scholarly journals In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking

2018 ◽  
Vol 115 (24) ◽  
pp. E5477-E5486 ◽  
Author(s):  
Chungyu Chang ◽  
Brendan R. Amer ◽  
Jerzy Osipiuk ◽  
Scott A. McConnell ◽  
I-Hsiu Huang ◽  
...  
Keyword(s):  
Cross Linking ◽  
Structural Elements ◽  
Gram Positive Bacteria ◽  
Activating Mutations ◽  
In Vitro Reconstitution ◽  
Isopeptide Bonds ◽  
Signature Sequence ◽  
Class C ◽  
Enzyme Intermediate

Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction, first cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate and then joining the terminal Thr to the nucleophilic Lys residue residing within the pilin motif of another pilin protomer. To date, the determinants of class C enzymes that uniquely enable them to construct pili remain unknown. Here, informed by high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and utilizing a structural variant of the enzyme (SrtA2M), whose catalytic pocket has been unmasked by activating mutations, we successfully reconstituted in vitro polymerization of the cognate major pilin (SpaA). Mass spectrometry, electron microscopy, and biochemical experiments authenticated that SrtA2Msynthesizes pilus fibers with correct Lys–Thr isopeptide bonds linking individual pilins via a thioacyl intermediate. Structural modeling of the SpaA–SrtA–SpaA polymerization intermediate depicts SrtA2Msandwiched between the N- and C-terminal domains of SpaA harboring the reactive pilin and LPXTG motifs, respectively. Remarkably, the model uncovered a conserved TP(Y/L)XIN(S/T)H signature sequence following the catalytic Cys, in which the alanine substitutions abrogated cross-linking activity but not cleavage of LPXTG. These insights and our evidence that SrtA2Mcan terminate pilus polymerization by joining the terminal pilin SpaB to SpaA and catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile platform for protein engineering and bio-conjugation that has major implications for biotechnology.

scholarly journals Transglutaminase 5 is regulated by guanine–adenine nucleotides1

2004 ◽  
Vol 381 (1) ◽  
pp. 313-319 ◽  
Author(s):  
Eleonora CANDI ◽  
Andrea PARADISI ◽  
Alessandro TERRINONI ◽  
Valentina PIETRONI ◽  
Sergio ODDI ◽  
...  
Keyword(s):  
Amino Acid ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Cross Linking ◽  
Bifunctional Enzyme ◽  
Amino Acid Sequence Alignment ◽  
Gtp Binding ◽  
Epidermal Tissue ◽  
Isopeptide Bonds

Transglutaminases (TGases) are Ca2+-dependent enzymes capable of catalysing transamidation of glutamine residues to form intermolecular isopeptide bonds. Nine distinct TGases have been described in mammals, and two of them (types 2 and 3) are regulated by GTP/ATP. TGase2 hydrolyses GTP and is therefore a bifunctional enzyme. In the present study, we report that TGase5 is also regulated by nucleotides. We have identified the putative TGase5 GTP-binding pocket by comparative amino acid sequence alignment and homology-derived three-dimensional modelling. GTP and ATP inhibit TGase5 cross-linking activity in vitro, and Ca2+ is capable of completely reversing this inhibition. In addition, TGase5 mRNA is not restricted to epidermal tissue, but is also present in different adult and foetal tissues, suggesting a role for TGase5 outside the epidermis. These results reveal the reciprocal actions of Ca2+ and nucleotides with respect to TGase5 activity. Taken together, these results indicate that TGases are a complex family of enzymes regulated by calcium, with at least three of them, namely TGase2, TGase3 and TGase5, also being regulated by ATP and GTP.

scholarly journals An Unusual Mechanism of Isopeptide Bond Formation Attaches the Collagenlike Glycoprotein BclA to the Exosporium of Bacillus anthracis

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Li Tan ◽  
Mei Li ◽  
Charles L. Turnbough
Keyword(s):  
Bacillus Anthracis ◽  
Bond Formation ◽  
Basal Layer ◽  
Side Chain ◽  
Cross Linking ◽  
Amino Group ◽  
Content Type ◽  
Isopeptide Bond ◽  
Isopeptide Bonds

