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J. Biol. Chem., Vol. 282, Issue 9, 6571-6581, March 2, 2007

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Engineering the Substrate Specificity of Staphylococcus aureus Sortase A

THE 6/7 LOOP FROM SrtB CONFERS NPQTN RECOGNITION TO SrtA^*

Matthew L. Bentley, Helena Gaweska, Joseph M. Kielec, and Dewey G. McCafferty¹

From the Department of Biochemistry and Biophysics and the Johnson Research Foundation, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and the Department of Chemistry, Duke University, Durham, North Carolina 27708

Received for publication, November 13, 2006 , and in revised form, December 21, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
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 6/7 loop in SrtA with the corresponding domain from SrtB. The chimeric 6/7 loop swap enzyme (SrtLS) conferred the ability to acylate NPQTN-containing substrates, with a 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 () of SrtLS was altered 700,000-fold from SrtA. These results indicate that the 6/7 loop is an important site for substrate recognition in sortases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Recently, much attention has been focused on the role of surface protein virulence factors in the pathogenesis of Gram-positive bacteria (1–3). These covalently anchored proteins mediate such diverse processes as adherence to and invasion of host endothelial tissues, immune system evasion, and iron acquisition (4, 5). Inhibition of surface protein anchoring has thus emerged as a possible new therapeutic strategy for treatment of Gram-positive infections (6).

Sortases are a family of transpeptidases that catalyze the covalent attachment of these surface proteins to the peptidoglycan layer of the Gram-positive cell wall (4). Several lines of evidence support a role for sortases in the pathogenicity of infections. Strains of Staphylococcus aureus in which srtA was genetically deleted are unable to anchor surface proteins in vivo and show a reduced ability to form renal abscesses in mice (7). The srtA knock-out in Listeria monocytogenes has been shown to be less invasive in vitro, fails to anchor internalin to the cell wall in vivo, and is reduced in its ability to colonize the liver and spleen in a mouse model of infection (8, 9). In Streptococcus gordonii, srtA inactivation leads to decreased surface protein display, reduced fibronectin binding in vitro, and a reduced ability to colonize the oral mucosa of mice (10). Likewise, srtA knockouts from Streptococcus family members S. mutans (11), S. pneumonia (12), S. pyogenes (13), S. suis (14), S. sanguinis (15), and S. agalactiae (16) have been shown to have attenuated pathogenicity.

Most Gram-positive bacterial species whose genomes have been sequenced to date contain at least one sortase homolog, and many species contain multiple sortase genes (17). In many cases, putative sortase genes are located within operons that contain genes encoding potential sortase substrates (proteins with C-terminal LPXTG motifs). This raises the possibility that many of these additional sortase genes are co-expressed along with their substrates, suggesting that they might play discrete functional roles by anchoring different subsets of surface proteins (17).

Within the genome of S. aureus, there are two sortase isoforms, termed Sortase A (SrtA) and Sortase B (SrtB). SrtA is responsible for anchoring proteins containing a C-terminal tripartite sorting signal, which consists of 1) an LPXTG pentapeptide followed by 2) a less well conserved hydrophobic domain and 3) a basic charged tail (18, 19). Both the hydrophobic domain and the charged tail help to retain the putative surface protein in the membrane prior to sortase-catalyzed anchoring. SrtA cleaves the LPXTG sequence between the threonine and glycine residues by use of a nucleophilic active site cysteine (Cys¹⁸⁴) (20). An acyl-enzyme intermediate is formed, which is then resolved by nucleophilic attack from an amino group on the Gly₅ cross-bridge of branched Lipid II. This leads to covalent attachment of the N-terminal portion of the surface protein to Lipid II, from which it is incorporated into the bacterial cell wall (21).

SrtB functions analogously to SrtA with two key differences. First, SrtB has a specificity profile different from that of SrtA. It selectively anchors the NPQTN-containing protein IsdC, and it does not anchor LPXTG-containing proteins (22). Second, the anchor structure for SrtB is not Lipid II (a peptidoglycan biosynthetic precursor) but is the Gly₅ cross-bridge of mature peptidoglycan from the staphylococcal cell wall (23). In addition, the gene for SrtB is located along with its substrate in an iron-responsive operon, which is only transcribed under conditions of iron starvation (22). Since the acquisition of iron is an essential process for bacterial colonization of mammalian host tissues (24), it is likely that SrtB carries out an essential function for virulence by anchoring a protein with a specific role in iron acquisition to a unique anchor motif in the cell wall (25). Although SrtA and SrtB are highly specific for their unique recognition motifs, to date little is known about how these enzymes discriminate between their substrates. Herein we report the application of mutagenesis, domain swapping, and kinetic analysis to identify specificity-determining regions of SrtA and SrtB.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Materials—Buffer salts were purchased from Sigma. Standard Fmoc² amino acids (Novabiochem), Fmoc-Dap-(DNP)-OH (Bachem), NH₂-Gly₅-OH (Bachem), and t-butoxycarbonyl-Abz-OH (Bachem) were purchased and were used with no further purification. Oligonucleotide primers were purchased from Integrated DNA Technologies (Coralville, IA) and used without further purification. Protein purification was performed on an AKTA fast protein liquid chromatography system with a fraction collector (Amersham Biosciences). Chelating Sepharose fast flow chromatography resin (Amersham Biosciences) and a HiPrep 26/60 Sephacryl S-200 high resolution gel filtration column (Amersham Biosciences) were used according to the manufacturer's recommendations. HPLC was performed using an Agilent 1200 series system equipped with an autosampler, fraction collector, diode array UV detector, fluorimeter, and either a semipreparative Jupiter^TM octadecyl silica column (Phenomenex) or a fast analytical (4.6 x 50 mm, 3 µm) octadecyl silica column (Vydac). MALDI-MS was performed on a Voyager^TM MALDI-time-of-flight mass spectrometer (Applied Biosystems). Electrospray ionization mass spectrometry (ESI-MS) was performed on an Agilent 1100 series LC/MSD ion trap electrospray mass spectrometer.

