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Elucidation of the recognition sequence of Sortase B from Bacillus anthracis by using a newly developed LC-MS based method Chasper Puorger, Salvatore Di Girolamo, and Georg Lipps Biochemistry, Just Accepted Manuscript • Publication Date (Web): 05 May 2017 Downloaded from http://pubs.acs.org on May 8, 2017

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Biochemistry

Elucidation of the recognition sequence of Sortase B from Bacillus anthracis by using a newly developed LC-MS based method Chasper Puorger, Salvatore Di Girolamo, Georg Lipps* University of Applied Sciences and Arts, Institute for Chemistry and Bioanalytics, Gründenstrasse 40, 4132 Muttenz, Switzerland *

Corresponding author: [email protected]

Abstract Sortases are enzymes, which are responsible for the attachment of secreted proteins to the cell wall of Gram positive bacteria. Hereby the sortases recognize short, five residues long amino acid sequences present in the target proteins and fuse them to the peptidoglycan layer via a transpeptidation reaction, creating a new peptide bond between the C-terminus of the recognition sequence and the cell wall. The transpeptidation activity of sortases is highly used in protein engineering for modification of target proteins. The majority of protocols rely on the high activity of the well characterized Staphylococcus aureus SrtA and variants thereof, while sortases from other classes are not used for this purpose. This can be attributed to the lower activity of other sortases but also to the limited data on sequence specificity available on the different sortases. We set out to determine the sequence specificity of the Bacillus anthracis SrtB. To this end, we developed a new method for sequence specificity determination of sortases or other bond forming enzymes, which recognize an amino acid sequence. Using mixtures of recognition peptides of limited complexity, which are reacted with biotinylated substrates, the biotinylated transpeptidation products are isolated with magnetic streptavidin beads and analyzed with LC-MS. With this, peptide sequences, which are recognized by the sortase and function as substrates can be determined and quantified. The method, developed with the highly active evolved SrtA from S. aureus, allowed for the first time unbiased in depth analysis of the sequence specificity for SrtB from B. anthracis, which is 104-fold less active than SrtA from S. aureus.

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Introduction Gram positive bacteria have evolved a number of strategies for surface presentation of extracellular proteins. These extracellular proteins have functions ranging from nutrient uptake, cell-cell attachment and communication, detoxification or killing of potential competitors. Surface proteins are attached to the cell surface by transmembrane domains,1 through lipid modification,2 noncovalently by cell wall binding repeats3 or by covalent attachment to the cell wall.4 Covalent attachment to the bacterial cell wall is executed by sortase enzymes which are almost exclusively found in Gram-positive bacteria.5,6 Sortases can be divided into different classes, A through E, depending on their primary sequences and their functions.7,8 Class A sortases are generally involved in anchoring a large number of proteins to the cell wall, while the members of the other classes have more specific substrates like proteins for heme uptake and pilus proteins (class B), pilus protein polymerization (class C), proteins involved in spore formation (class D), or pilus attachment and proteins involved in aerial hyphae formation (class E). Target proteins recognized by sortases possess a C-terminal cell wall sorting signal, consisting of a pentapeptide recognition sequence, a hydrophobic region of about 20 amino acids for transient membrane anchoring and a positively charged lysine/arginine rich tail.9 After secretion via the Sec pathway the cell wall sorting signals of the target proteins are recognized by the corresponding sortases, which then process and attach the substrates to the cell wall.10 The sortases from the different classes are thought to share a common ping-pong bi-bi transpeptidation reaction mechanism, according to which the sortase first binds the five residue recognition sequence located in the C-terminal region of the substrate protein.11,12 The sortases then form transient acylenzyme intermediates with the substrates, which are resolved by a nucleophilic attack from components of the bacterial cell wall. In case of Staphylococcus aureus sortase A (SaSrtA) the nucleophile consists of the N-terminal amine from the pentaglycine of lipid II and its nucleophilic attack of the acyl-enzyme intermediate results in formation of a new peptide bond between the fourth residue of the recognition sequence and the pentaglycine.13 This nucleophile might vary in different Gram positive strains as the composition of the peptidoglycan layers differs from strain to strain. Diaminopimelic acid, for example, which cross-bridges the peptidoglycans in Bacillus anthracis, is thought to be the point of attachment for the sortase substrate proteins in Bacillus strains.14–16 In absence of a dedicated nucleophile the acylenzyme intermediate was shown to be hydrolyzed, resulting in cleavage of the recognition sequence without formation of a new peptide bond.12


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Biochemistry

Sortases of the particular classes have been shown to recognize varying sequences in their substrate proteins. For class A sortases (conserved domain cd06165), like SaSrtA, the recognition sequence LPXTG was determined, where the third residue was shown to be variable.4,9,17 The recognition sequences of the other sortase classes are less well characterized but a consensus can be found for class D subclass 4 (conserved domain cd05828) LPNTA18 and class D subclass 5 (conserved domains cd06166 and cd05830) LAXTG.19 In contrast, the specificity for the different members of classes B15,20–24 (conserved domain cd05826) and C25,26 (conserved domain cd05827) is less well defined. For sortases of class B the recognition sequences NPKTG and NPQTN were reported for sortase B enzymes from Bacillus anthracis and Staphylococcus aureus, respectively. No data on substrate or sequence specificity of class E sortases (conserved domain cd05829) is available. The information on the recognition sequences of the different sortases relies largely on scanning of the sequences of known substrate proteins for putative recognition sequences. The sequences were then verified for a few example sortases with in vitro experiments, where peptides with the recognition sequences were used as substrates to analyze the sortase activity by fluorescence un-quenching, SDS-PAGE analysis, HPLC analysis or by mass spectrometry.18,23,27–33 The only sortase for which an exhaustive sequence specificity screening was performed is wild type SrtA from S. aureus.17 Investigation of other sortases is generally complicated by the low activity of the sortase enzymes. In recent years sortases have drawn increasing interest as tools for protein engineering (so-called sortase mediated ligation or sortagging), for example in protein-protein ligation,34 protein or peptide cyclization,35–37 site-specific protein labelling38,39 or protein immobilization.40,41 However these applications are again hampered by the low activity of sortases, but gained significance after the development of a SaSrtA variant with five mutations (eSaSrtA), which resulted in a 140-fold increased activity.42 In this work we perform an in depth analysis of the sequence specificity of Bacillus anthracis (BaSrtB) to which end we developed a method for the determination of the sequence specificities of sortases with low transpeptidation activity. Relying on the available information on recognition sequences, we use mixtures of peptides with individual variable positions as sortase substrates. These peptides are reacted in presence of the designated sortase with a biotinylated nucleophile, with the result that peptides which are recognized as substrates are biotinylated in the transpeptidation reaction (Figure 1). Products are then isolated from the reaction mixture with magnetic streptavidin beads and are, after elution, analyzed with LC-MS. The method was developed with the highly active eSaSrtA and then applied to BaSrtB for which we calculated a more than 104-fold lower activity compared to eSaSrtA. We were able ACS Paragon Plus Environment

