共查询到20条相似文献,搜索用时 31 毫秒
1.
Ethan M. Weiner Scott Robson Melanie Marohn Robert T. Clubb 《The Journal of biological chemistry》2010,285(30):23433-23443
The pathogen Bacillus anthracis uses the Sortase A (SrtA) enzyme to anchor proteins to its cell wall envelope during vegetative growth. To gain insight into the mechanism of protein attachment to the cell wall in B. anthracis we investigated the structure, backbone dynamics, and function of SrtA. The NMR structure of SrtA has been determined with a backbone coordinate precision of 0.40 ± 0.07 Å. SrtA possesses several novel features not previously observed in sortase enzymes including the presence of a structurally ordered amino terminus positioned within the active site and in contact with catalytically essential histidine residue (His126). We propose that this appendage, in combination with a unique flexible active site loop, mediates the recognition of lipid II, the second substrate to which proteins are attached during the anchoring reaction. pKa measurements indicate that His126 is uncharged at physiological pH compatible with the enzyme operating through a “reverse protonation” mechanism. Interestingly, NMR relaxation measurements and the results of a model building study suggest that SrtA recognizes the LPXTG sorting signal through a lock-in-key mechanism in contrast to the prototypical SrtA enzyme from Staphylococcus aureus. 相似文献
2.
Chandrabose Selvaraj Gurudeeban Selvaraj Randa Mohamed Ismail Rajendran Vijayakumar Alaa Baazeem Dong-Qing Wei Sanjeev Kumar Singh 《Saudi Journal of Biological Sciences》2021,28(7):3650-3659
Bacillus anthracis is a gram positive, deadly spore forming bacteria causing anthrax and these bacteria having the complex mechanism in the cell wall envelope, which can adopt the changes in environmental conditions. In this, the membrane bound cell wall proteins are said to progressive drug target for the inhibition of Bacillus anthracis. Among the cell wall proteins, the SrtA is one of the important mechanistic protein, which mediate the ligation with LPXTG motif by forming the amide bonds. The SrtA plays the vital role in cell signalling, cell wall formation, and biofilm formations. Inhibition of SrtA leads to rupture of the cell wall and biofilm formation, and that leads to inhibition of Bacillus anthracis and thus, SrtA is core important enzyme to study the inhibition mechanism. In this study, we have examined 28 compounds, which have the inhibitory activity against the Bacillus anthracis SrtA for developing the 3D-QSAR and also, compounds binding selectivity with both open and closed SrtA conformations, obtained from 100 ns of MD simulations. The binding site loop deviate in forming the open and closed gate mechanism is investigated to understand the inhibitory profile of reported compounds, and results show the closed state active site conformations are required for ligand binding specificity. Overall, the present study may offer an opportunity for better understanding of the mechanism of action and can be aided to further designing of a novel and highly potent SrtA inhibitors. 相似文献
3.
Dasgupta S Samantaray S Sahal D Roy RP 《The Journal of biological chemistry》2011,286(27):23996-24006
The housekeeping transpeptidase sortase A (SrtA) from Staphyloccocus aureus catalyzes the covalent anchoring of surface proteins to the cell wall by linking the threonyl carboxylate of the LPXTG recognition motif to the amino group of the pentaglycine cross-bridge of the peptidoglycan. SrtA-catalyzed ligation of an LPXTG containing polypeptide with an aminoglycine-terminated moiety occurs efficiently in vitro and has inspired the use of this enzyme as a synthetic tool in biological chemistry. Here we demonstrate the propensity of SrtA to catalyze "isopeptide" ligation. Using model peptide sequences, we show that SrtA can transfer LPXTG peptide substrates to the ε-amine of specific Lys residues and form cyclized and/or a gamut of branched oligomers. Our results provide insights about principles governing isopeptide ligation reactions catalyzed by SrtA and suggest that although cyclization is guided by distance relationship between Lys (ε-amine) and Thr (α-carboxyl) residues, facile branched oligomerization requires the presence of a stable and long-lived acyl-enzyme intermediate. 相似文献
4.