Expression and Purification of SrtA_N24—N-terminally His₆-tagged SrtA_N24 was expressed in Escherichia coli BL21(DE3) cells containing the plasmid pET15bSrtA_N24 (26). Cells were grown in LB medium containing 100 µg/ml ampicillin at 37 °C to an A₆₀₀ of 0.6. 1 mM isopropyl 1-thio--D-galactopyranoside was added to induce expression of SrtA_N24, and cells were harvested by centrifugation after 3 h. Cells were resuspended in 150 mM NaCl, 50 mM Tris-Cl, 5 mM imidazole, 10% glycerol, pH 7.5, and lysed with an EmulsiFlex-C5 high pressure homogenizer (Avestin, Inc.). Lysate was clarified by centrifugation and then applied to a chelating Sepharose fast flow column. The column was washed with 50 mM imidazole for 30 min, SrtA_N24 was eluted using a linear gradient of 50–500 mM imidazole over 1 h, and fractions were collected. SrtA-containing fractions were pooled, concentrated, and dialyzed overnight into 150 mM NaCl, 50 mM Tris-Cl, 5 mM CaCl₂, 0.1% -mercaptoethanol, 10% glycerol, pH 7.5. Purified SrtA_N24 was concentrated to 683 µM using 10,000 molecular weight cut-off Centriplus centrifugal filters (Amicon, Inc.). SrtA_N24 concentration was determined using a calculated extinction coefficient (₂₈₀ = 17,420 M^-1 cm^-1). Protein molecular weight was confirmed by MALDI-MS.

Mutagenesis of SrtA—The plasmid pET15bSrtA_N24 was used as a template for introduction of single amino acid substitutions by PCR, using the QuikChange® method (Stratagene, Inc.). All PCRs contained 5% Me₂SO. The mutant I182S was amplified using the primers 5'-CAATTAACATTAAGTACTTGTGATGATTAC-3' and 5'-GTAATCATCACAAGTACTTAATGTTAATTG-3'. Mutant A118Y was amplified using primers 5'-CAAAATATTTCAATTTATGGACACACTTTC-3' and 5'-GAAAGTGTGTCCATAAATTGAAATATTTTG-3'. All mutations were confirmed by DNA sequencing.

Cloning, Expression, and Purification of SrtB_N21—SrtB₂₁ was amplified from the genomic DNA of S. aureus strain N315, using the primers SrtB₂₁fwd (5'-CCCGAATTCCATATGGGTTACAAAATTGTTCAAACATATATT-3') and SrtB₂₁rev (5'-CATTAGCGTGGATCCCTCGAGTTAACTTACCTTAATTATTTTTGC-3'). The resulting DNA fragment was doubly digested with EcoRI and BamHI and ligated with T4 DNA ligase into the subcloning vector pUC19, which had previously been digested with EcoRI and BamHI, to generate the plasmid pUC19SrtB_N21. This plasmid was transformed into DH5 cells and isolated with a QIAprep^TM spin miniprep kit, and the desired sequence was confirmed by DNA sequencing. BamHI and NdeI were used to liberate the gene from the plasmid, and it was then cloned into a similarly treated pET15b expression vector to generate the plasmid pET15bSrtB_N21. This construct was transformed into BL21(DE3) cells, and cells were grown in LB medium with 100 µg/ml ampicillin. Cells were grown to an A₆₀₀ of 0.7, and then 1 mM isopropyl 1-thio--D-galactopyranoside was added to induce expression of SrtB_N21; cells were grown overnight at 25 °C and then harvested by centrifugation. Cells were resuspended in the same buffer, and protein was purified in the same manner as for SrtA_N24. Purified SrtB_N21 was concentrated to 948 µM using 10,000 molecular weight cut-off Centriplus centrifugal filters (Amicon). SrtB_N21 concentration was determined spectrophotometrically, using the extinction coefficient ₂₈₀ = 26,360 M^-1 cm^-1. Protein molecular weight was confirmed by MALDI-MS.

Sequence and MEME Analysis of Sortase Family Members—The sequences of S. aureus SrtA and SrtB were used as starting points for a bioinformatic analysis. PSI-BLAST (NCBI) (27) searches were performed on each to identify similar sequences. Five iterations of PSI-BLAST identified 214 similar sequences to SrtA, whereas two iterations of PSI-BLAST produced 42 sequences with similarity to SrtB. Combining these and removing all proteins with E values of greater than 3.0 x 10^-5 left 175 hits. Further analysis reduced this number to 135 sequences that showed conservative identities with either SrtA or SrtB. ClustalW (EMBL-EBI) (28) was used to generate a large scale multiple sequence alignment of these 135 sequences. T-Coffee (EMBL-EBI) (29) was used to generate an alignment of a smaller subset of 25 sequences (supplemental Fig. S1).

Motifs were discovered using MEME (SDSC) (30). MEME analysis was performed on a small scale alignment of 25 sequences (17 SrtA homologs, 8 SrtB homologs). Discovered motifs were mapped onto a T-Coffee multiple sequence alignment using GeneDoc (31) and then onto the existing structures of SrtA (Protein Data Bank code 1T2W) and SrtB (Protein Data Bank code 1NG5 [PDB] ) (Fig. 1). In light of previous sortase activity studies (26), this sequence-structural analysis helped identify putative sequence motifs and residues likely to be involved in substrate binding, recognition, and catalysis.