Biochemistry

to determine the specific recognition sequences for both sortases including additionally allowed amino acids at the different positions. With BaSrtB we determined for the first time the detailed sequence

Srt, T = 25 °C

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FITC-LPXTGE + GGGK-biotin

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LPXTGE GGGK-biotin GGGK-biotin LPXTGE LPXTGE GGGK-biotin

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binding

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Reaction mixture

wash with PBS and H2O

LPXTGGGK-biotin GGGK-biotin

GGGK-biotin GGGK-biotin LPXTGE GGGK-biotin LPXTGE GGGK-biotin GGGK-biotin GGGK-biotin LPXTGE LPXTGE

GGGK-biotin

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LPXTGE

Magnetic streptavidin beads

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elution with H2O

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at 80 °C

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LPXTGGGK-biotin GGGK-biotin LPXTGGGK-biotin

specificity with all possible variations of a sortase which is not a member of class A and which has a significantly decreased in vitro activity compared to SrtA. Figure 1. Schematic overview of the strategy for determination of the sortase substrate specificity. (a) FITC-labeled recognition peptides with single amino acid variation are mixed with biotinylated GGGK nucleophile and the reaction is started by addition of sortase enzyme. The sortase performs a transpeptidation reaction, where the C-terminal residues of the recognition sequence are cleaved off (indicated by the red arrow) and replaced by the nucleophile, forming a biotinylated product. (b) Isolation of biotinylated transpeptidation product is performed by mixing the reaction mixture with magnetic streptavidin beads, which specifically bind the product and non-reacted nucleophile. After washing, bound molecules can be eluted with water by incubation at 80 °C. Isolated products can then be analyzed with RP-HPLC or LC-MS.


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Biochemistry

Materials and Methods Gene synthesis and plasmid construction The amino acid sequence of Bacillus anthracis sortase B (BaSrtB) was analyzed with TMpred, PredictProtein and Probius revealing an N-terminal signal sequence and a transmembrane region of 37 residues. Consequently the gene encoding only the soluble domain of BaSrtB (i.e. residues 38-217 of BaSrtB) was codon optimized for E. coli expression, synthesized (GenScript) and cloned into the p7XC3H vector containing a C-terminal C3-protease site and His10-tag by using the FX-cloning strategy.43

Protein expression and purification BL21 (DE3) cells carrying the plasmid p7X-BaSrtB were grown at 37 °C in 2 L kanamycin containing LB medium until reaching an OD600 of 0.6, induced with 1 mM IPTG and grown for additional 3 h. Cells were then harvested by centrifugation, resuspended in 20 ml 100 mM KH2PO4, pH 8.0, 300 mM KCl and lysed by lysozyme treatment and ultrasonication. The lysate was centrifuged at 12000 g to remove insoluble cell debris and the cleared lysate was applied to a 5 ml TALON metal affinity column equilibrated in 100 mM KH2PO4, pH 8.0, 300 mM KCl, 10 mM imidazole. Bound proteins were eluted with a step gradient of 100 mM KH2PO4, pH 8.0, 300 mM KCl, 250 mM imidazole. The purification progress was analyzed with SDS-PAGE and fractions containing 95 % pure protein were pooled and dialyzed against 50 mM Tris-HCl pH 8.0, 60 % glycerol. The protein yield was 20 mg L-1 cell culture. A sample of evolved SrtA from Staphylococcus aureus (eSaSrtA42) was a gift from NBE Therapeutics (Basel, Switzerland).

Transpeptidation reaction N-terminally FITC-labelled peptides containing the putative recognition sequences were synthesized by randomizing one position (e.g. LPXTGE) at a time (Genscript, USA) and dissolved in DMF (Fluka, Switzerland). As nucleophile, a peptide with the sequence GGGK was synthesized with a biotin attached to the ε amino group of the side chain of lysine (Genecust, Belgium). The transpeptidation reactions were performed at 25 °C in 50 mM Tris-HCl, pH 8.2, 150 mM NaCl, 10 mM CaCl2 with peptide concentrations of 250 µM (for both recognition peptide and nucleophile) and an enzyme concentration of 5 µM for SaSrtA or concentrations of 1 mM for recognition peptide, 250 µM for nucleophile and 50 µM for enzyme in case of BaSrtB. ACS Paragon Plus Environment

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Product isolation The reaction samples were diluted 20-fold with PBS, pH 7.4 and 100 µl of diluted sample were mixed with 0.2 mg magnetic streptavidin beads (Dynabeads MyOne Streptavidin C1, Invitrogen) previously treated according to the manufacturer's manual. After 40 min incubation at 25 °C and shaking with 450 rpm (with sporadic manual mixing) beads were retained with a magnet and the supernatant was collected as a control. The beads with bound product were washed three times with 100 µl PBS pH 7.4 to remove non-bound substrate and four times with 100 µl H2O to remove salt. Products (as well as nucleophile) were eluted in 100 µl H2O by incubation for 10 min at 80 °C.

