Structural and Computational Studies of the Staphylococcus Aureus

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 13, pp. 8891–8902, March 28, 2014 © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. Structural and Computational Studies of the Staphylococcus aureus Sortase B-Substrate Complex Reveal a Substrate-stabilized Oxyanion Hole*□S Received for publication, August 10, 2013, and in revised form, January 29, 2014 Published, JBC Papers in Press, February 11, 2014, DOI 10.1074/jbc.M113.509273 Alex W. Jacobitz‡, Jeff Wereszczynski§, Sung Wook Yi‡, Brendan R. Amer‡, Grace L. Huang‡, Angelyn V. Nguyen‡, Michael R. Sawaya¶, Michael E. Jung‡, J. Andrew McCammon§, and Robert T. Clubb‡¶ʈ1 From the ‡Department of Chemistry and Biochemistry, ¶UCLA-DOE Institute of Genomics and Proteomics, and ʈMolecular Biology Institute, University of California, Los Angeles, Los Angeles, California 90095, and the §Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093 Background: Sortase enzymes catalyze a transpeptidation reaction that displays bacterial surface proteins. Results: Structural and computational studies reveal how the sortase B enzyme recognizes its sorting signal substrate. Downloaded from Conclusion: Sortase enzymes catalyze transpeptidation using a substrate-stabilized oxyanion hole. Significance: The results of this work could facilitate the rational design of sortase inhibitors. Sortase cysteine transpeptidases covalently attach proteins to Surface proteins in bacteria play key roles in the infection the bacterial cell wall or assemble fiber-like pili that promote process by promoting microbial adhesion to host tissues, nutri- http://www.jbc.org/ bacterial adhesion. Members of this enzyme superfamily are ent acquisition, host cell entry, and the suppression of the widely distributed in Gram-positive bacteria that frequently uti- immune response. In Gram-positive bacteria, virulence factors lize multiple sortases to elaborate their peptidoglycan. Sortases are displayed by sortase enzymes, a superfamily of cysteine catalyze transpeptidation using a conserved active site His-Cys- transpeptidases that join proteins bearing a cell wall sorting Arg triad that joins a sorting signal located at the C terminus of signal to the cell wall or to other proteins to construct pili (1–6). at UCLA-Louise Darling Biomed. Lib. on July 3, 2015 their protein substrate to an amino nucleophile located on Sortases have proven to be useful molecular biology tools to the cell surface. However, despite extensive study, the catalytic site-specifically attach proteins to a variety of biomolecules and mechanism and molecular basis of substrate recognition are considered a potential drug target because they display remains poorly understood. Here we report the crystal structure virulence factors. The clinically important pathogen, Staphylo- of the Staphylococcus aureus sortase B enzyme in a covalent coccus aureus, displays surface proteins using two sortase complex with an analog of its NPQTN sorting signal substrate, enzymes, sortase A (SrtA)2 and sortase B (SrtB). srtAϪ strains revealing the structural basis through which it displays the IsdC of S. aureus are significantly attenuated in virulence, whereas protein involved in heme-iron scavenging from human hemo- srtBϪ strains establish less persistent infections (7–9). SrtA globin. The results of computational modeling, molecular plays a “housekeeping” role in the cell, covalently mounting a dynamics simulations, and targeted amino acid mutagenesis variety of proteins to the cell wall, whereas SrtB anchors the indicate that the backbone amide of Glu224 and the side chain of heme transporter IsdC, a key component of the iron-regulated Arg233 form an oxyanion hole in sortase B that stabilizes surface determinant system that captures heme-iron from high energy tetrahedral catalytic intermediates. Surprisingly, a hemoglobin (10–12). The mechanism of catalysis is best under- highly conserved threonine residue within the bound sorting stood for SrtA, the archetypal member of the sortase superfam- signal substrate facilitates construction of the oxyanion hole by ily. Proteins anchored by SrtA possess a C-terminal cell wall stabilizing the position of the active site arginine residue via sorting signal that consists of an LPXTG motif that is processed hydrogen bonding. Molecular dynamics simulations and pri- by the enzyme, followed by a hydrophobic transmembrane seg- mary sequence conservation suggest that the sorting signal-sta- ment and a positively charged C-terminal tail (13). SrtA oper- bilized oxyanion hole is a universal feature of enzymes within ates through a ping-pong mechanism that begins when its the sortase superfamily. active site cysteine residue nucleophilically attacks the carbonyl