Generation of a Chimeric 6/7 Loop Swap (SrtLS)—To generate the novel sequence of SrtLS_N24, the technique of splicing by overlap extension was used (32). The plasmids pET15bSrtA_N24 (26) and pET15bSrtB_N21 were used as starting templates for PCR. Two rounds of this technique were needed to generate SrtLS_N24, which contains the sequence of SrtA_N24 from the 5' end to the 6/7 loop, the sequence of SrtB_N21 for the loop, and then the sequence of SrtA_N24 from the loop end to the 3' end of the gene. Splicing by overlap extension-PCR was performed according to the method of Horton et al. (32). Reaction products were gel-purified following each successful amplification and used as templates for the next rounds of PCR. The following primers were used (underlined sequences are those derived from srtB): Primer a, 5'-CCCGCGAAATTAATACGACTCACTATAGGG-3'; Primer b, 5'-CTTTAGTAGTAGTTTTATCTCTTATACTTG-3'; Primer c, 5'-CAAGTATAAGAGATAAAACTACTACTAAAG-3'; Primer d/g, 5'-CCGTTTAGAGGCCCCAAGGGGTTATGC-3'; Primer e, 5'-GTAATTAATGTTAATTGTTTATCTTTTACCG-3'; Primer f, 5'-CGGTAAAAGATAAACAATTAACATTAATTAC-3'.

The final PCR product was digested with NdeI and BamHI, ligated into pET15b vector that had previously been digested with NdeI and BamHI, and then transformed into DH5 cells. DNA sequencing confirmed generation of the vector pET15bSrtLS_N24.

Expression and Purification of SrtLS_N24—pET15bSrtLS_N24 (26) was expressed in BL21(DE3) cells, which were grown in LB medium with 100 µg/ml ampicillin. Cells were grown to an A₆₀₀ of 0.6, induced with 0.1 mM isopropyl 1-thio--D-galactopyranoside, and then grown overnight at 25 °C. Cells were pelleted by centrifugation at 5000 x g for 10 min, resuspended in 150 mM NaCl, 50 mM Tris-Cl, 5 mM imidazole, 10% glycerol, pH 7.5, and lysed with an EmulsiFlex-C5 high pressure homogenizer (Avestin, Inc.). The lysate was then clarified by centrifugation and applied to a chelating Sepharose fast flow column. The column was washed with 5 mM imidazole, and SrtLS_N24 was eluted using a linear gradient of 5–500 mM imidazole. SrtLS_N24-containing fractions were pooled, concentrated, and loaded onto a HiPrep 26/60 Sephacryl S-200 gel filtration column (previously equilibrated with 150 mM NaCl, 50 mM Tris (pH 7.5), 0.1% -mercaptoethanol, 10% glycerol). SrtLS_N24-containing fractions were collected and concentrated to 490 µM using 10,000 molecular weight cut-off Centriplus centrifugal filters (Amicon). SrtLS_N24 concentration was determined spectrophotometrically, using the extinction coefficient ₂₈₀ = 18,490 M^-1 cm^-1.

Mutagenesis of SrtLS—Single point mutations A118Y and I208S and the double mutant A118Y/I208S were introduced into the plasmid pET15bSrtLS_N24 by PCR, using the same methods and primers used for mutagenesis of SrtA. Mutations were confirmed by DNA sequencing.

Circular Dichroism Spectroscopy—Aliquots (300 µl) of SrtA_N24, SrtB_N21, and SrtLS_N24 were dialyzed overnight at 4 °C against buffer containing 10 mM Tris (pH 7.5), 30 mM NaCl, and 5 mM CaCl₂. Samples were diluted in the same buffer until final protein concentrations were 10 µM. Protein concentrations of the final solutions were obtained spectrophotometrically (SrtA_{N24 280} = 17,420 M^-1 cm^-1, SrtB_{N21 280} = 26,360 M^-1 cm^-1, SrtLS_{N24 280} = 18,490 M^-1 cm^-1). For each protein, 400 µl was transferred to a quartz cuvette with a 0.1-cm path length for CD data collection. CD spectra were measured on an Aviv Instruments model 202 circular dichroism spectrometer. Data were collected at 25 °C, from 260 to 190 nm in 1-nm steps, with a scanning speed of 4 s/nm. Three scans were taken for each sample and were averaged and corrected for background absorbance. The observed CD signal (millidegrees) was converted to mean residue ellipticity (_m, degrees cm² dmol^-1 residue^-1) using Equation 1,

(Eq. 1)

where _obs represents the observed ellipticity in degrees, c is the protein concentration (M), d is the optical path length in cm, and n is the number of amino acid residues in the protein (202 for SrtA_N24, 244 for SrtB_N21, and 228 for SrtLS_N24).

Solid Phase Synthesis of Peptide Substrates—Substrates Abz-LPETG-Dap(DNP)-NH₂ and Abz-KVENPQTNAGT-Dap-(DNP)-NH₂ were synthesized by the Fmoc/piperidine strategy on PAL resin on a 0.25-mmol scale using an Applied Biosystems 433A synthesizer. Peptides were cleaved from the resin via incubation with a 95:2.5:2.5 trifluoroacetic acid/water/triisopropylsilane mixture for 2.5 h. The peptides were precipitated using cold diethyl ether following the removal of excess trifluoroacetic acid via rotary evaporation. After filtration, the precipitate was dissolved in a 50:50 water/acetonitrile mixture and lyophilized to yield crude peptide. A semipreparative C₁₈ Jupiter^TM column was used to purify the peptides to 98% purity via reverse phase HPLC. MALDI-time-of-flight MS was used to verify the identities of the purified products (Abz-LPETG-Dap-(DNP)-NH₂, m/z = 885.3; Abz-KVENPQTNAGT-Dap(DNP)-NH₂, m/z = 1527.7).