RP-HPLC analysis Products eluted from magnetic streptavidin beads were analyzed with RP-HPLC at basic pH (allowing better separation of the different peptide species and higher fluorescence signal from FITC, which almost completely loses fluorescence at acidic pH in organic solvent) with an XBridge C18 column (Waters) with 0.1 % NH4OH (pH ̴ 10) in 5 % MeOH as solvent A and 0.1 % NH4OH in 95 % MeOH as solvent B. Elution was performed with a linear gradient from 0 to 100 % B over 10 ml, eluted peptides were detected by absorbance at 497 nm and fluorescence at 522 nm (excitation at 497 nm). Peptide substrates eluted in three regions according to the nature of the variable residue: acidic side chain -> early elution; arginine, lysine, polar and small hydrophobic side chain -> intermediate elution; large hydrophobic side chain -> late elution. The transpeptidation products were more hydrophobic than the substrate peptides, thus the products eluted later than the corresponding substrates.

LC-MS analysis: The same conditions for liquid chromatography as for the RP-HPLC analysis were used. MS-analysis was performed on an Agilent LC/MSD Trap XCT instrument run in negative mode. LC-MS data were analyzed with the Openchrom software (http://www.openchrom.net). The ion intensity for each product was determined and corrected by the abundance of the corresponding substrate in the starting material. Data were assembled in a LOGO presentation by using the Seq2Logo server (http://www.cbs.dtu.dk/biotools/Seq2Logo/). ACS Paragon Plus Environment

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Biochemistry

Results Development of a protocol for recognition sequence determination With exception of the sortase SrtA from Staphylococcus aureus, limited experimental data on sequence specificity and especially variability of the various sortases from different families is available up to date. Most information was obtained by genomic search for possible sortase recognition sequences in putative substrate proteins. While these data are valuable as a starting point for the search for the recognition sequence of a given sortase, it is not evident that the found sequence is the best and if other sequences are also recognized by the sortase. We set out to develop a method which would allow us to determine the sequence specificity of a given sortase and which would also work for sortases with low activity. Starting from a sequence predicted by genomic analysis or known to be a sortase substrate, our strategy consisted of using a mixture of recognition sequence peptides, where one position was randomized and the four other positions were kept constant. For better detection, we N-terminally labeled the recognition peptides with fluorescein. As nucleophile we chose a short peptide with the sequence GGGK where the ε-amine of the lysine side chain was modified with biotin (Figure 1a). In the transpeptidation reaction peptides, which are recognized by the sortase are reacted with biotinylated nucleophile, allowing isolation of the products, along with non-reacted nucleophile, by using streptavidin coated magnetic beads. After elution the isolated product could then be analyzed with RP-HPLC and LC-MS in order to detect if product is formed and to determine the nature of such product. For method development, we initially used the evolved variant of the well characterized sortase A from Staphylococcus aureus (eSaSrtA), since it has a high activity and the recognition sequence is known to be LPXTG, where the X at position 3 designates no preference for a specific amino acid side chain 17,42. The reactions were set up with equal amounts of both substrates (recognition sequence and nucleophile) and the time course of the reaction was first determined with the sequence LPXTGE by analyzing the raw reactions with RP-HPLC under basic conditions (Figure S1). The chromatogram of substrate peptides can be roughly divided into three peak regions according to the hydrophobicity of the variable amino acid, with the shortest retention time for acidic side chains (D, E), intermediate retention times for noncharged, polar and small hydrophobic side chains (A, R, N, Q, G, H, K, M, P, S, T, Y, V) and finally the longest retention time for larger hydrophobic side chains (I, L, F, W). The profiles show that significant product formation occurred within one hour (indicated by dashed lines). Compared to the retention times of the substrates these product peaks are shifted to longer retention times by about 1.7 (+/-0.2) minutes. Additional peaks, shifted by less than a minute in their retention time compared to substrates,


Biochemistry

appeared after further incubation. These additional peaks could be attributed to hydrolysis of recognition peptide and transpeptidation products as was verified by performing the reaction in absence of GGGK-biotin nucleophile (Figure S2). Our analysis of the different peaks in the RP-HPLC profiles showed that very limited hydrolysis product is formed after one hour of reaction in presence of GGGKbiotin nucleophile (Figure S3). We therefore decided to limit the reaction time for eSaSrtA to one hour. We separated the products from the reaction mixture by using streptavidin coated magnetic beads and again analyzed the obtained samples with RP-HPLC. The eluate fractions show that biotinylated

a 450 400 Absorbance at 497 nm

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eluate

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b Gly Ala

FITC-LPXTGGGK-biotin

Ser

Asn Gln Pro

His 745.8015

725.8412

transpeptidation products can indeed by captured and eluted using streptavidin beads (Figure 2a). It also Figure 2. RP-HPLC and LC-MS analysis of eSaSrtA transpeptidation products. (a) RP-HPLC chromatograms of reactions of LPXTGE peptides with (blue) or without (grey) eSaSrtA after 1 h incubation at 25 °C. A sample after 1 h reaction was applied to magnetic


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Biochemistry

streptavidin beads and supernatant (green) and eluate (red) were analyzed. Dashed lines indicate the position of product peaks as seen in the eluate fraction. Peak positions with transpeptidation product are indicated with a dashed line. (b) Example spectrum of the LC-MS analysis of the second peak of the chromatogram of the reaction with LPXTGE peptides with noncharged and small hydrophobic residues at the variable position X.

