Functional analysis of Clostridium difficile sortase B reveals key residues for catalytic activity and substrate specificity

Abstract

Most of Gram-positive bacteria anchor surface proteins to the peptidoglycan cell wall by sortase, a cysteine transpeptidase that targets proteins displaying a cell wall sorting signal. Unlike other bacteria, Clostridium difficile, the major human pathogen responsible for antibiotic-associated diarrhea, has only a single functional sortase (SrtB). Sortase's vital importance in bacterial virulence has been long recognized, and C. difficile sortase B (Cd-SrtB) has become an attractive therapeutic target for managing C. difficile infection. A better understanding of the molecular activity of Cd-SrtB may help spur the development of effective agents against C. difficile infection. In this study, using site-directed mutagenesis, biochemical and biophysical tools, LC-MS/MS, and crystallographic analyses, we identified key residues essential for Cd-SrtB catalysis and substrate recognition. To the best of our knowledge, we report the first evidence that a conserved serine residue near the active site participates in the catalytic activity of Cd-SrtB and also SrtB from Staphylococcus aureus The serine residue indispensable for SrtB activity may be involved in stabilizing a thioacyl-enzyme intermediate because it is neither a nucleophilic residue nor a substrate-interacting residue, based on the LC-MS/MS data and available structural models of SrtB-substrate complexes. Furthermore, we also demonstrated that residues 163-168 located on the β6/β7 loop of Cd-SrtB dominate specific recognition of the peptide substrate PPKTG. The results of this work reveal key residues with roles in catalysis and substrate specificity of Cd-SrtB.

Keywords: Clostridium difficile; crystal structure; cysteine transpeptidase; enzyme catalysis; fluorescence resonance energy transfer (FRET); protein chemistry; protein purification; protein sorting; protein structure; sortase B; substrate specificity.

Figure 1.

Effect of mutation at residue Ser-207 on the enzymatic activity of Cd-SrtB_ΔN26 assessed by FRET-based assay. A, purified Cd-SrtB_ΔN26,WT and Cd-SrtB_ΔN26,S207A (120 μm) were incubated with PPKTG-containing peptide (20 μm) at 37 °C. The fluorescence signals were measured every hour during the first 8 h and at 24 h. B, the ratios of fluorescence units of Cd-SrtB_ΔN26,S207A/Cd-SrtB_ΔN26 at 24 h are shown. C, purified Cd-SrtB_ΔN26,WT, Cd-SrtB_ΔN26,S207A, Cd-SrtB_ΔN26,C209A, and Cd-SrtB_{ΔN26,S207A C209A} (120 μm) were incubated with PPKTG-containing peptide (20 μm) at 37 °C. The fluorescence signals were measured every hour during the first 8 h and at 24 h. D, the ratios of fluorescence units Cd-SrtB_ΔN26,S207A/Cd-SrtB_ΔN26,WT, Cd-SrtB_ΔN26,C209A/Cd-SrtB_ΔN26,WT, and Cd-SrtB_{ΔN26,S207A C209A}/Cd-SrtB_ΔN26,WT at 24 h are shown. The data represent the means ± S.D. of three independent experiments. **, p < 0.01; ****, p < 0.0001; unpaired t test.

Figure 2.

LC-MS/MS analysis for peptide cleavage site. A, MS extracted ion chromatographs (EIC) are shown for cleavage of Dabcyl-PVPPKTGDSTTIIGE-Edans by Cd-SrtB_ΔN26,WT (upper panel) and Cd-SrtB_ΔN26,C209A (lower panel). B, MS/MS spectra of the cleaved substrate peptide from the reaction mixture of Cd-SrtB_ΔN26,WT with PPKTG-containing peptide shows a doubly charged ion at m/z 570.7421 for MH₂²⁺ corresponding to the mass of the cleaved peptide product GDSTTIIGE-Edans. C, the MS/MS spectrum shows a doubly charged ion at m/z 730.8409 for MH₂²⁺ corresponding to the mass of the scrambled peptide PVGSSTPDSTTIIGE incubated with Cd-SrtB_ΔN26,WT. The labeled peaks correspond to masses of y and b ions of the peptide.

Figure 3.