HPLC Assays for Sortase Activity—The assay procedure of Kruger et al. (33) for SrtA was employed to measure SrtB activity, with the modifications indicated (see Fig. 3). For SrtB, initial activity assays were performed with varying concentrations of enzyme (1, 10, 50, or 94.8 µM) and the peptide substrates Abz-KVENPQTNAGT-Dap(DNP)-NH₂ (2.4 mM) and NH₂-Gly₅-OH (2 mM). Reactions were performed in a 100-µl volume in standard assay buffer (150 mM NaCl, 5 mM CaCl₂, 300 mM Tris-Cl, pH 7.5) at 37 °C, and the enzyme volume added to each assay was kept under one-tenth of the final assay volume. Assays were initiated by the addition of the enzyme; were incubated for 0.5, 1, 2, or 4 h; and were quenched by the addition of 50 µl of 1.2 N HCl. 50 µl of the quenched reaction mixture was then injected onto a Vydac reverse phase fast analytical C₁₈ column (4 ml/min) and separated using a linear gradient from 0 to 45% CH₃CN, 0.1% trifluoroacetic acid over 5 min. Peaks corresponding to elution of the substrate (Abz-KVENPQTNAGT-Dap(DNP)-NH₂) and product (NH₂-G-Dap(DNP)-NH₂) were monitored at 355 nm. At the same time, peaks corresponding to elution of the substrate and the transpeptidation product (Abz-KVENPQTGGGGG-OH) were monitored by fluorescence, with excitation at 318 nm and emission at 420 nm. Integration of the areas of the substrate and product peaks at 355 nm allowed for determination of the percentage of substrate converted to product. Linearity of SrtB was confirmed by plotting product formation as a function of time over the 4-h assay period.

For determination of the kinetic parameters of SrtB, 94.8 µM enzyme was used in a 3-h assay, since these conditions were expected to give product conversions of 1–5% during the assay. The substrate Abz-KVENPQTNAGT-Dap(DNP)-NH₂ was varied from 74.8 µM to 21.9 mM, whereas NH₂-Gly₅-OH was held constant at 2 mM. GraFit version 4.0 (Erithacus Software) was used to fit the raw data to the Michaelis-Menten equation, producing estimates of the kinetic parameters k_cat and K_m.

Initial assays for comparison of SrtA, SrtB, and SrtLS activity with Abz-KVENPQTNAGT-Dap(DNP)-NH₂ were conducted using a 94.8 µM concentration of each enzyme and 2.7 mM substrate. Assays were incubated for 3, 6, 9, and 12 h, and the amount of product was quantitated as described above for SrtB. Estimates of the second order parameter were obtained using the relation,

(Eq. 2)

where v_o is the initial velocity, [E] is the concentration of enzyme, and [S] is the concentration of substrate. Initial activity assays for each enzyme with Abz-LPETG-Dap(DNP)-NH₂ were performed and analyzed similarly.

Mass Spectrometry of Reaction Products—To confirm the identity of reaction products, individual product peaks from the HPLC assay were collected with a fraction collector and concentrated using a speed vacuum system. Dried, concentrated products were resuspended in 100 µl of 50% CH₃CN in H₂O and injected onto an Agilent 1100 series LC/MSD ion trap electrospray mass spectrometer. MS data were collected in positive ion mode, and ions corresponding to putative reaction products were selected, fractionated, and analyzed by MS/MS.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Despite carrying out similar biological functions, SrtA and SrtB have different specificity profiles. SrtA anchors LPXTG-containing proteins, whereas SrtB is specific for NPQTN-containing proteins (22, 26). In order to better understand the basis for this specific recognition, we used sequence analysis to look at SrtA and SrtB isoforms from various Gram-positive bacteria. We looked for sequence positions that were highly conserved among SrtA and SrtB sequences but were isoform-specific. We then performed global analysis of the structures of S. aureus SrtA (Protein Data Bank code 1T2W) and SrtB (Protein Data Bank code 1NG5 [PDB] ) to identify larger domains that differed structurally between the two enzymes (34, 35). Mutagenesis and domain-swapping were then used to explore the effect of each identified region on SrtA specificity.

Bioinformatic and MEME Analysis of Sortase A and B Isoforms—A large scale multiple sequence alignment of 135 sequences of Sortase A and B isoforms from different Gram-positive species was performed using the program ClustalW. Analysis of this alignment highlighted the presence of several conserved residues and domains. The active site cysteine (Cys¹⁸⁴ in SrtA) was conserved among all sortase homologs, as were the nearby histidine and arginine residues (His¹²⁰ and Arg¹⁹⁷ in SrtA). These three conserved residues are located within two conserved domains along the 4, 7, and 8 strands, which make up the sortase active site.

MEME analysis of a smaller alignment of 25 sequences revealed the presence of seven conserved motifs (Table 1). Four of these motifs were common to both SrtA and SrtB isoforms. When these motifs were mapped onto the existing crystal structures of SrtA and SrtB from S. aureus, they corresponded to distinct structural features. Motifs 1 and 3 corresponded to the 4 and 7 strands, respectively, which make up the floor of the sortase active site. Motif 2 maps onto the 1 and 2 strands of both proteins, where it forms part of the underlying structure. The N terminus of both proteins is membrane-associated, and this sequence region also corresponds to a conserved motif (Motif 5).