becomes evident that with the chosen conditions not the entire product was captured as some product could still be found in the supernatant. The captured product allowed us to further investigate the product composition with LC-MS. With exception of the peptides having a leucine or isoleucine at the variable position all peptides present in the reaction have a distinct mass, which enabled us to assign the detected masses to the different transpetidation products (see Figure 2b for an example). Thus, we were able to determine which sequences from the initial mixture of 20 different peptides are recognized by the sortase and used in the transpeptidation reaction. To calculate the preference for single amino acids we compared the abundances of the twenty peptides of the substrates and the products using the ion counts of the individual masses. Our analysis confirmed previous results showing that SaSrtA has no clear preference for a particular amino acid at position three of the recognition sequence (Figure 3a, Figure S4c).17 At positions one and two we find predominantly leucine/isoleucine and proline, respectively (Figure S4a and b, Table S1). Since peptides with leucine and

a

b

Figure 3. Logo representations of the sequence specificity of eSaSrtA (a) and BaSrtB (b). Both sortases show no specificity at position three while the other positions are more stringent. Non-charged polar amino acids are depicted in green, basic amino acids in blue, hydrophobic amino acids in black, acidic amino acids in red, proline in orange, cysteine and methionine in yellow. J (purple) represents isoleucine and leucine, which could not be distinguished in the LC-MS analysis because of identical masses. Glycine at position five in the recognition sequence of BaSrtB is depicted in grey because it could not be determined directly due to the low substrate turn over (see main text). Figures were prepared by using the Seq2Logo server (http://www.cbs.dtu.dk/biotools/Seq2Logo/)


Biochemistry

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isoleucine have exactly the same mass, we were not able to discriminate between the two with our analysis. Position four shows the highest signal for serine and threonine, followed by reduced occurrence of product with alanine, leucine/isoleucine, valine and a minor intensity for glycine (Figure S4d). For position five we had to modify our analysis method due to the fact that the fifth amino acid of the recognition sequence is cleaved off in the transpeptidation reaction and is not present in the transpeptidation products, which therefore have the same sequence independent of the substrate sequence. We therefore analyzed the consumption of the different substrate sequences in the reaction mixture, which did not give as accurate results as analysis of the formed product, but nevertheless allowed discrimination between substrate peptides and peptides which are not recognized by eSaSrtA. The only peptide which was significantly consumed in the transpetidation reaction contained a glycine at position 5 (Figure S4e). Overall, our results for eSaSrtA are in good agreement with the previously published recognition sequences of wild type SaSrtA4,17 and of evolved SaSrtA44 (see supplementary material for a more detailed comparison), proving the validity of our methodology. Determination of the sequence specificity of BaSrtB, a sortase with marginal activity The developed protocol for the determination of sortase specificity works for the highly active eSaSrtA. We next applied the protocol for sortase B from Bacillus anthracis (BaSrtB), a sortase with much lower activity. The structure of BaSrtB was solved and superimposes well with the structure of Staphylococcus aureus SrtB.15 BaSrtB was found to recognize the sequence NPKTG present in the substrate protein IsdC, a surface protein necessary for heme-iron uptake in B. anthracis.20 The BaSrtB recognition sequence deviates from the SaSrtA sequence at the first position (Asn versus Leu) but the specificity has not been investigated in detail. As a starting point for the determination of the BaSrtB recognition sequence we used longer peptides of the form DNPXTGDE derived from IsdC (the recognition sequence is underlined) to make sure we are not missing residues involved in recognition by BaSrtB. As nucleophile we continued to use GGGK-biotin peptide as for eSaSrtA, although in accordance with the peptidoglycan composition in the B. anthracis, the nucleophile is thought to be diaminopimelic acid as opposed to pentaglycine found for S. aureus sortases. We tested non-biotinylated diaminopimelic acid as a nucleophile since we did not have the biotinylated form, but could not discern any product formation when analyzing the raw reactions with RP-HPLC (data not shown). Thus, diaminopimelic acid is not a significantly better nucleophile for BaSrtB compared to the GGGK-biotin nucleophile. The reaction of BaSrtB was initially performed with the same conditions as for SaSrtA (i.e. 5 µM BaSrtB and 250 µM substrate, 50 mM Tris-HCl pH 8.2, 150 mM NaCl, 10 mM CaCl2), which resulted in no


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detectable product formation (data not shown). After increasing the enzyme concentration to 50 µM and the concentration of recognition peptide to 1 mM it was possible to isolate detectable amounts of transpeptidation product after 24 h of incubation (Figure 4a, Figure S5). The products formed by BaSrtB are shifted in the RP-HPLC chromatograms by about 2.8 minutes to longer retention times compared to the corresponding substrates. From the peak intensities of the isolated BaSrtB products and taking into consideration the higher concentrations of enzyme and recognition peptides as well as the longer

a

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Biochemistry

0

12

b Asn

FITC-DNPXTGGGK-biotin

Ser Gln 799.27

Gly

His 803.3

763.67

incubation time and an about 10 – 20 % loss of product during isolation, we calculated a more than 104fold lower activity for BaSrtB (v0 ≈ 3.3 * 10-11 M s-1 with 50 µM BaSrtB, kapp = 6.6 * 10-7 s-1) compared to eSaSrtA (v0 = 2.4 * 10-7 M s-1 with 5 µM eSaSrtA, kapp = 0.048 s-1). ACS Paragon Plus Environment

Biochemistry

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Figure 4. RP-HPLC and LC-MS analysis of BaSrtB transpeptidation products. (a) RP-HPLC chromatograms of reactions of DNPXTGDE peptides with (blue) or without (grey) BaSrtB after 24 h incubation at 25 °C. A sample after 24 h reaction was applied to magnetic streptavidin beads and supernatant (green) and eluate (red) were analyzed. The blue, grey and green chromatograms (left hand axis) are scaled different than the chromatogram of the eluate (red, right hand axis). Dashed lines indicate the position of product peaks as seen in the eluate fraction. Peak positions with transpeptidation product are indicated with a dashed line. (b) Example spectrum of the LC-MS analysis of the second peak of the chromatogram of the reaction with DNPXTGDE containing peptides with non-charged residues at the variable position X. Some masses could not be assigned (e. g. 808.8 Da) and possibly derive from streptavidin fragments dissociated from the magnetic beads.