Crystal structure of the Cd-SrtB_ΔN26,S207A and structural comparison with Cd-SrtB_ΔN26,WT. A, overall crystal structure of Cd-SrtB_ΔN26,S207A is shown in blue (left panel). The β6/β7 loop region (residues 162–167) and the β6/β7 loop region (residues 210–216) are disordered in this structure. A zooming structure of Cd-SrtB_ΔN26,S207A around the residue Ser-207 with a F_o − F_c omit electron density map at the 2 σ level is shown in gray chicken wire (right panel). B, superposition of Cd-SrtBΔN26,WT (PDB 5GYJ, yellow) and Cd-SrtBΔN26,S207A (PDB 6KYC, blue) yields an RMSD value of 0.174 for 176 Cα atoms. The side chains of Ser-207 in Cd-SrtB_ΔN26,WT and Ala-207 of Cd-SrtB_ΔN26,S207A are shown as sticks on β7-strand.

Figure 4.

Mutational effects at residues Cys-194 and Ser-192 on enzymatic activity of Sa-SrtB_ΔN29. A, the fluorescence signals were monitored after incubating the purified Sa-SrtB_ΔN29,WT, Sa-SrtB_ΔN26,C194A, Sa-SrtB_ΔN29,S192A, and Sa-SrtB_{ΔN29,S192A C194A} (120 μm) with NPQTN-containing peptide (20 μm), respectively, at 37 °C for every hour during the first 8 h and at 24 h. B, the relative fluorescence signals of Sa-SrtB_ΔN26,C194A, Sa-SrtB_ΔN29,S192A, and Sa-SrtB_{ΔN29, S192A C194A} are significantly lower than WT Sa-SrtB_ΔN29 at 24 h. The data represent the means ± S.D. of three independent experiments. ****, p < 0.0001; unpaired t test.

Figure 5.

Effects of mutation at His-116 and Arg-217 on enzymatic activity of Cd-SrtB. A, purified Cd-SrtB_ΔN26,WT, Cd-SrtB_ΔN26,C209A, Cd-SrtB_ΔN26,R217A, and Cd-SrtB_ΔN26,H116A (120 μm) were incubated with PPKTG-containing peptide (20 μm), respectively, at 37 °C. The fluorescence signals were measured every hour during the first 8 h and at 24 h. B, the ratios of fluorescence units of Cd-SrtB_ΔN26,C209A/Cd-SrtB_ΔN26,WT, Cd-SrtB_ΔN26,R217A/Cd-SrtB_ΔN26,WT, and Cd-SrtB_ΔN26,H116A/Cd-SrtB_ΔN26,WT at 24 h are shown. The data represent the means ± S.D. of three independent experiments. n.s., nonsignificant. **, p < 0.01; ****, p < 0.0001; unpaired t test.

Figure 6.

Crystal structure of Cd-SrtB_ΔN26,R17A and structural comparison with WT Cd-SrtB_ΔN26,WT. A, overall crystal structure of Cd-SrtB_ΔN26,R217A is displayed in green (left panel). The β6/β7 loop region (residues 162–167) and the β6/β7 loop region (residues 210–216) are disordered in this structure. A zooming structure of Cd-SrtB_ΔN26,R217A around residue Arg-217 with a F_o − F_c omit electron density map at the 2 σ level is shown in gray chicken wire (right panel). B, superposition of C_α atoms on the structure of WT Cd-SrtB_ΔN26,WT (PDB code 5GYJ) and Cd-SrtB_ΔN26,R217A are presented in yellow and green, with RMSD = 0.239 for 176 C_α atoms, respectively. The side chains of Arg-217 of Cd-SrtB_ΔN26,WT and Ala217 of Cd-SrtB_ΔN26,R217A are shown as sticks on the β8-strand.

Figure 7.

Substrate specificity of Cd-SrtB_ΔN26,WT and Sa-SrtB_ΔN29,WT. The fluorescence signals were monitored to calculate the enzymatic activity of Cd-SrtB_ΔN26,WT and Sa-SrtB_ΔN29,WT for every hour during the first 8 h and at 24 h at 37 °C with 20 μm peptide substrate PPKTG (A) or NPQTN (B). The data represent the means ± S.D. of three independent experiments. **, p < 0.01; ***, p < 0.001; unpaired t-test.

Figure 8.

Effect of β6/β7 loop substitution on the enzymatic activity of Cd-SrtB_ΔN26,WT by FRET-based assay. Residues 163–168 on β6/β7 loop in Cd-SrtB_ΔN26,WT was replaced with the corresponding residues 177–182 from Sa-SrtB β6/β7 loop. Purified Cd-SrtB_ΔN26,LS was incubated with a peptide containing PPKTG motif (A) or NPQTN motif (B). The fluorescence signals were measured every hour during the first 8 h and at 24 h at 37 °C. The data represent the means ± S.D. of three independent experiments. ***, p < 0.001; ****, p < 0.0001; unpaired t test.