View this table: [in this window] [in a new window]   TABLE 1 MEME-discovered motifs in SrtA and SrtB sequences MEME was used to search for conserved motifs in 25 sequences of SrtA and SrtB isoforms from Gram-positive bacteria, as described under "Experimental Procedures." Four motifs common to both SrtA and SrtB were observed (motifs 1, 2, 3, and 5). Each of these maps onto conserved structural elements. Motifs 1 and 3 correspondtothe 4 and 7 strands, which make up the basis for the sortase active site, and contain the catalytic His and Cys residues, respectively. Motif 6 was found only among SrtB isoforms and maps onto a structural feature (2/3 hairpin loop) not found in SrtA. Motifs 4 and 7 are unique to SrtB and SrtA, respectively, but map onto the same structural feature (6 strand, 6/7 loop).

View larger version (34K): [in this window] [in a new window]   FIGURE 1. MEME overlay of crystal structures of SrtA and SrtB. X-ray structures of SrtA (Protein Data Bank code 1T2W) and SrtB (Protein Data Bank code 1NG5) from S. aureus, overlaid with MEME-determined motifs. The sortase active site is made up of the parallel 4 (purple) and 7 (red) strands, which contain conserved sequence motifs found in all sortase family members (see supplemental Fig. S1). A motif found only in SrtB sequences (green) corresponds to a unique hairpin fold in the SrtB structure. The orange and cyan motifs (SrtB-only and SrtA-only, respectively) contain the front end of the 6/7 loop, which differs in size, position, and predicted hydrophobicity between SrtA and SrtB. The position of the LPETG substrate in the x-ray structure of SrtA is shown in stick form in blue for reference.

Similarly, the position 2 residues N-terminal to the catalytic histidine (His¹²⁰ in S. aureus SrtA, His¹³⁰ in S. aureus SrtB) is differentially conserved, yielding an AXH motif in SrtA isoforms and YXH in SrtB. Since the active sites of sortases are composed of extended -sheets, these residues (2 residues away from the catalytic Cys-His pair) would be expected to have their side chains oriented nearly parallel to the catalytic residues and would be positioned to contact and orient the peptide substrate. The presence of the more polar Tyr¹²⁸-Ser²²¹ pair in SrtB, as compared with the Ala¹¹⁸-Ile¹⁸² pair in SrtA, might help to stabilize the binding of the more polar NPQTN sequence versus the LPXTG of SrtA substrates. In addition, the Tyr¹²⁸ and Ser²²¹ residues in the SrtB active site are both capable of forming side chain hydrogen bonds, which might better stabilize the substrate for catalysis by Cys²²³. To test these hypotheses, we selected the residues Ala¹¹⁸ and Ile¹⁸² from SrtA for mutagenesis.

Activity Assays of SrtA Point Mutants—To determine if the SrtA A/Y and I/S mutations affected substrate specificity, we prepared the point mutants SrtA A118Y and I182S as well as the double mutant SrtA A118Y/I182S. Initial activity assays showed that the mutants I182S and A118Y were inactive against an NPQTN peptide (results not shown). The double mutant, SrtA A118Y/I182S, was inactive against both LPETG and NPQTN peptides. Thus, the effect of the point mutations was to reduce native SrtA activity and not to alter substrate preference.

Identification of the 6/7 Loop as a Potential Specificity Determinant—Since point mutations had little effect on the specificity of SrtA, we sought to identify larger domains that differed between SrtA and SrtB and thus might be expected to be functionally important for specificity. A comparison of the crystal structures of SrtA and SrtB was performed to identify domains that differed in structure between the two proteins. Although sequence alignments have shown that S. aureus SrtA and SrtB have very low sequence homology (40% homology, 23% identity over 184 equivalent residues (36)), their structures show similar overall folds (Fig. 1). The overall structures align with a root mean square deviation of 3.30 Å over 127 equivalent residues. However, within the core -barrel, the two proteins are very similar, aligning with a root mean square deviation of only 1.31 Å. Thus, most of the structural differences between SrtA and SrtB lie in surface-exposed loops and helices, not in the -sheet core.

Most notable among these structural differences are the presence of an extra domain in SrtB (the 2/3 hairpin loop) and the vastly different sequences and conformations of the 6/7 loops in the two enzymes. In SrtA, this region is a short, 15-amino acid unstructured loop (Val¹⁶¹–Asp¹⁷⁶), whereas in SrtB, it is 41 amino acids long (Lys¹⁷⁴–Asp²¹⁵) and consists of a short loop, an helix, and another loop. In SrtA, the 6/7 loop contains several highly conserved hydrophobic residues, such as Val¹⁶⁸ and Leu¹⁶⁹, which are replaced by more polar residues in SrtB, such as Asn¹⁸⁰, Tyr¹⁸¹, and Arg¹⁸³. Although the size and composition of the 6/7 loops are different in SrtA and SrtB, they occupy equivalent structural positions, with the points of connectivity of the loops being the same in both structures (Asp¹⁶⁰/Tyr¹⁷³ at the N termini in SrtA/SrtB, respectively, Lys¹⁷⁷/Lys²¹⁶ at the C termini). The loop in SrtA and the first loop of the domain in SrtB are both positioned at the back of the active site cavity and appear to be ideally located to contact the N-terminal residues of the recognition motif (Leu-Pro in SrtA substrates, Asn-Pro in SrtB substrates) (Fig. 2).