We next tested different buffer compositions to find the optimal conditions for the transpeptidation reaction with BaSrtB. A pH screen between 5.0 and 9.5 revealed the highest product formation between pH 8.0 and 9.0 with no activity at pH 5.0 (data not shown). In another screen the reaction showed a slight dependence on the presence of bivalent ions like Ca2+ or Mg2+, in absence of bivalent ions the product yield was about halved (data not shown). Thus, the buffer screening showed that the optimum conditions for the reaction with BaSrtB are similar to the conditions for eSaSrtA, however with a smaller influence of bivalent cations on the product yields. We went on to analyze the isolated products with LC-MS in order to determine the recognition sequence of BaSrtB. Although the amount of isolated product for BaSrtB was about 30 times lower compared to SaSrtA we were able to determine the sequence preference. Figure 4b shows an excerpt from the analysis of the reaction with the DNPXTGDE peptide mixture, with variable amino acid composition at position three (X) of the recognition sequence (underlined). We estimated the lowest amount of peptide detected and identified to be in the range of 2 pmol. The abundance of the different products was again calculated by taking into account the distribution of peptide concentration in the starting material. Similar to eSaSrtA, BaSrtB has no clear amino acid preference at position three, sequences with all amino acids except for cysteine and threonine could be detected in the isolated products. Since asparagine turned out to be the most prominent in our analysis, sequences with Asn at position 3 were chosen for all further peptides. For the other positions of the recognition sequence, BaSrtB has a distinct preference (Figure 3b, Figure S6): At position one we found mainly asparagine and to a minor extent histidine, while at positions two and four proline and threonine were the sole amino acids found, respectively. In contrast to SaSrtA, we were not able to determine the preferred residue at position five for BaSrtB because the consumption of substrate in the reaction was too low to be detected. Nevertheless, since we used peptides with glycine at position five when determining the specificity for the other positions we can assert that glycine is at least among the allowed amino acids at this position. Furthermore, no significant increase of isolated product could be detected for the peptides with variable position five


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compared to the other positions, showing that none of the other amino acids is strongly preferred over glycine. The peptide sequence we chose for the determination of the specificity of BaSrtB is three residues longer than the usual core recognition sequence published. We therefore wanted to test if a sequence shortened by two amino acids would give similar product yields as the longer sequence. To this end, we used two peptides with the optimized recognition sequence, DNPNTGDE and NPNTGD, to perform the transpeptidation reaction, isolated the products and analyzed the amount of formed product with RPHPLC. Evaluation of the peak areas revealed that about 30 % less product was formed with the shorter peptide compared to the longer peptide (Figure 5), thus additional interactions extending over the classical recognition sequence might play a role in substrate recognition by BaSrtB. We tested if the amino acid side chain preceding the recognition sequence has an influence on the transpeptidation activity by using a peptide mixture with all 20 amino acids at this position (i. e. XNPNTGDE) in BaSrtB catalyzed transpetidation reactions. Similar to position 3 of the recognition sequence no clear preference for an amino acid was observed, all amino acids except for proline and cysteine (the latter was however not present in the initial substrate peptide mixture) were found among the isolated transpeptidation products. Finally, we set out to verify the obtained recognition sequence by testing the first position, where predominantly product with asparagine was found but also about 2.5 % of product with histidine. We synthesized the short sequences NPNTGD and HPNTGD for this analysis with RP-HPLC and LC-MS. The peptide with asparagine at position one yielded about 60-fold more product compared to its histidine analog (Figure 5). This is in excellent agreement with the results obtained with the mixture of the longer Fluorescence intenisty (a. u.)

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Biochemistry

eluate HPNTGD

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peptides, where 40-times more product with asparagine was observed. This last result additionally confirms the validity of the developed method for the detailed analysis of sortase recognition sequences from peptide mixtures. Figure 5. RP-HPLC analysis of transpeptidation products of BaSrtB with the peptides DXPNTGDE (grey), DNPNTGDE (red), NPNTGD (dark blue) and HPNTGD (green) eluted from magnetic streptavidin beads. The inset shows the chromatogram for the peptide HPNTGD followed by the fluorescence signal, which allowed detection with higher sensitivity. Arrows indicate the peaks with transpeptidation product as confirmed by LC-MS.


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Biochemistry

Discussion In recent years sortase enzymes have become popular tools in protein engineering. Recognition sequences can be added to protein sequences by genetic approaches and thus allow site specific modification of protein targets. Applications range from protein-protein ligation, modification of proteins with fluorescent or effector molecules, protein immobilization or protein cyclization. A major shortcoming of sortase enzymes is the low activity and along with that the low substrate turnover. Recently an evolved sortase with a two orders of magnitude increased activity has been developed on the basis of Staphylococcus aureus sortase A.42 This sortase was later evolved to sortases with orthogonal sequence specificity.44 These new enzymes are valuable for the above-mentioned applications in protein engineering. An important prerequisite for the application of sortase enzymes in protein engineering is knowledge of its specificity towards substrate sequences especially if more than one site is to be modified with different molecules in a single protein. Work on determination of substrate specificity is limited to date, as it mainly relies on analysis of natural substrate sequences and some in vitro investigation of sortase reactions with individual peptide sequences.18,23,27–33 As sole example the recognition sequence of S. aureus SrtA was investigated in depth.17 Among the sortases which were not analyzed in detail some may be of interest for protein engineering, because of their distinct substrate specificities or other beneficial properties. The data presented in this article show the successful detailed in vitro determination of substrate specificity of a sortase with low activity by using a newly developed method where the transpeptidation products are isolated from the reaction mixture and analyzed with LC-MS. Starting from a known possible recognition sequence, the optimal sequence could be determined by varying individual positions of the sequence at a time. We found that variation of more than one position at once is not feasible since the number of different sequences increases from 20 for a single position to 400 for two positions if all 20 amino acids are considered. The amount of the individual peptides is as a consequence very small and does not allow analysis of complex mixtures due to sensitivity limitation. Furthermore, the substrate concentrations cannot be increased at will because of limits in peptide solubility. The restriction of the sequence variability to one position of a possible recognition sequence at a time also means that one cannot start the analysis from a completely randomized sequence. Usually this is not necessary since recognition sequences for various sortases have been predicted or even confirmed experimentally and can thus be taken as a starting point for the specificity determination. Furthermore, we are aware that the presented method allows investigation of each position individually but may fail


