Localization and mutagenesis of the sorting signal binding site on sortase A from <i>Staphylococcus aureus</i>

Liew, Chu Kong; Smith, Brenton T.; Pilpa, Rosemarie; Suree, Nuttee; Ilangovan, Udayar; Connolly, Kevin M.; Jung, Michael E.; Clubb, Robert T.

doi:10.1016/j.febslet.2004.06.070

Cited by 48 publications

(61 citation statements)

References 32 publications

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“…Previously, we assigned the backbone chemical shifts of the SrtA ⌬N59 protein in the complex (29). In this report, to solve the structure of the complex, we assigned nearly all of the 1 H, 13 C, and 15 N chemical shifts of the protein and the 1 H chemical shifts of the bound peptide.…”

Section: Resultsmentioning

confidence: 99%

“…Wild-type SrtA from S. aureus containing amino acid residues 60 -206 (SrtA ⌬N59 ) was produced as described previously (29). Uniformly 15 N-and 13 C-or 15 N-labeled SrtA ⌬N59 protein was covalently attached to an analog of the LPXTG sorting signal, Cbz-LPAT* (where T* is (2R,3S)-3-amino-4-mercapto-2-butanol, and Cbz is a carbobenzyloxy protecting group).…”

Section: Preparation Of the Covalent Complex For Nmr Studies-mentioning

confidence: 99%

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The Structure of the Staphylococcus aureus Sortase-Substrate Complex Reveals How the Universally Conserved LPXTG Sorting Signal Is Recognized

Suree¹,

Liew²,

Villareal³

et al. 2009

Journal of Biological Chemistry

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181

369

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In Gram-positive bacteria, sortase enzymes assemble surface proteins and pili in the cell wall envelope. Sortases catalyze a transpeptidation reaction that joins a highly conserved LPXTG sorting signal within their polypeptide substrate to the cell wall or to other pilin subunits. The molecular basis of transpeptidation and sorting signal recognition are not well understood, because the intermediates of catalysis are short lived. We have overcome this problem by synthesizing an analog of the LPXTG signal whose stable covalent complex with the enzyme mimics a key thioacyl catalytic intermediate. Here we report the solution structure and dynamics of its covalent complex with the Staphylococcus aureus SrtA sortase. In marked contrast to a previously reported crystal structure, we show that SrtA adaptively recognizes the LPXTG sorting signal by closing and immobilizing an active site loop. We have also used chemical shift mapping experiments to localize the binding site for the triglycine portion of lipid II, the second substrate to which surface proteins are attached. We propose a unified model of the transpeptidation reaction that explains the functions of key active site residues. Since the sortase-catalyzed anchoring reaction is required for the virulence of a number of bacterial pathogens, the results presented here may facilitate the development of new anti-infective agents.Bacterial surface proteins function as virulence factors that enable pathogens to adhere to sites of infection, evade the immune response, acquire essential nutrients, and enter host cells (1). Gram-positive bacteria use a common mechanism to covalently attach proteins to the cell wall. This process is catalyzed by sortase transpeptidase enzymes, which join proteins bearing a highly conserved Leu-Pro-X-Thr-Gly (LPXTG, where X is any amino acid) sorting signal to the cross-bridge peptide of the peptidylglycan (2-4). Sortases also polymerize proteins containing sorting signals into pili, filamentous surface exposed structures that promote bacterial adhesion (5, 6). The search for small molecule sortase inhibitors is an active area of research, since these enzymes contribute to the virulence of a number of important pathogens, including among others Staphylococcus aureus, Listeria monocytogenes, Streptococcus pyogenes, and Streptococcus pneumoniae (reviewed in Refs. 7 and 8). Sortase enzymes are also promising molecular biology reagents that can be used to site-specifically attach proteins to a variety of biomolecules (9 -14, 72).The sortase A (SrtA) 7 enzyme from S. aureus is the prototypical member of the sortase enzyme family (15, 16). It anchors proteins to the murein sacculus that possess a COOH-terminal cell wall sorting signal that consists of a LPXTG motif, followed by a hydrophobic segment of amino acids and a tail composed of mostly positively charged residues (17). SrtA is located on the extracellular side of the membrane. After partial secretion of its protein substrate across the cell membrane, SrtA cleaves the LPXTG motif between...

