous expression system and the biochemical characterization of MimAC, the complex of MimA and MimC and MimG to understand their exact roles. MimAC and MimG were fused with His-tags and purified using affinity chromatography in a homogenous state on SDS-PAGE. Blue native PAGE demonstrated that the quaternary structure of MimG was almost identical to that of chaperonin GroEL, indicating that its function was also similar to GroEL. Size-exclusion chromatography and ICP-AES analysis demonstrated that MimAC was assembled in the dimer of two sort of subunits and exhibited two iron atoms and at least one zinc atom per two subunits. This result indicated that MimAC possessed a dinuclear iron center, similar to other soluble diiron monooxygenase hydroxylases.</p>

scholarly journals Isolation and biochemical characterization of the α- and β-subunits of glycoprotein IIb of human platelet plasma membrane

1986 ◽  
Vol 240 (1) ◽  
pp. 155-161 ◽  
Author(s):  
J J Calvete ◽  
J González-Rodríguez
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Human Platelet ◽  
Size Exclusion ◽  
Beta Subunits ◽  
Exclusion Chromatography ◽  
Stepwise Reduction ◽  
Acidic Nature

The alpha- and beta-subunits of glycoprotein IIb (GPIIb) of human platelet plasma membrane were isolated in fully reduced, partially reduced and alkylated, and fully alkylated forms, by size-exclusion chromatography after reduction of pure GPIIb. The sugar moiety of GPIIb alpha accounts for 16.4% of its total weight, whereas that of GPIIb beta accounts for only 10.2%. The molar percentages (per 100 mol of total amino acids) of neuraminic acid and galactose in the alpha-subunit more than double those in the beta-subunit, whereas galactosamine is present only in GPIIb alpha. From the amino acid and sugar compositions the acidic nature of both subunits was confirmed. The Mr values obtained, 114,000 for GPIIb alpha and 22,200 for GPIIb beta, are in very good agreement with those obtained by physical methods. We found by stepwise reduction of pure GPIIb with dithioerythritol that GPIIb alpha and GPIIb beta are joined by a single interchain disulphide bridge, while the remaining half-cystine residues participate in intrachain bonds, six in GPIIb alpha and one in GPIIb beta, the intersubunit disulphide bond being that reduced first. Neither of the two subunits is liberated from isolated plasma membranes when this GPIIb interchain bond is reduced in isolated membranes.

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