Biochemistry

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to detect more optimal coupled pairs of amino acids. However, no such effect has been reported so far for recognition sequence binding of sortases. In the analysis of BaSrtB we were not only able to determine the sequence specificity with the developed method but also to analyze the sortase activity in relation to the buffer conditions. We performed a pH screen and a screen for bivalent cations and could determine the dependence of BaSrtB activity on these conditions. Additional parameters like temperature, ion strength etc. could be tested as well. Furthermore, with some adaptations the method could also be used to find suitable nucleophiles, which resolve the acyl-enzyme intermediates and are incorporated into the final product. This could be achieved by either using mixtures of biotinylated nucleophiles or as an alternative by using a biotinylated recognition peptide to isolate and analyze products with possible nucleophiles. The advantage of using biotinylated recognition peptides is that one is not restricted in the availability of biotinylated nucleophiles. Finally, the presented method enables the determination of cross-reactivity of a sortase with the recognition sequence of another sortase, especially if trace amounts of formed product have to be detected. This information is important if two sortases are used for the distinct modification of two positions of the same target protein. If one sortase recognizes even to a minor extent both recognition sequences in the target, specificity of the modification is lowered, leading to heterogeneity of the product. By determining the sequence specificity as presented here, it can be detected easily if the second sequence is recognized by a given sortase. Our method has some similarity with a previously developed method where a biotinylated peptide glycolate ester is fused to protein N-termini by using the enzyme subtiligase with the goal to determine N-terminal modifications.45,46 However due to the different necessity for our research we could restrict the work-flow to fewer steps, i. e. we did not need a proteolytic step and analysis could be performed with LC-MS instead of using an LC-MS/MS approach. In addition, recovery of biotinylated peptides from streptavidin beads was achieved by incubation in H2O at 80 °C compared to proteolytic cleavage with TEV protease. Thus, we achieved an efficient workflow, with a limited amount of steps and hands-on time for the entire analysis. We developed the method for the determination of the substrate specificity of sortases by employing the S. aureus SrtA, which was evolved to be highly active. This sortase was chosen since it is well characterized and the substrate specificity is known. The results obtained with the presented method are in good agreement with published data of wild type and evolved SaSrtA,4,17,44 showing the validity of the experimental setup. We were able to characterize the SrtB enzyme from B. anthracis, for which only


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Biochemistry

limited data on the sequence specificity is found in the literature.15,20 This sortase has an estimated 104 fold lower activity than the SrtA variant used for method development. Nevertheless, we were successful in determining the sequence specificity of BaSrtB, indicating the broad applicability of the method. By isolating the transpeptidation products from the substrate and side products, it is possible to dramatically reduce the background, facilitating the analysis of sortases with low activity. Many of the sortases are thought to have much lower activities then the ubiquitous Class A sortases and the developed method offers a simple and robust way to analyze them in detail.


Biochemistry

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Funding This research was funded by the Commission for Technology and Innovation (CTI) of the Swiss federal council, Grant 16312.1 PFLS-LS.

Conflict of Interest The authors declare no competing financial interest.

Supporting Information Comparison of eSaSrtA substrate profile from different studies (Table S1), time dependence of the transpeptidation reaction of LPXTGE with eSaSrtA (Figure S1), time dependence of the hydrolysis reaction of LPXTGE with eSaSrtA (Figure S2), analysis of time dependence of eSaSrtA reactions (Figure S3), detailed view on substrate specificity of eSaSrtA (Figure S4), time dependence of the transpeptidation reaction of DNPXTGDE with BaSrtB (Figure S5), detailed view on substrate specificity of BaSrtB (Figure S6). This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgements We thank the CTI and NBE Therapeutics for funding and the members of the team and the institute for helpful discussions and support with mass spectrometry.