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Section: Resultsmentioning

confidence: 99%

Section: Preparation Of the Covalent Complex For Nmr Studies-mentioning

confidence: 99%

The Structure of the Staphylococcus aureus Sortase-Substrate Complex Reveals How the Universally Conserved LPXTG Sorting Signal Is Recognized

Suree¹,

Liew²,

Villareal³

et al. 2009

Journal of Biological Chemistry

Self Cite

181

369

View full text Add to dashboard Cite

show abstract

“…This cation, present in millimolar amounts in host tissues, activates sortase activity eightfold (84). Analysis of the sortase NMR spectra in the presence and absence of calcium revealed that Glu 105 , Glu 108 , and Asp 108 side chains of the ␤3-␤4 loop interact with the cation. In contrast, the ␤6-␤7 loop forms a flap that is disordered in the absence of calcium (131,227).…”

Section: Sortase a Structurementioning

confidence: 99%

“…Leucine and proline residues of the LPETG peptide are bound in the C-terminal region of ␤7, surrounded by several highly hydrophobic residues (228). NMR analysis of the 1 H-15 N chemical shifts of sortase in the presence or absence of ligand allowed identification of residues that comprise the LPXTG binding surface (108). Residues perturbed after ligand binding also mapped to the C-terminal region of the ␤7 strand (Thr 180 and Ile 182 ) and to the vicinity of the loop connecting strands ␤3 and ␤4 (Ala 118 ).…”

Section: Sortase a Structurementioning

confidence: 99%

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Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria

Marraffini

DeDent²,

Schneewind³

2006

Microbiol Mol Biol Rev

608

600

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SUMMARY The cell wall envelopes of gram-positive bacteria represent a surface organelle that not only functions as a cytoskeletal element but also promotes interactions between bacteria and their environment. Cell wall peptidoglycan is covalently and noncovalently decorated with teichoic acids, polysaccharides, and proteins. The sum of these molecular decorations provides bacterial envelopes with species- and strain-specific properties that are ultimately responsible for bacterial virulence, interactions with host immune systems, and the development of disease symptoms or successful outcomes of infections. Surface proteins typically carry two topogenic sequences, i.e., N-terminal signal peptides and C-terminal sorting signals. Sortases catalyze a transpeptidation reaction by first cleaving a surface protein substrate at the cell wall sorting signal. The resulting acyl enzyme intermediates between sortases and their substrates are then resolved by the nucleophilic attack of amino groups, typically provided by the cell wall cross bridges of peptidoglycan precursors. The surface protein linked to peptidoglycan is then incorporated into the envelope and displayed on the microbial surface. This review focuses on the mechanisms of surface protein anchoring to the cell wall envelope by sortases and the role that these enzymes play in bacterial physiology and pathogenesis.

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Bilden und Brechen von Peptidbindungen: Protein‐Engineering mithilfe von Sortase

Popp

Ploegh

2011

Angewandte Chemie

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Sortasen gehören zu einer Klasse bakterieller Enzyme, die Transpeptidaseaktivität aufweisen. Die Fähigkeit, Peptidbindungen ortsspezifisch zu brechen und neue Bindungen mit einem angreifenden Nukleophil zu bilden, macht Sortase zu einem hervorragenden Hilfsmittel für das Protein‐Engineering. Diese Methode wurde für eine Reihe von Anwendungen angepasst, die von der Chemie über die Zellbiologie bis hin zu technischen Gebieten reichen. Wir wollen hier einen kurzen Überblick über die Biologie der Sortaseenzyme und ihre Anwendungen im Protein‐Engineering geben, auf Gebiete für zukünftige Innovationen hinweisen und neue Anwendungen vorschlagen.

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Localization and mutagenesis of the sorting signal binding site on sortase A from Staphylococcus aureus

Cited by 48 publications

References 32 publications

The Structure of the Staphylococcus aureus Sortase-Substrate Complex Reveals How the Universally Conserved LPXTG Sorting Signal Is Recognized

The Structure of the Staphylococcus aureus Sortase-Substrate Complex Reveals How the Universally Conserved LPXTG Sorting Signal Is Recognized

Sortases and the Art of Anchoring Proteins to the Envelopes of Gram-Positive Bacteria

Bilden und Brechen von Peptidbindungen: Protein‐Engineering mithilfe von Sortase

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