Ca2+‐independent sortase‐A exhibits high selective protein ligation activity in the cytoplasm of Escherichia coli
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A Staphylococcus aureus transpeptidase, sortase A (SrtA), which catalyzes a peptide ligation with high substrate specificity, is a useful tool to site‐specifically attach proteinaceous/peptidic functional molecules to target proteins. However, its strong Ca2+ dependency makes SrtA difficult for use under low Ca2+ concentrations and in the presence of Ca2+‐binding substances. To overcome this problem, we designed a SrtA mutant that Ca2+‐independently demonstrates a high catalytic activity. The heptamutant (P94R/E105K/E108A/D160N/D165A/K190E/K196T), which resulted from a combination of known mutations at the Ca2+‐binding site and around the substrate‐binding site, successfully catalyzed a selective protein‐protein ligation in the cytoplasm of Escherichia coli. Selective protein modification in living cells is a promising approach for investigating cellular events and regulating cell functions. This SrtA mutant may prove to be a versatile tool for adding new functionalities to proteins of interest by incorporating functional proteins and chemically modified peptides in living cells, which usually retain low Ca2+ concentrations. 相似文献
5.
Sortagging is a versatile method for site‐specific modification of proteins as applied to a variety of in vitro reactions. Here, we explore possibilities of adapting the sortase method for use in living cells. For intracellular sortagging, we employ the Ca2+‐independent sortase A transpeptidase (SrtA) from Streptococcus pyogenes. Substrate proteins were equipped with the C‐terminal sortase‐recognition motif (LPXTG); we used proteins with an N‐terminal (oligo)glycine as nucleophiles. We show that sortase‐dependent protein ligation can be achieved in Saccharomyces cerevisiae and in mammalian HEK293T cells, both in the cytosol and in the lumen of the endoplasmic reticulum (ER). ER luminal sortagging enables secretion of the reaction products, among which circular polypeptides. Protein ligation of substrate and nucleophile occurs within 30 min of translation. The versatility of the method is shown by protein ligation of multiple substrates with green fluorescent protein‐based nucleophiles in different intracellular compartments. 相似文献
6.
Albert H. Chan Sung Wook Yi Austen L. Terwilliger Anthony W. Maresso Michael E. Jung Robert T. Clubb 《The Journal of biological chemistry》2015,290(42):25461-25474
The endospore forming bacterium Bacillus anthracis causes lethal anthrax disease in humans and animals. The ability of this pathogen to replicate within macrophages is dependent upon the display of bacterial surface proteins attached to the cell wall by the B. anthracis Sortase A (BaSrtA) enzyme. Previously, we discovered that the class A BaSrtA sortase contains a unique N-terminal appendage that wraps around the body of the protein to contact the active site of the enzyme. To gain insight into its function, we determined the NMR structure of BaSrtA bound to a LPXTG sorting signal analog. The structure, combined with dynamics, kinetics, and whole cell protein display data suggest that the N terminus modulates substrate access to the enzyme. We propose that it may increase the efficiency of protein display by reducing the unproductive hydrolytic cleavage of enzyme-protein covalent intermediates that form during the cell wall anchoring reaction. Notably, a key active site loop (β7/β8 loop) undergoes a disordered to ordered transition upon binding the sorting signal, potentially facilitating recognition of lipid II. 相似文献
7.
Quinn DJ Cunningham S Walker B Scott CJ 《Biochemical and biophysical research communications》2008,370(1):77-81
We have examined the potential of displaying a protease species in vitro using ribosome display and demonstrate specific capture on the basis of its catalytic activity. Using a model bacterial cysteine protease, sortase A (SrtA), we show that this enzyme can be functionally expressed in vitro. By overlap PCR we constructed ribosome display templates with the SrtA open reading frame fused to a C terminal glycine-serine rich flexible linker and a tether derived from eGFP. Using the broad range cysteine protease irreversible inhibitor E-64 linked to acrylic beads, we show that we can isolate SrtA ribosome display ternary complexes, and recover their encoding mRNA by RT-PCR. This recovery was lost when applied to a SrtA catalytically inactive mutant, or could be alleviated by competition with free inhibitor. This sensitive technique could be further developed to allow the screening of proteases against putative inhibitors and/or the identification of novel proteolytic species. 相似文献
8.
Xiaochen Hou Meining Wang Yi Wen Tengfeng Ni Xiangna Guan Lefu Lan Naixia Zhang Ao Zhang Cai-Guang Yang 《Bioorganic & medicinal chemistry letters》2018,28(10):1864-1869
Sortase A (SrtA) anchors surface proteins to the cell wall and aids biofilm formation during infection, which functions as a key virulence factor of important Gram-positive pathogens, such as Staphylococcus aureus. At present researchers need a way in which to validate whether or not SrtA is a druggable target alternative to the conventional antibiotic targets in the mechanism. In this study, we performed a high-throughput screening and identified a new class of potential inhibitors of S. aureus SrtA, which are derived from natural products and contain the quinone skeleton. Compound 283 functions as an irreversible inhibitor that covalently alkylates the active site Cys184 of SrtA. NMR analysis confirms the direct interaction of the small-molecule inhibitor towards SrtA protein. The anchoring of protein A (SpA) to the cell wall and the biofilm formation are significantly attenuated when the S. aureus Newman strain is cultured in the presence of inhibitor. Our study indicates that compound 283 could be a potential hit for the development of new anti-virulence agents against S. aureus infections by covalently targeting SrtA. 相似文献
9.
