Structural and Functional Insights Into Skl and Pal Endolysins, Two Cysteine-Amidases With Anti-pneumococcal Activity. Dithiothreitol (DTT) Effect on Lytic Activity

Abstract

We have structurally and functionally characterized Skl and Pal endolysins, the latter being the first endolysin shown to kill effectively Streptococcus pneumoniae, a leading cause of deathly diseases. We have proved that Skl and Pal are cysteine-amidases whose catalytic domains, from CHAP and Amidase_5 families, respectively, share an α3β6-fold with papain-like topology. Catalytic triads are identified (for the first time in Amidase_5 family), and residues relevant for substrate binding and catalysis inferred from in silico models, including a calcium-binding site accounting for Skl dependence on this cation for activity. Both endolysins contain a choline-binding domain (CBD) with a β-solenoid fold (homology modeled) and six conserved choline-binding loci whose saturation induced dimerization. Remarkably, Pal and Skl dimers display a common overall architecture, preserved in choline-bound dimers of pneumococcal lysins with other catalytic domains and bond specificities, as disclosed using small angle X-ray scattering (SAXS). Additionally, Skl is proved to be an efficient anti-pneumococcal agent that kills multi-resistant strains and clinical emergent-serotype isolates. Interestingly, Skl and Pal time-courses of pneumococcal lysis were sigmoidal, which might denote a limited access of both endolysins to target bonds at first stages of lysis. Furthermore, their DTT-mediated activation, of relevance for other cysteine-peptidases, cannot be solely ascribed to reversal of catalytic-cysteine oxidation.

Interrogation of the contribution of (endo)lysin domains to tune their bacteriolytic efficiency provides a novel clue to design superior antibacterials

“…The construction and cloning of the genes encoding for the chimeras are detailed in Section 1.1 of Supplementary material. Lysin expression and purification were carried out as previously described for parental enzymes [33,38]. Purified proteins were analyzed by SDS-PAGE and mass spectrometry [15] and then stored at − 20 • C in the elution buffer.…”

“…Choline titration curves were obtained by collecting spectra at increasing ligand concentration and plotting the ellipticity values at 222 nm as a function of choline concentration [40]. Ligand dissociation constants were calculated by non-linear least square fitting of the Hill equation with one or two sets of binding sites to the titration data [33]. All measurements were performed in PB (20 mM phosphate buffer), pH 7.5, at 20 • C using 4-15 μM lysin.…”

“…S1) and choline binding promotes the formation of catalytically-relevant dimers of similar size and shape [33][34][35]. LytA and Skl also have similar antibacterial activities, although LytA binds choline with higher affinity and forms dimers at lower choline concentrations than Skl [33,36]. Altogether, these traits indicated that the efficiencies of the EADs and the CBDs might be balancing each other.…”

“…The applicability of this strategy to develop more potent antimicrobials was assessed here using Streptococcus pneumoniae (in urgent need of novel antimicrobials according to the WHO [2]) as a bacterial target, and two well-characterized lysins with high structural similarity, identical average net charge, and comparable anti-pneumococcal activity as parental enzymes: LytA (the major pneumococcal autolysin) and Skl (the endolysin from Streptococcus mitis prophage φSK137) [33]. LytA and Skl are N-acetylmuramoyl-L-alanine amidases with an EAD of similar size (Amidase_2 and CHAP type, respectively; Fig.…”

“…The residues defining the choline-binding locus are conserved in both lysins (Fig. S1) and choline binding promotes the formation of catalytically-relevant dimers of similar size and shape [33][34][35]. LytA and Skl also have similar antibacterial activities, although LytA binds choline with higher affinity and forms dimers at lower choline concentrations than Skl [33,36].…”