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Biochemistry

References

(1) Tjalsma, H., Antelmann, H., Jongbloed, J. D., Braun, P. G., Darmon, E., Dorenbos, R., Dubois, J. Y., Westers, H., Zanen, G., Quax, W. J., Kuipers, O. P., Bron, S., Hecker, M., and van Dijl, J. M. (2004) Proteomics of protein secretion by Bacillus subtilis: separating the “secrets” of the secretome. Microbiol. Mol. Biol. Rev. 68, 207–233. (2) Tjalsma, H., Bolhuis, A., Jongbloed, J. D., Bron, S., and van Dijl, J. M. (2000) Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol. Mol. Biol. Rev. 64, 515–547. (3) Fernandez-Tornero, C., Lopez, R., Garcia, E., Gimenez-Gallego, G., and Romero, A. (2001) A novel solenoid fold in the cell wall anchoring domain of the pneumococcal virulence factor LytA. Nat. Struct. Biol. 8, 1020–1024. (4) Navarre, W. W., and Schneewind, O. (1994) Proteolytic cleavage and cell wall anchoring at the LPXTG motif of surface proteins in gram-positive bacteria. Mol. Microbiol. 14, 115–121. (5) Pallen, M. J., Lam, A. C., Antonio, M., and Dunbar, K. (2001) An embarrassment of sortases - a richness of substrates? Trends Microbiol. 9, 97–102. (6) Comfort, D., and Clubb, R. T. (2004) A comparative genome analysis identifies distinct sorting pathways in gram-positive bacteria. Infect. Immun. 72, 2710–2722. (7) Bradshaw, W. J., Davies, A. H., Chambers, C. J., Roberts, A. K., Shone, C. C., and Acharya, K. R. (2015) Molecular features of the sortase enzyme family. FEBS J. 282, 2097–2114. (8) Spirig, T., Weiner, E. M., and Clubb, R. T. (2011) Sortase enzymes in Gram-positive bacteria. Mol. Microbiol. 82, 1044–1059. (9) Fischetti, V. A., Pancholi, V., and Schneewind, O. (1990) Conservation of a hexapeptide sequence in the anchor region of surface-proteins from Gram-positive cocci. Mol. Microbiol. 4, 1603–1605. (10) Ton-That, H., Marraffini, L. A., and Schneewind, O. (2004) Protein sorting to the cell wall envelope of Gram-positive bacteria. Biochim. Biophys. Acta 1694, 269–278. (11) Frankel, B. A., Tong, Y., Bentley, M. L., Fitzgerald, M. C., and McCafferty, D. G. (2007) Mutational analysis of active site residues in the Staphylococcus aureus transpeptidase SrtA. Biochemistry 46, 7269–7278. (12) Frankel, B. A., Kruger, R. G., Robinson, D. E., Kelleher, N. L., and McCafferty, D. G. (2005) Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism. Biochemistry 44, 11188–11200. (13) Ton-That, H., and Schneewind, O. (1999) Anchor structure of staphylococcal surface proteins. IV. Inhibitors of the cell wall sorting reaction. J. Biol. Chem. 274, 24316–24320. (14) Severin, A., Tabei, K., and Tomasz, A. (2004) The structure of the cell wall peptidoglycan of Bacillus cereus RSVF1, a strain closely related to Bacillus anthracis. Microb. Drug Resist. 10, 77–82. (15) Zhang, R., Wu, R., Joachimiak, G., Mazmanian, S. K., Missiakas, D. M., Gornicki, P., Schneewind, O., and Joachimiak, A. (2004) Structures of sortase B from Staphylococcus aureus and Bacillus anthracis reveal catalytic amino acid triad in the active site. Structure 12, 1147–1156.


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(16) Weiner, E. M., Robson, S., Marohn, M., and Clubb, R. T. (2010) The Sortase A enzyme that attaches proteins to the cell wall of Bacillus anthracis contains an unusual active site architecture. J. Biol. Chem. 285, 23433–23443. (17) Kruger, R. G., Otvos, B., Frankel, B. A., Bentley, M., Dostal, P., and McCafferty, D. G. (2004) Analysis of the substrate specificity of the Staphylococcus aureus sortase transpeptidase SrtA. Biochemistry 43, 1541–1551. (18) Marraffini, L. A., and Schneewind, O. (2007) Sortase C-mediated anchoring of BasI to the cell wall envelope of Bacillus anthracis. J. Bacteriol. 189, 6425–6436. (19) Elliot, M. A., Karoonuthaisiri, N., Huang, J., Bibb, M. J., Cohen, S. N., Kao, C. M., and Buttner, M. J. (2003) The chaplins: a family of hydrophobic cell-surface proteins involved in aerial mycelium formation in Streptomyces coelicolor. Genes Dev. 17, 1727–1740. (20) Maresso, A. W., Chapa, T. J., and Schneewind, O. (2006) Surface protein IsdC and Sortase B are required for heme-iron scavenging of Bacillus anthracis. J. Bacteriol. 188, 8145–8152. (21) Marraffini, L. A., and Schneewind, O. (2005) Anchor structure of staphylococcal surface proteins. V. Anchor structure of the sortase B substrate IsdC. J. Biol. Chem. 280, 16263–16271. (22) Bierne, H., Garandeau, C., Pucciarelli, M. G., Sabet, C., Newton, S., Garcia-del, P. F., Cossart, P., and Charbit, A. (2004) Sortase B, a new class of sortase in Listeria monocytogenes. J. Bacteriol. 186, 1972–1982. (23) Mariscotti, J. F., Garcia-del, P. F., and Pucciarelli, M. G. (2009) The Listeria monocytogenes sortase-B recognizes varied amino acids at position 2 of the sorting motif. J.Biol.Chem. 284, 6140–6146. (24) Mazmanian, S. K., Ton-That, H., Su, K., and Schneewind, O. (2002) An iron-regulated sortase anchors a class of surface protein during Staphylococcus aureus pathogenesis. Proc. Natl. Acad. Sci. U. S. A. 99, 2293–2298. (25) Budzik, J. M., Oh, S. Y., and Schneewind, O. (2009) Sortase D forms the covalent bond that links BcpB to the tip of Bacillus cereus pili. J. Biol. Chem. 284, 12989–12997. (26) Shaik, M. M., Maccagni, A., Tourcier, G., Di Guilmi, A. M., and Dessen, A. (2014) Structural basis of pilus anchoring by the ancillary pilin RrgC of Streptococcus pneumoniae. J. Biol. Chem. 289, 16988– 16997. (27) Ton-That, H., Mazmanian, S. K., Faull, K. F., and Schneewind, O. (2000) Anchoring of surface proteins to the cell wall of Staphylococcus aureus. Sortase catalyzed in vitro transpeptidation reaction using LPXTG peptide and NH(2)-Gly(3) substrates. J. Biol. Chem. 275, 9876–9881. (28) Jacobitz, A. W., Wereszczynski, J., Yi, S. W., Amer, B. R., Huang, G. L., Nguyen, A. V., Sawaya, M. R., Jung, M. E., McCammon, J. A., and Clubb, R. T. (2014) Structural and computational studies of the Staphylococcus aureus sortase B-substrate complex reveal a substrate-stabilized oxyanion hole. J. Biol. Chem. 289, 8891–8902. (29) Robson, S. A., Jacobitz, A. W., Phillips, M. L., and Clubb, R. T. (2012) Solution structure of the sortase required for efficient production of infectious Bacillus anthracis spores. Biochemistry 51, 7953–7963. (30) Suryadinata, R., Seabrook, S. A., Adams, T. E., Nuttall, S. D., and Peat, T. S. (2015) Structural and biochemical analyses of a Clostridium perfringens sortase D transpeptidase. Acta Crystallogr. D. Biol. Crystallogr. 71, 1505–1513.