We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography—utilizing the Strep-tag II appended to gelonin—and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins. 相似文献
10.
Staphylococcus aureus sortase A is a transpeptidase that is extensively used in various protein research applications. Sortase A is highly selective and does not require any cofactors for the catalysis of protein ligation and, importantly, can be produced in high yields. However, the primary disadvantage of this transpeptidase is its inability to access the recognition site within the highly structured regions of folded substrates. To overcome this problem, we developed an Escherichia coli expression system that produces milligram quantities of circularly closed sortase A; efficient enzyme cyclization was achieved by Synechocystis sp. PCC6803 intein-mediated post-translational splicing. The structural integrity of circular sortase A and its biochemical characteristics were compared to those of the linear enzyme analog and were found to be similar under native conditions. Additionally, the modified sortase was active at concentrations of urea up to 3 M and was capable of efficient catalytic protein–protein coupling, as shown by the ligation of purified glutathione-S-transferase and green fluorescence protein. In contrast to the circular enzyme, linear sortase A was unable to mediate the ligation of substrate proteins under the same conditions. Therefore, the proposed circular sortase A has improved enzymatic properties and has applications in advanced protein engineering and design. 相似文献
11.
Lipoate-protein ligases are used to scavenge lipoic acid from the environment and attach the coenzyme to its cognate proteins, which are generally the E2 components of the 2-oxoacid dehydrogenases. The enzymes use ATP to activate lipoate to its adenylate, lipoyl-AMP, which remains tightly bound in the active site. This mixed anhydride is attacked by the ϵ-amino group of a specific lysine present on a highly conserved acceptor protein domain, resulting in the amide-linked coenzyme. The Streptomyces coelicolor genome encodes only a single putative lipoate ligase. However, this protein had only low sequence identity (<25%) to the lipoate ligases of demonstrated activity and appears to be a circularly permuted version of the known lipoate ligase proteins in that the canonical C-terminal domain seems to have been transposed to the N terminus. We tested the activity of this protein both by in vivo complementation of an Escherichia coli ligase-deficient strain and by in vitro assays. Moreover, when the domains were rearranged into a protein that mimicked the arrangement found in the canonical lipoate ligases, the enzyme retained complementation activity. Finally, when the two domains were separated into two proteins, both domain-containing proteins were required for complementation and catalysis of the overall ligase reaction in vitro. However, only the large domain-containing protein was required for transfer of lipoate from the lipoyl-AMP intermediate to the acceptor proteins, whereas both domain-containing proteins were required to form lipoyl-AMP. 相似文献
12.
Staphylococcus aureus Sortase A (SrtA) is an important Gram-positive membrane enzyme which catalyzes the anchoring of many cell surface proteins conserved with the LPXTG sequence. Recently SrtA has been demonstrated to be a dimer with a Kd of 55 microM in vitro. Herein, we show that a single point mutation of amino acid residue on the surface of SrtA can completely disrupt the dimerization. Native polyacrylamide gel electrophoresis and analytical gel filtration chromatography were used to detect the dimer-monomer equilibrium of SrtA mutants. Circular dichroism spectrum experiments were performed to study the conformational change of each SrtA mutant. An enzyme activity assay confirmed that all the SrtA mutants were active in vitro. Our results not only are important for understanding the SrtA protein self-associating mechanism but also provided the necessary starting materials for the study of sortase A pathway in vivo, which may have significant implications for discovering microbial physiology and give a potential target for novel Gram-positive antibiotics. 相似文献
13.