View larger version (64K): [in this window] [in a new window]   FIGURE 2. Comparison of the active sites of S. aureus SrtA (top) and SrtB (bottom). Highlighted sequences (right) show the conservation differences between SrtA and SrtB sequences from various Gram-positive species. The conserved alanine-isoleucine and tyrosine-serine pairs are highlighted in green and pink, respectively. These positions are also highlighted in the structures (left), showing the proximity of the residues to the catalytic cysteine, histidine, and arginine. The 6/7 loop is visible at the back of each structure. The SrtA substrate peptide LPETG is shown in stick form. Conserved hydrophobic residues Val168 and Leu169 from the 6/7 loop of SrtA are shown, highlighting their proximity to the leucine from the LPETG peptide. The equivalent structural positions in SrtB are occupied by more polar residues, such as Asn180, Tyr181, and Arg183, which may interact with the more polar asparagine of the NPQTN substrate of SrtB. S.aur, S. aureus; L.inn, Listeria innocua; L.mon, Listeria monocytogenes; B.cer, Bacillus cereus; B.ant, Bacillus anthracis; B.hal, Bacillus halodurans.

By analogy with SrtA, SrtB also contains a putative calcium-binding site on the 3/4 loop (Asp¹¹³, Asn¹¹⁶, Glu¹¹⁷, and Asn¹²²) and a conserved acidic residue (Asp¹⁸⁵) on the 6/7 loop. To date, a detailed analysis of the calcium-binding properties of SrtB has not been carried out. However, given the similarities between the structures and functions of SrtA and SrtB, it seems reasonable to assume that SrtB also utilizes bound Ca²⁺ to stabilize the conformation of its 6/7 loop, allowing for productive substrate binding and catalysis.

Design of a 6/7 Loop Swap Chimera, SrtLS—Based on the above lines of evidence, we hypothesized that the 6/7 loops might be playing an important role in contacting substrate and determining the different specificities of SrtA and SrtB. In order to test this hypothesis, we designed a loop swap enzyme (SrtLS_N24), wherein we exchanged the 6/7 loop of SrtA with the corresponding loop from SrtB. This chimeric protein contains the sequence of SrtA for residues Lys²⁶–Asp¹⁶⁰ and then the sequence of SrtB for the next 42 residues (Lys¹⁷⁴–Asp²¹⁵ in SrtB, renumbered Lys¹⁶¹–Asp²⁰² in this enzyme), and then the sequence of SrtA for the remainder (Lys¹⁷⁷–Lys²⁰⁶ in SrtA, renumbered Lys²⁰³–Lys²³² in this enzyme). Since the 6/7 loop is longer in SrtB than in SrtA by 26 residues, this causes the C-terminal residues in SrtLS to be renumbered +26 from their corresponding residues in SrtA (i.e. Cys¹⁸⁴ from SrtA becomes Cys²¹⁰ in SrtLS).

Kinetics of SrtB with Abz-KVENPQTNAGT-Dap(DNP)-NH₂—To serve as a benchmark for activity comparisons, we first characterized SrtB using an HPLC-based assay, similar to one developed by Kruger et al. to measure the activity of SrtA (33). In place of the SrtA substrate Abz-LPETG-Dap(DNP)-NH₂, we used the NPQTN-containing peptide Abz-KVENPQTNAGT-Dap(DNP)-NH₂. SrtB is less active than SrtA, and correspondingly greater concentrations of enzyme (94.8 µM SrtB versus 1 µM SrtA) and longer assay times (3 h versus 30 min) were required to obtain similar levels of product formation. Estimates of the steady-state kinetic parameters were obtained, with SrtB exhibiting a k^app_cat of 5.4 ± 0.5 x 10^-4 s^-1 and a K^app_m for Abz-KVENPQTNAGT-Dap(DNP)-NH₂ of 7.8 ± 2 mM (Fig. 3). For comparison, previous studies have reported a k_cat of 0.28 ± 0.02 s^-1 for SrtA_N24 and a K_m of 7.3 ± 1 mM for SrtA_N24 with Abz-LPETG-Dap(DNP)-NH₂ (39). These values show that SrtB has a K_m for this substrate that is within the error of that measured for SrtA but a k_cat that is 500-fold lower.

View larger version (22K): [in this window] [in a new window]   FIGURE 3. HPLC sortase assay using Abz-KVENPQTNAGT-Dap(DNP)-NH2. A, general scheme of transpeptidation reaction using Abz-KVENPQTNAGT-Dap(DNP)-NH2 and pentaglycine. B, HPLC trace showing the substrate Abz-KVENPQTNAGT-Dap(DNP)-NH2 and the cleavage product NAGT-Dap(DNP)-NH2. Trace corresponds to a 100-µl assay quenched with HCl after 3 h. Initial velocities were calculated from the linear portion of each assay, and peak areas were converted to concentrations as outlined under "Experimental Procedures." C, determination of kinetic parameters of SrtBN21 with Abz-KVENPQTNAGT-Dap(DNP)-NH2. Assays were performed at 37 °C with pentaglycine (2 mM), Abz-KVENPQTNAGT-Dap(DNP)-NH2 (74.8µM to 21.9 mM), and SrtBN21 (94.8µM). Estimates of the kinetic parameters kcat = 5.4 ± 0.5 x 10-4 s-1 and Km = 7.8 ± 2 mM were obtained.