carbon of the threonine in the LPXTG motif. This results in a tetrahedral intermediate that, after cleavage of the threonine- glycine peptide bond, generates a semistable enzyme-substrate * This work was supported, in whole or in part, by National Institutes of Health Grants AI52217 (to R. T. C. and M. E. J.), GM31749 (to J. A. M.), and T32 thioacyl intermediate (14, 15). The protein is then transferred GM007185 (to A. W. J. and G. L. H.). This work was also supported by the by SrtA to the cell wall precursor, lipid II, when the amino National Science Foundation, the Howard Hughes Medical Institute, the group in this molecule nucleophilically attacks the thioacyl Center for Theoretical Biological Physics, the National Biomedical Compu- tation Resource, and the National Science Foundation Supercomputer linkage creating a second tetrahedral intermediate that col- Centers. lapses to form the covalently linked protein-lipid II product The atomic coordinates and structure factors (code 4LFD) have been deposited in (16–18). Transglycosylation and transpeptidation reactions the Protein Data Bank (http://wwpdb.org/). □S This article contains supplemental Video S1. 1 To whom correspondence should be addressed. Tel.: 310-206-2334; Fax: 2 The abbreviations used are: SrtA, S. aureus sortase A; SrtB, S. aureus sortase B; 310-206-4749; E-mail: [email protected]. MD, molecular dynamics. MARCH 28, 2014•VOLUME 289•NUMBER 13 JOURNAL OF BIOLOGICAL CHEMISTRY 8891 Structural and Computational Studies of SrtB-NPQT Complex that synthesize the cell wall then incorporate this product into into a pE-SUMO vector (LifeSensors) and transformed into the peptidoglycan. SrtB anchors the IsdC protein to the cell wall Escherichia coli Rosetta (DE3) pLysS cells (Novagen). Protein through a similar mechanism. However, unlike SrtA, SrtB rec- expression was induced by addition of 1 mM isopropyl ␤-D-1- ognizes a unique NPQTN sorting signal, and it attaches IsdC to thiogalactopyranoside and allowed to continue for 16 h at un-cross-linked peptidoglycan instead of heavily cross-linked 16 °C. Protein was purified by affinity purification using HisPur peptidoglycan (19, 20). cobalt resin (Thermo) per the manufacturer’s instructions. ␣ ␤ Sortase enzymes adopt an / sortase fold that contains The His6-SUMO tag was then cleaved by incubating the protein three proximally positioned active site residues: His130, Cys223, overnight at 4 °C with recombinant ULP1 protease and and Arg233 (SrtB numbering). Although the nucleophilic role of removed by reapplying the protein mixture to the HisPur cobalt the cysteine residue is well established, various catalytic func- resin. Cbz-NPQT* (where T* is (2R,3S)-3-amino-4-mercapto- tions have been proposed for the histidine and arginine resi- 2-butanol, and Cbz is a carbobenzyloxy protecting group) was dues. His130 was originally thought to activate the cysteine by synthesized as in Ref. 34 and added to purified SrtB in modifi- forming a histidine-cysteine ion pair (21), but more recent data cation buffer (10 mM Tris-HCl, pH 7.0, 20 mM NaCl, 1 ML-pro- suggest that it instead functions as a general acid/base (14, 22). line) at a ratio of 10:1 for a final concentration of 1 mM Cbz- 233 Arg has been proposed to either stabilize substrate binding NPQT* to 100 ␮M SrtB. The reaction was first reduced with 1 (23–26), function as a general base (27), or directly stabilize the mM DTT for 4 h, then oxidized by addition of 10 mM CuCl2, Downloaded from tetrahedral catalytic intermediates (14, 22, 23). Other residues and allowed to rock gently at room temperature for 7 days. within the active sites of sortases have also been proposed to Production of stable complex was confirmed by MALDI mass participate in catalysis, including Asp225 in SrtB, which was spectrometry. postulated to participate in a His130-Cys223-Asp225 catalytic Crystals of the SrtB-NPQT* complex were produced from a triad (28). The catalytic mechanism has remained poorly stock of 150 ␮M SrtB-NPQT* in 10 mM Tris-HCl, pH 7.0, 20 mM http://www.jbc.org/ understood because the tetrahedral and acyl intermediates of NaCl. Crystals were grown using the hanging drop, vapor dif- catalysis are too short lived to be characterized by either NMR fusion method in 2.8 M ammonium sulfate, 70 mM sodium cit- or x-ray crystallography. Several sortase structures have been rate, pH 5.0. Data were collected on Beamline 24-ID-C at 100 K determined in the absence of their substrates (28–32) or cova- at the Advanced Photon Source (␭ ϭ 0.964 Å). Three data sets lently bound to generic sulfhydryl modifiers (33). However, were scaled, integrated, and merged using XDS and XSCALE at UCLA-Louise Darling Biomed. Lib. on July 3, 2015 only a single structure of a sortase

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