Sortase A‐mediated synthesis of ligand‐grafted cyclized peptides for modulating a model protein‐protein interaction
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Specific ligand‐grafted cyclic peptides are promising drug candidates that can modulate protein‐protein interactions (PPIs) with increased proteolytic stability. In this study, we aimed to demonstrate that Sortase A (SrtA)‐mediated peptide transpeptidation can be applied to produce bioactive sequence‐grafted, stable, cyclic peptides. A naturally occurring cyclic peptide, sunflower trypsin inhibitor 1 (SFTI‐1), was selected as the scaffold, and a tetrapeptide motif, Glu‐Ser‐Asp‐Val (ESDV), was grafted into the scaffold as a model ligand. The linear precursor of the grafted peptide with SrtA‐recognition motifs at the N‐ and C‐termini was cyclized in good yield simply by co‐incubation with SrtA. The ESDV‐grafted cyclic SFTI‐1 obtained was confirmed to have high stability against proteolysis by human serum and bound to the target PDZ2 domain of postsynaptic density‐95 protein. An optimized sequence‐grafted cyclic SFTI‐1 could competitively suppress the interaction of PDZ2 with its natural ligand, the C‐terminal peptide of the NR2B subunit of the N‐methyl‐D‐aspartate receptor. These results show that a strategy combining peptide grafting into the SFTI‐1 scaffold with SrtA‐catalyzed cyclization can be a simple and effective method for producing stable peptide drugs. 相似文献
14.
Huang X Aulabaugh A Ding W Kapoor B Alksne L Tabei K Ellestad G 《Biochemistry》2003,42(38):11307-11315
Staphylococcus aureus sortase (SrtA) is a thiol transpeptidase. The enzyme catalyzes a cell wall sorting reaction in which a surface protein with a sorting signal containing a LPXTG motif is cleaved between the threonine and glycine residues. The resulting threonine carboxyl end of this protein is covalently attached to a pentaglycine cross-bridge of peptidoglycan. The transpeptidase activity of sortase has been demonstrated in in vitro reactions between a LPETG-containing peptide and triglycine. When a nucleophile is not available, sortase slowly hydrolyzes the LPETG peptide at the same site. In this study, we have analyzed the steady-state kinetics of these two types of reactions catalyzed by sortase. The kinetic results fully support a ping-pong mechanism in which a common acyl-enzyme intermediate is formed in transpeptidation and hydrolysis. However, each reaction has a distinct rate-limiting step: the formation of the acyl-enzyme in transpeptidation and the hydrolysis of the same acyl-enzyme in the hydrolysis reaction. We have also demonstrated in this study that the nucleophile binding site of S. aureus sortase SrtA is specific for diglycine. While S1' and S2' sites of the enzyme both prefer a glycine residue, the S1' site is exclusively selective for glycine. Lengthening of the polyglycine acceptor nucleophile beyond diglycine does not further enhance the binding and catalysis. 相似文献
15.
Nakata M Kimura KR Sumitomo T Wada S Sugauchi A Oiki E Higashino M Kreikemeyer B Podbielski A Okahashi N Hamada S Isoda R Terao Y Kawabata S 《The Journal of biological chemistry》2011,286(43):37566-37577
The human pathogen Streptococcus pyogenes produces diverse pili depending on the serotype. We investigated the assembly mechanism of FCT type 1 pili in a serotype M6 strain. The pili were found to be assembled from two precursor proteins, the backbone protein T6 and ancillary protein FctX, and anchored to the cell wall in a manner that requires both a housekeeping sortase enzyme (SrtA) and pilus-associated sortase enzyme (SrtB). SrtB is primarily required for efficient formation of the T6 and FctX complex and subsequent polymerization of T6, whereas proper anchoring of the pili to the cell wall is mainly mediated by SrtA. Because motifs essential for polymerization of pilus backbone proteins in other Gram-positive bacteria are not present in T6, we sought to identify the functional residues involved in this process. Our results showed that T6 encompasses the novel VAKS pilin motif conserved in streptococcal T6 homologues and that the lysine residue (Lys-175) within the motif and cell wall sorting signal of T6 are prerequisites for isopeptide linkage of T6 molecules. Because Lys-175 and the cell wall sorting signal of FctX are indispensable for substantial incorporation of FctX into the T6 pilus shaft, FctX is suggested to be located at the pilus tip, which was also implied by immunogold electron microscopy findings. Thus, the elaborate assembly of FCT type 1 pili is potentially organized by sortase-mediated cross-linking between sorting signals and the amino group of Lys-175 positioned in the VAKS motif of T6, thereby displaying T6 and FctX in a temporospatial manner. 相似文献
16.
17.