ABSTRACTThe outermost exosporium layer of spores ofBacillus anthracis, the causative agent of anthrax, is comprised of a basal layer and an external hairlike nap. The nap includes filaments composed of trimers of the collagenlike glycoprotein BclA. Essentially all BclA trimers are tightly attached to the spore in a process requiring the basal layer protein BxpB (also called ExsFA). Both BclA and BxpB are incorporated into stable, high-molecular-mass complexes, suggesting that BclA is attached directly to BxpB. The 38-residue amino-terminal domain of BclA, which is normally proteolytically cleaved between residues 19 and 20, is necessary and sufficient for basal layer attachment. In this study, we demonstrate that BclA attachment occurs through the formation of isopeptide bonds between the free amino group of BclA residue A20 and a side chain carboxyl group of an acidic residue of BxpB. Ten of the 13 acidic residues of BxpB can participate in isopeptide bond formation, and at least three BclA polypeptide chains can be attached to a single molecule of BxpB. We also demonstrate that similar cross-linking occursin vitrobetween purified recombinant BclA and BxpB, indicating that the reaction is spontaneous. The mechanism of BclA attachment, specifically, the formation of a reactive amino group by proteolytic cleavage and the promiscuous selection of side chain carboxyl groups of internal acidic residues, appears to be different from other known mechanisms for protein cross-linking through isopeptide bonds. Analogous mechanisms appear to be involved in the cross-linking of other spore proteins and could be found in unrelated organisms.IMPORTANCEIsopeptide bonds are protein modifications found throughout nature in which amide linkages are formed between functional groups of two amino acids, with at least one of the functional groups provided by an amino acid side chain. Isopeptide bonds generate cross-links within and between proteins that are necessary for proper protein structure and function. In this study, we discovered that BclA, the dominant structural protein of the external nap ofBacillus anthracisspores, is attached to the underlying exosporium basal layer protein BxpB via isopeptide bonds formed through a mechanism fundamentally different from previously described mechanisms of isopeptide bond formation. The most unusual features of this mechanism are the generation of a reactive amino group by proteolytic cleavage and promiscuous selection of acidic side chains. This mechanism, which apparently relies only on short peptide sequences in protein substrates, could be a general mechanismin vivoand adapted for protein cross-linkingin vitro.

scholarly journals NuSAP, a Mitotic RanGTP Target That Stabilizes and Cross-links Microtubules

2006 ◽  
Vol 17 (6) ◽  
pp. 2646-2660 ◽  
Author(s):  
Katharina Ribbeck ◽  
Aaron C. Groen ◽  
Rachel Santarella ◽  
Markus T. Bohnsack ◽  
Tim Raemaekers ◽  
...  
Keyword(s):  
Cross Linking ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Large Numbers ◽  
In Vitro Reconstitution ◽  
Cross Links ◽  
Xenopus Egg ◽  
Meiotic Spindles ◽  
Interfering Rna

Nucleolar and spindle-associated protein (NuSAP) was recently identified as a microtubule- and chromatin-binding protein in vertebrates that is nuclear during interphase. Small interfering RNA-mediated depletion of NuSAP resulted in aberrant spindle formation, missegregation of chromosomes, and ultimately blocked cell proliferation. We show here that NuSAP is enriched on chromatin-proximal microtubules at meiotic spindles in Xenopus oocytes. When added at higher than physiological levels to Xenopus egg extract, NuSAP induces extensive bundling of spindle microtubules and causes bundled microtubules within spindle-like structures to become longer. In vitro reconstitution experiments reveal two direct effects of NuSAP on microtubules: first, it can efficiently stabilize microtubules against depolymerization, and second, it can cross-link large numbers of microtubules into aster-like structures, thick fibers, and networks. With defined components we show that the activity of NuSAP is differentially regulated by Importin (Imp) α, Impβ, and Imp7. While Impα and Imp7 appear to block the microtubule-stabilizing activity of NuSAP, Impβ specifically suppresses aspects of the cross-linking activity of NuSAP. We propose that to achieve full NuSAP functionality at the spindle, all three importins must be dissociated by RanGTP. Once activated, NuSAP may aid to maintain spindle integrity by stabilizing and cross-linking microtubules around chromatin.

Kinetics of in vitro reconstitution of oligomeric enzymes by cross-linking

Nature ◽  
1979 ◽  
Vol 277 (5693) ◽  
pp. 243-245 ◽  
Author(s):  
R. HERMANN ◽  
R. RUDOLPH ◽  
R. JAENICKE
Keyword(s):  
Cross Linking ◽  
In Vitro Reconstitution ◽  
Kinetics Of

scholarly journals Semi-in vitro Reconstitution of Roseocin, a Two-Component Lantibiotic from an Actinomycete

2018 ◽  
Author(s):  
Mangal Singh ◽  
Dipti Sareen
Keyword(s):  
Genome Mining ◽  
Disulfide Bridge ◽  
Biosynthetic Gene Cluster ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
C Terminus ◽  
In Vitro Reconstitution ◽  
Two Component ◽  
Modified Peptides

ABSTRACTLantibiotics are lanthionine containing peptide natural products that belong to the class of ribosomally synthesized and post-translationally modified peptides (RiPPs). Recent expansion in the availability of microbial genomic data and in silico analysis tools have accelerated the discovery of these promising alternatives to antibiotics. Following the genome-mining approach, a biosynthetic gene cluster for a putative two-component lantibiotic roseocin was identified in the genome of an Actinomycete, Streptomyces roseosporus NRRL 11379. Post-translationally modified lanthipeptides of this cluster were obtained by heterologous expression of the genes in E. coli, and were in vitro reconstituted to their bioactive form. The two lanthipeptides displayed antimicrobial activity against Gram-positive bacteria only synergistically, a property reminiscent of two-component lantibiotics. Structural analysis of the α-component identified a disulfide bridge flanking two of its four thioether bridges and the β-component having six thioether bridges with its C-terminus extended than the previously known two-component lantibiotics.

