Isopeptide ligation catalyzed by quintessential sortase A: mechanistic cues from cyclic and branched oligomers of indolicidin

Abstract

The housekeeping transpeptidase sortase A (SrtA) from Staphyloccocus aureus catalyzes the covalent anchoring of surface proteins to the cell wall by linking the threonyl carboxylate of the LPXTG recognition motif to the amino group of the pentaglycine cross-bridge of the peptidoglycan. SrtA-catalyzed ligation of an LPXTG containing polypeptide with an aminoglycine-terminated moiety occurs efficiently in vitro and has inspired the use of this enzyme as a synthetic tool in biological chemistry. Here we demonstrate the propensity of SrtA to catalyze "isopeptide" ligation. Using model peptide sequences, we show that SrtA can transfer LPXTG peptide substrates to the ε-amine of specific Lys residues and form cyclized and/or a gamut of branched oligomers. Our results provide insights about principles governing isopeptide ligation reactions catalyzed by SrtA and suggest that although cyclization is guided by distance relationship between Lys (ε-amine) and Thr (α-carboxyl) residues, facile branched oligomerization requires the presence of a stable and long-lived acyl-enzyme intermediate.

FIGURE 1.

Sortase-mediated transpeptidation reaction. Surface proteins harboring the LPXTG pentapeptide motif are captured as acyl-enzyme intermediates by sortases. A, the intermediate in the case of housekeeping sortase (SrtA) is resolved by terminal amine of the pentaglycine cross-bridge of the peptidoglycan (peptide ligation) leading to cell wall anchoring. B, the intermediate formed from a pilin sortase and a pilus protein is attacked by ϵ-amine of a conserved lysine residue (isopeptide ligation) in the pilin motif of another pilus subunit.

FIGURE 2.

SrtA-catalyzed isopeptide ligation of LPXTG-embedded indolicidin sequence (P4). The reaction of P4 with SrtA was carried out as described under “Materials and Methods.” The reaction mixture was analyzed by RP-HPLC (C18 column, Phenomenex, 100 Å, 5 μ, 4.6 × 250 mm, gradient: 4–72% acetonitrile in 0.1% TFA over 130 min, flow rate: 1 ml/min). The product peaks are numbered 1–6 in order of elution from the column.

FIGURE 3.

Mass spectrometric characterization of peak 1 obtained from reaction of SrtA and P4 (see Fig. 2). A, ES-MS analysis of peak 1 provided an experimental mass of 20338.2 Da, which corresponds to a covalent adduct of SrtA (17854.08 Da) and hydrolyzed peptide (ILPWKWPWWPWRRGGGLPNT, 2502.32 Da). B, MALDI-TOF analysis of the tryptic digest of peak 1 obtained from the RP-HPLC fractionation of the reaction of SrtA with P4 (see supplemental Table S4 for the mass data).

FIGURE 4.

Time course of the reaction of SrtA with P4. The reaction was carried out as described under “Materials and Methods.” An aliquot of the sample was taken at various time points and analyzed by RP-HPLC (conditions same as mentioned in Fig. 2). The product peak identifications (1–6) are based on Fig. 2.

FIGURE 5.

Characterization of the acyl-enzyme intermediate (peak 1) by electrophoresis and Western blotting. A, analysis on a 15% SDS-PAGE followed by Coomassie staining. Lane 1, reaction of SrtA with P4; lane 2, SrtA standard (nickel-nitrilotriacetic acid purified); lane 3, SrtA obtained from RP-HPLC analysis of the reaction of SrtA with P4; lane 4, RP-HPLC purified acyl-enzyme intermediate (peak 1) from reaction of SrtA with P4. B, Western blotting with anti-polyhistidine antibody. Lane 1, reaction of SrtA with P4; lane 2, SrtA standard (nickel-nitrilotriacetic acid purified); lane 3, SrtA obtained from RP-HPLC analysis of the reaction of SrtA with P4; lane 4, RP-HPLC purified acyl-enzyme intermediate (peak 1) from reaction of SrtA with P4.

FIGURE 6.

Reaction of SrtA with Ala analogs of P4. The analogs P5 (W4A), P6 (W6A), and P7 (W4A/W6A), respectively, were reacted with SrtA and analyzed by RP-HPLC (conditions same as mentioned in Fig. 2). A, reaction of SrtA with P5. B, reaction of SrtA with P6. C, reaction of SrtA with P7. The numbers in parentheses indicate the experimental mass (Da) of the respective reaction product.

FIGURE 7.

CD spectra of peptides. The spectra of peptides, P2 (solid line) and P4 (broken line), were recorded at a concentration of about 40 μm using a 0.2-cm path-length cell at 25 °C. The spectra are represented as the mean residue ellipticity (MRE), expressed in degree cm² dmol⁻¹.

FIGURE 8.

Reaction of SrtA with P4 in the presence and absence of the dimer. A, standard reaction of SrtA (0.1 mm) with P4 (0.2 mm). B, reaction of SrtA with P4 as in A but in the presence of 0.4 mm of the dimer. The numbers in parentheses indicate the experimental mass (Da) of the respective reaction products.

FIGURE 9.

Mechanism of the SrtA-catalyzed isopeptide-linked oligomerization of P4. The active site cysteine of SrtA performs a nucleophilic attack on the Thr-Gly bond of the LPXTG motif of a molecule of P4 (red circle) to form an acyl-enzyme intermediate. A, the intermediate undergoes hydrolysis to form the hydrolyzed peptide (green circle). B, intramolecular transpeptidation leads to the formation of a cyclic peptide. C, nucleophilic attack by the ϵ-amine of Lys⁵ of P4 leads to the formation of a branched dimer. D, the acyl-enzyme intermediate is attacked by the ϵ-amine of Lys⁵ of the dimer to produce a trimer. Likewise, the reaction continues to produce tetramer and higher branched oligomers. In general, the number of branched chain (n) may depend on the nature of the substrate and steric factors.