Bentley ML Gaweska H Kielec JM McCafferty DG 《The Journal of biological chemistry》2007,282(9):6571-6581
The Staphylococcus aureus transpeptidase Sortase A (SrtA) anchors virulence and colonization-associated surface proteins to the cell wall. SrtA selectively recognizes a C-terminal LPXTG motif, whereas the related transpeptidase Sortase B (SrtB) recognizes a C-terminal NPQTN motif. In both enzymes, cleavage occurs after the conserved threonine, followed by amide bond formation between threonine and the pentaglycine cross-bridge of cell wall peptidoglycan. Genetic and biochemical studies strongly suggest that SrtA and SrtB exhibit exquisite specificity for their recognition motifs. To better understand the origins of substrate specificity within these two isoforms, we used sequence and structural analysis to predict residues and domains likely to be involved in conferring substrate specificity. Mutational analyses and domain swapping experiments were conducted to test their function in substrate recognition and specificity. Marked changes in the specificity profile of SrtA were obtained by replacing the beta6/beta7 loop in SrtA with the corresponding domain from SrtB. The chimeric beta6/beta7 loop swap enzyme (SrtLS) conferred the ability to acylate NPQTN-containing substrates, with a k(cat)/K(m)(app) of 0.0062 +/- 0.003 m(-1) s(-1). This enzyme was unable to perform the transpeptidation stage of the reaction, suggesting that additional domains are required for transpeptidation to occur. The overall catalytic specificity profile (k(cat)/K(m)(app)(NPQTN)/k(cat)/K(m)(app)(LPETG)) of SrtLS was altered 700,000-fold from SrtA. These results indicate that the beta6/beta7 loop is an important site for substrate recognition in sortases. 相似文献
18.
Listeria monocytogenes is one of the most virulent foodborne pathogens. L. monocytogenes Sortase A (SrtA) enzyme, which catalyzes the cell wall anchoring reaction of the leucine, proline, X, threonine, and glycine proteins (LPXTG, where X is any amino acid), is a target for the development of antilisteriosis drugs. In this study, the structure of the L. monocytogenes SrtA enzyme-substrate complex was obtained using homology modeling, molecular docking and molecular dynamics simulations. Explicit enzyme-substrate interactions in the inactive and active forms of the enzyme were compared, based on 30 ns simulations on each system. The active site arginine (Arg 197) was found to be able change its hydrogen donor interactions from the LP backbone carbonyl groups of the LPXTG substrate in the inactive form, to the TG backbone carbonyls in the active form, which could be of importance for holding the substrate in position for the catalytic process. 相似文献
19.
Zong Y Bice TW Ton-That H Schneewind O Narayana SV 《The Journal of biological chemistry》2004,279(30):31383-31389
The cell wall envelope of staphylococci and other Gram-positive pathogens is coated with surface proteins that interact with human host tissues. Surface proteins of Staphylococcus aureus are covalently linked to the cell wall envelope by a mechanism requiring C-terminal sorting signals with an LPXTG motif. Sortase (SrtA) cleaves surface proteins between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between threonine at the C-terminal end of polypeptides and cell wall cross-bridges. The active site architecture and catalytic mechanism of sortase A has hitherto not been revealed. Here we present the crystal structures of native SrtA, of an active site mutant of SrtA, and of the mutant SrtA complexed with its substrate LPETG peptide and describe the substrate binding pocket of the enzyme. Highly conserved proline (P) and threonine (T) residues of the LPXTG motif are held in position by hydrophobic contacts, whereas the glutamic acid residue (E) at the X position points out into the solvent. The scissile T-G peptide bond is positioned between the active site Cys(184) and Arg(197) residues and at a greater distance from the imidazolium side chain of His(120). All three residues, His(120), Cys(184), and Arg(197), are conserved in sortase enzymes from Gram-positive bacteria. Comparison of the active sites of S. aureus sortase A and sortase B provides insight into substrate specificity and suggests a universal sortase-catalyzed mechanism of bacterial surface protein anchoring in Gram-positive bacteria. 相似文献
20.
The Klenow fragment (KF) has been used to make the blunt end as a tool enzyme. Its 5′-3′ polymerase activity can extend the 5′ overhanging sticky end to the blunt end, and 3′-5′ exonuclease activity can cleave the 3′ overhanging sticky end to the blunt end. The blunt end is useful for cloning. Here, we for the first time determined that a sticky end can be made by using the 3′-5′ exonuclease activity of KF. We found that KF can cleave the blunt end into certain sticky ends under controlled conditions. We optimized enzyme cleavage conditions, and characterized the cleaved sticky ends to be mainly 2 nt 5′ overhang. By using these sticky ends, we realized ligation reaction in vitro, and accomplished cloning short oligonucleotides directionally with high cloning efficiency. In some cases, this method can provide sticky end fragments in large scale for subsequent convenient cloning at low cost. 相似文献