Tridegin, a Novel Peptidic Inhibitor of Factor XIIIa from the Leech, Haementeria ghilianii, Enhances Fibrinolysis In Vitro

1997 ◽  
Vol 77 (05) ◽  
pp. 0959-0963 ◽  
Author(s):  
Lisa Seale ◽  
Sarah Finney ◽  
Roy T Sawyer ◽  
Robert B Wallis
Keyword(s):  
Potent Inhibitor ◽  
Platelet Rich Plasma ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Fibrinolytic Enzymes ◽  
Small Polypeptide ◽  
Tissue Plasminogen ◽  
Protein Cross Linking

SummaryTridegin is a potent inhibitor of factor Xllla from the leech, Haementeria ghilianii, which inhibits protein cross-linking. It modifies plasmin-mediated fibrin degradation as shown by the absence of D-dimer and approximately halves the time for fibrinolysis. Plasma clots formed in the presence of Tridegin lyse more rapidly when either streptokinase, tissue plasminogen activator or hementin is added 2 h after clot formation. The effect of Tridegin is markedly increased if clots are formed from platelet-rich plasma. Platelet-rich plasma clots are lysed much more slowly by the fibrinolytic enzymes used and if Tridegin is present, the rate of lysis returns almost to that of platelet- free clots. These studies indicate the important role of platelets in conferring resistance to commonly used fibrinolytic enzymes and suggest that protein cross-linking is an important step in this effect. Moreover they indicate that Tridegin, a small polypeptide, may have potential as an adjunct to thrombolytic therapy.

Synthesis, Spectral and Antimicrobial Investigation of 2-(Naphthalenel-ylamino)-2- Phenylacetonitrile and 1, 10-Phenanthroline with Five Divalent Transition Metal Ions

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Mohammed Al-Amery1 ◽  
Ashraf Saad Rasheed ◽  
Dina A. Najeeb
Keyword(s):  
Transition Metal ◽  
Metal Ions ◽  
Transition Metal Ions ◽  
Molar Conductance ◽  
Octahedral Geometry ◽  
Mixed Ligand ◽  
Gram Negative Bacteria ◽  
Magnetic Studies ◽  
Gram Positive Bacteria

Five new mixed ligand metal complexes have been synthesized by the reaction of divalent transition metal ions (Hg, Ni, Zn, Cu and Cd) with 2-(naphthalen-l-ylamino)-2-phenylacetonitrile (L1 ) and 1,10-phenanthroline (L2). The coordination likelihood of the two ligands toward metal ions has been suggested in the light of elemental analysis, UV-Vis spectra, FTIR, 1H-NMR, flam atomic absorption, molar conductance and magnetic studies. Results data suggest that the octahedral geometry for all the prepared complexes. Antibacterial examination of synthesized complexes in vitro was performed against four bacterias. Firstly, Gram-negative bacteria namely, Pseudomonas aerugin and Escherichia. Secondly, Gram-positive bacteria namely, Bacillus subtilis, Staphylococcuaurouss. Results data exhibit that the synthesized complexes exhibited more biological activity than tetracycline pharmaceutical.

Synthesis, SAR, In-Silico appraisal and Anti-Microbial Study of substituted 2-aminobenzothiazoles derivatives

2019 ◽  
Vol 15 ◽  
Author(s):  
Devidas G. Anuse ◽  
Suraj N. Mali ◽  
Bapu R. Thorat ◽  
Ramesh S. Yamgar ◽  
Hemchandra K. Chaudhari
Keyword(s):  
Antimicrobial Activity ◽  
In Silico ◽  
Docking Study ◽  
Moderate Activity ◽  
Molecular Docking Study ◽  
Mass Spectral ◽  
Gram Positive Bacteria ◽  
Ft Ir ◽  
In Silico Molecular Docking

Background: Antimicrobial resistance is major global health problem, which is being rapidly deteriorating the quality of human health. Series of substituted N-(benzo[d]thiazol-2-yl)-2-(4-(6-fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)acetamide (3a-j) were synthesized from substituted N-(benzo[d]thiazol-2-yl)-2-chloroacetamide/bromopropanamide (2a-j) and 6-fluoro-3-(piperidin-4-yl)benzo[d]isoxazole (2) and further evaluated for their docking properties and antimicrobial activity. Methods: All synthesized compounds were characterized by FT-IR, NMR and Mass spectral analysis. All compounds were allowed to dock against different antimicrobial targets having PDB ID: 1D7U and against common antifungal target having PDB ID: 1EA1. Results: The compounds 3d and 3h were showed good activity against Methicillin-resistant Staphylococcus aureus (MRSA, resistance Gram-positive bacteria). All synthesized compounds showed good to moderate activity against selected bacterial and fungal microbial strains. If we compared the actual in-vitro antimicrobial activity and in-silico molecular docking study, we found that molecules 3i and 3h were more potent than the others. Conclusion: Our current study would definitely pave the new way towards designing and synthesis of more potent 2-aminobenzothiazoles derivatives.

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