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(31) Duong, A., Capstick, D. S., Di, B. C., Findlay, K. C., Hesketh, A., Hong, H. J., and Elliot, M. A. (2012) Aerial development in Streptomyces coelicolor requires sortase activity. Mol. Microbiol. 83, 992– 1005. (32) van Leeuwen, H. C., Klychnikov, O. I., Menks, M. A., Kuijper, E. J., Drijfhout, J. W., and Hensbergen, P. J. (2014) Clostridium difficile sortase recognizes a (S/P)PXTG sequence motif and can accommodate diaminopimelic acid as a substrate for transpeptidation. FEBS Lett. 588, 4325–4333. (33) Kang, H. J., Paterson, N. G., Kim, C. U., Middleditch, M., Chang, C., Ton-That, H., and Baker, E. N. (2014) A slow-forming isopeptide bond in the structure of the major pilin SpaD from Corynebacterium diphtheriae has implications for pilus assembly. Acta Crystallogr. D. Biol. Crystallogr. 70, 1190–1201. (34) Amer, B. R., Macdonald, R., Jacobitz, A. W., Liauw, B., and Clubb, R. T. (2016) Rapid addition of unlabeled silent solubility tags to proteins using a new substrate-fused sortase reagent. J. Biomol. NMR 64, 197–205. (35) Antos, J. M., Popp, M. W., Ernst, R., Chew, G. L., Spooner, E., and Ploegh, H. L. (2009) A straight path to circular proteins. J. Biol. Chem. 284, 16028–16036. (36) Zhang, J., Yamaguchi, S., and Nagamune, T. (2015) Sortase A-mediated synthesis of ligand-grafted cyclized peptides for modulating a model protein-protein interaction. Biotechnol. J. 10, 1499–1505. (37) Stanger, K., Maurer, T., Kaluarachchi, H., Coons, M., Franke, Y., and Hannoush, R. N. (2014) Backbone cyclization of a recombinant cystine-knot peptide by engineered Sortase A. FEBS Lett. 588, 4487–4496. (38) Antos, J. M., Miller, G. M., Grotenbreg, G. M., and Ploegh, H. L. (2008) Lipid modification of proteins through sortase-catalyzed transpeptidation. J. Am. Chem. Soc. 130, 16338–16343. (39) Beerli, R. R., Hell, T., Merkel, A. S., and Grawunder, U. (2015) Sortase Enzyme-Mediated Generation of Site-Specifically Conjugated Antibody Drug Conjugates with High In Vitro and In Vivo Potency. PLOS ONE (Hagemeyer, C. E., Ed.) 10, e0131177. (40) Heck, T., Pham, P. H., Hammes, F., Thony-Meyer, L., and Richter, M. (2014) Continuous monitoring of enzymatic reactions on surfaces by real-time flow cytometry: sortase a catalyzed protein immobilization as a case study. Bioconjug. Chem. 25, 1492–1500. (41) Raeeszadeh-Sarmazdeh, M., Parthasarathy, R., and Boder, E. T. (2015) Site-specific immobilization of protein layers on gold surfaces via orthogonal sortases. Colloids Surf. B Biointerfaces. 128, 457–463. (42) Chen, I., Dorr, B. M., and Liu, D. R. (2011) A general strategy for the evolution of bond-forming enzymes using yeast display. Proc. Natl. Acad. Sci. U. S. A. 108, 11399–11404. (43) Geertsma, E. R. (2013) FX cloning: a versatile high-throughput cloning system for characterization of enzyme variants. Methods Mol. Biol. 978, 133–148. (44) Dorr, B. M., Ham, H. O., An, C., Chaikof, E. L., and Liu, D. R. (2014) Reprogramming the specificity of sortase enzymes. Proc. Natl. Acad. Sci. U. S. A. 111, 13343–13348. (45) Mahrus, S., Trinidad, J. C., Barkan, D. T., Sali, A., Burlingame, A. L., and Wells, J. A. (2008) Global Sequencing of Proteolytic Cleavage Sites in Apoptosis by Specific Labeling of Protein N Termini. Cell 134, 866–876. (46) Yoshihara, H. A. I., Mahrus, S., and Wells, J. A. (2008) Tags for labeling protein N-termini with subtiligase for proteomics. Bioorg. Med. Chem. Lett. 18, 6000–6003.


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For Table of Contents use only

Elucidation of the recognition sequence of Sortase B from Bacillus anthracis by using a newly developed LC-MS based method Chasper Puorger, Salvatore Di Girolamo, Georg Lipps*


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Biochemistry

Figure 1 a b

FITC-LPXTGE + GGGK-biotin

Srt, T = 25 °C

FITC-LPXTGGGK-biotin + GE LPXTGE

LPXTGE

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LPXTGE Srt GGGK-biotin GGGK-biotin LPXTGE LPXTGE GGGK-biotin

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Srt

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Reaction mixture

wash with PBS and H2O

LPXTGE

LPXTGE

GGGK-biotin LPXTGE Srt GGGK-biotin LPXTGE GGGK-biotin GGGK-biotin GGGK-biotin LPXTGE LPXTGE GGGK-biotin

GGGK-biotin

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Biochemistry

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Figure 2 a 450 400 Absorbance at 497 nm

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Biochemistry

Figure 3 a

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b Asn FITC-DNPXTGGGK-biotin Ser Gln 799.277

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Figure 4 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

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Biochemistry

Figure 5 Fluorescence intenisty (a. u.)

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Bacillus anthracis - ACS Publications - American Chemical

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