SrtLS has a CD spectrum that is a combination of the features of the spectra of SrtA and SrtB (Fig. 4). It has the same general shape as the SrtA spectrum, although it has more -helical character in the far UV. The 6-7 domain from SrtB contains an -helix that is not ordinarily found in SrtA. Thus, the presence of this swapped domain in SrtLS would be expected to add more -helical character to the secondary structure, which is what was observed. Based on these results, it appears that SrtLS exhibits structural features common to both the folded structures of SrtA and SrtB, in a predicted manner. We therefore assayed SrtLS for enzymatic activity and altered substrate specificity.

Activity Comparisons of Sortase Mutants—To assess the role of the 6/7 loop swap on substrate recognition, we performed activity assays of SrtA, SrtB, and SrtLS with LPXTG and NPQTN substrates to directly compare rates and efficiencies. We also assayed the single point mutants SrtLS A118Y and SrtLS I208S, as well as the double mutant SrtLS A118Y/I208S. Mutants were compared based on their catalytic utilization ratios (), since their intrinsically low activities prevented individual determinations of k_cat and K_m. Slightly higher substrate concentrations (2.7 mM) were used than would ideally be the case; however, given the low levels of activity of several of the enzymes being measured, it was necessary to use low millimolar concentrations of substrate in order to obtain detectable levels of product using our HPLC-based assay. Estimates of k_cat/K^app_m obtained for SrtA (with LPETG) and SrtB (with NPQTN) were found to closely match k_cat/K_m values obtained by individual measurements of k_cat and K_m, and Equation 2 was therefore determined to be valid for our assay conditions.

View larger version (13K): [in this window] [in a new window]   FIGURE 4. CD wavelength scans of SrtA (black), SrtB (red), and SrtLS (green). Spectra were collected at 25 °C, and the observed signal was converted to mean residue ellipticity as described under "Experimental Procedures." SrtA shows a spectrum consistent with a secondary structure predominantly composed of -sheet and random coil. The spectrum of SrtB is more consistent with a secondary structure of roughly equal parts -helix and -sheet. SrtLS has a spectrum that is similar overall to that of SrtA, with some influence from SrtB, consistent with its hybrid nature. Separate scans at 98 °C (not shown) confirmed that the unfolded spectra for each protein differed from the 25 °C spectra, suggesting that each protein is properly folded.

View this table: [in this window] [in a new window]   TABLE 2 Kinetic parameters measured for sortase mutants Assays were performed at low [S] relative to the Km, allowing for estimates of as described under "Experimental Procedures." Specificity ratio = (). ND, not detectable to the limit of detection (>25 nmol of product formation). Replacement of the 6/7 loop from SrtA with the loop from SrtB generated an enzyme (SrtLS) with a for NPQTN only 11 times lower than SrtB and a specificity ratio shifted 700,000-fold from SrtA.

Activity Comparisons of SrtLS Active Site Mutants—In order to assess the impact of the conserved isoleucine and alanine residues identified by our bioinformatic analysis, we generated point mutants of SrtLS, I208S and A118Y, as well as the double mutant A118Y/I208S. Each of these mutants was expressed and purified and then assayed for LPETG and NPQTN activity. The single point mutant SrtLS A118Y was able to cleave the LPETG peptide, at a of 2.7 ± 0.1 x 10^-4 M^-1 s^-1, about 70% of the efficiency of SrtLS. SrtLS I208S was also active against LPETG, with a of 2.2 ± 0.1 x 10^-4, about 60% of the efficiency of SrtLS (Table 2). The double mutant SrtLS A118Y/I208S was completely inactive for LPETG cleavage, to the limits of detection in our assay (>25 nmol of product formed). For the substrate Abz-KVENPQTNAGT-Dap(DNP)-NH₂, the addition of the point mutations A118Y, I208S, and A118Y/I208S had no effect on the activity of SrtLS (Table 2). Although these point mutations did not increase the NPQTN cleavage activity of SrtLS, they did decrease the ability of SrtLS to recognize the LPETG substrate, leading to an overall increase in the specificity ratio of SrtLS (Table 2). The effects of these mutations suggest that the residues Ala¹¹⁸ and Ile¹⁸² may play an important role in LPETG recognition by SrtA, whereas the corresponding residues from SrtB do not appear to be important for NPQTN recognition.

View larger version (15K): [in this window] [in a new window]   FIGURE 5. ESI mass spectrometry of reaction products from SrtB and SrtLS assays with Abz-KVENPQTNAGT-Dap(DNP)-NH2. Products from the SrtB and SrtLS assays were isolated, concentrated, and subjected to ESI-MS to confirm their identities. A, ESI-MS spectrum of product (retention time = 1.46 min) from the SrtB assay, as monitored at 355 nm. The peak at m/z = 613.2 corresponds to the expected cleavage product NAGT-Dap(DNP)-NH2. Subsequent MS/MS analysis of this peak (not shown) confirmed its identity. The product of the SrtLS assay yielded a similar mass spectrum (not shown). B, ESI-MS spectrum of the product (retention time = 1.65 min) of the SrtB assay, as monitored using fluorescence detection (excitation = 318 nm, emission = 420 nm). The peak at m/z = 1219.5 corresponds to the transpeptidation product Abz-KVENPQTGGGGG-OH. The smaller peak at m/z = 934.4 corresponds to the hydrolysis product Abz-KVENPQT-OH. MS/MS analysis confirmed the identities of both peaks (not shown). C, ESI-MS spectrum of the same product (retention time = 1.46 min) from the SrtLS assay. Only the m/z = 934.4 peak, corresponding to the hydrolysis product, is present.

When the product peak of the SrtLS reaction (as monitored at 420 nm) was analyzed, only the ion at m/z = 934.4 was observed (Fig. 5C). MS/MS analysis of this ion confirmed its identity as the hydrolysis product Abz-KVENPQT-OH. Ions corresponding to the predicted transpeptidation product were not found. Thus, it appears that whereas SrtLS is capable of acylating the NPQTN sorting signal, it is unable to perform transpeptidation of the cleaved sorting signal onto the Gly₅ substrate. Instead, the acyl-enzyme intermediate is resolved by hydrolysis from water.

The fact that SrtB, but not SrtLS, is competent to carry out the transpeptidation stage of the sorting reaction in our in vitro assays suggests that there are differences in the binding sites for the cell wall peptidoglycan substrates of SrtA and SrtB. A recent study by Marraffini and Schneewind (23) found evidence for a distinct anchor structure for the IsdC protein (a SrtB substrate) in vivo. They suggest that SrtB-anchored surface proteins are attached to peptidoglycan that has shorter glycan chains and fewer cross-links than is the case for SrtA-anchored proteins. This implies that there are different peptidoglycan substrates for SrtA and SrtB. If this is the case, then there must be unique features in the structure of SrtB that allow it to specifically recognize its less cross-linked peptidoglycan substrates.

In our study, pentaglycine was able to serve as an anchor for NPQTN peptide cleaved by SrtB but not by our chimera, SrtLS. Pentaglycine was also able to serve as an anchor for LPETG that had been cleaved by SrtA (data not shown). It is possible that the presence of the swapped loop in SrtLS somehow prevents pentaglycine from accessing the active site and thus is the cause of the lack of transpeptidation. Another possible explanation is that there may be additional domains or features on the surface of SrtB that are not included in SrtLS and that the presence of these domains is necessary for transpeptidation of the cleaved sorting signal to occur. One candidate domain for this is the conserved SrtB-only domain found in our MEME analysis (Fig. 1), which corresponds to a hairpin between strands 2 and 3 on SrtB. This unique fold is not observed in the structure of SrtA and could serve as a recognition site for the peptidoglycan substrate of SrtB. Studies are currently in progress to swap this loop into SrtLS, to see if its addition allows for successful transpeptidation onto pentaglycine.

Redesign of existing enzyme frameworks for altered specificity has long been a means to study molecular recognition and catalysis. In the case of malate dehydrogenase, a single point mutation was sufficient to convert it into a lactate dehydrogenase, with a k_cat/K_m shift of 10⁷ (40). However, for many systems, larger numbers of mutations or more drastic changes are required to alter specificity (41, 42). In some of these cases, domain swapping has been validated as a tool for changing the specificity of closely related enzymes for which crystal structures are available (43, 44). Ma and Penning (43) used loop swaps as a tool to change mammalian 3-hydroxysteroid dehydrogenase into 20-hydroxysteroid dehydrogenase. In their study, point mutants had little effect on the specificity of 3-hydroxysteroid dehydrogenase, but replacement of surface loops with the corresponding loops from 20-hydroxysteroid dehydrogenase did change specificity, with a shift in k_cat/K_m of 2 x 10¹¹. Similarly, in their study on the conversion of trypsin to chymotrypsin, Hedstrom et al. (44) found that it was necessary to swap two of the trypsin surface loops in order to alter the specificity of the enzyme. A shift in k_cat/K_m of 10⁹ was observed upon swapping of these loops. We observed a k_cat/K_m shift for SrtA that was slightly smaller, only 7 x 10⁵. However, when one considers the very slow rates in vitro of SrtA and SrtB (k_cat/K_m on the order of 10¹ for SrtA and 10^-2 for SrtB), this is a significant change. Alteration of the 6/7 loop alone in SrtA was sufficient to convert it to an NPQTN-cleaving enzyme, with an efficiency of cleavage only 11 times lower than native SrtB.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Sequence analysis, mutagenesis, and domain swapping have been used as tools to explore specificity and molecular recognition in S. aureus sortase enzymes. Point mutants identified from analysis of SrtA and SrtB sequences did not affect the specificity profile of SrtA, suggesting that these residues play a role in stabilizing the folded structure of the SrtA active site, rather than contacting substrate. However, replacement of the 6/7 loop in SrtA with the corresponding loop from SrtB was sufficient to change the specificity profile of SrtA by over 700,000-fold. Additional point mutants were unable to boost the NPQTN-cleaving activity of SrtLS. Together, these results support the idea that the 6/7 loop is a primary substrate recognition site in sortase enzymes. Future studies are ongoing to identify additional sites that are involved in the molecular recognition and catalysis of sortase substrates and those that affect transpeptidation.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant AI46611 (to D. G. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

¹ To whom correspondence should be addressed: Dept. of Chemistry, Duke University, B120 LSRC Bldg., Box 90317, Durham, NC 27708-0317. Tel.: 919-660-1516; Fax: 919-668-5483; E-mail: dewey{at}duke.edu.

² The abbreviations used are: Fmoc, N-(9-fluorenyl)methoxycarbonyl; Abz, aminobenzoic acid; Dap, diaminopropionic acid; DNP, dinitrophenol; MEME, multiple expectation/maximization for motif elucidation; HPLC, high pressure liquid chromatography; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; ESI, electrospray ionization.

	ACKNOWLEDGMENTS

 
S. aureus strain N315 was provided by the Network on Antimicrobial Resistance in Staphylococcus aureus. We thank Dawn Schmidt, John Schmidt, and Amanda Lind for critical evaluation of the manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 

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