Regulation of a Phage Endolysin by Disulfide Caging

In contrast to canonical phage endolysins, which require holin-mediated disruption of the membrane to gain access to attack the cell wall, signal anchor release (SAR) endolysins are secreted by the host sec system, where they accumulate in an inactive form tethered to the membrane by their N-terminal SAR domains. SAR endolysins become activated by various mechanisms upon release from the membrane. In its inactive form, the prototype SAR endolysin, Lyz_P1, of coliphage P1, has an active-site Cys covalently blocked by a disulfide bond; activation involves a disulfide bond isomerization driven by a thiol in the newly released SAR domain, unblocking the active-site Cys. Here, we report that Lyz₁₀₃, the endolysin of Erwinia phage ERA103, is also a SAR endolysin. Although Lyz₁₀₃ does not have a catalytic Cys, genetic evidence suggests that it also is activated by a thiol-disulfide isomerization triggered by a thiol in the SAR domain. In this case, the inhibitory disulfide in nascent Lyz₁₀₃ is formed between cysteine residues flanking a catalytic glutamate, caging the active site. Thus, Lyz_P1 and Lyz₁₀₃ define subclasses of SAR endolysins that differ in the nature of their inhibitory disulfide, and Lyz₁₀₃ is the first enzyme found to be regulated by disulfide bond caging of its active site.In infections by double-stranded DNA phages, host lysis requires degradation of the peptidoglycan by a phage-encoded endolysin (17). By far the most intensively studied endolysin is the T4 lysozyme E (EC 3.2.1.17), which attacks the glycosidic bonds between GlcNAc and MurNAc in the murein (1). During the latent period, canonical endolysins are produced as fully active enzymes sequestered in the cytoplasm, thereby preventing premature lysis. Another phage protein, the holin, terminates the infection cycle by suddenly forming extremely large, nonspecific holes in the membrane that allow the endolysin to escape and attack the murein layer. Recently, studies of the lysis system of bacteriophage P1 have revealed that phage-encoded endolysins are not always dependent upon holins for export (19, 20). Although it is an ortholog of T4 E, the P1 lysozyme, Lyz_P1, is translocated across the cytoplasmic membrane by the host sec system by virtue of an N-terminal transmembrane domain (TMD) that is absent in E (Fig. ​(Fig.11 A). Since this transmembrane domain is not removed by signal peptidase, nascent Lyz_P1 remains tethered to the membrane with its catalytic residues already present in the periplasm. The Lyz_P1 TMD exits the membrane and becomes part of the soluble, periplasmic form of the protein, either at a low spontaneous rate or, more efficiently, when the holin triggers to depolarize the membrane (11). Because of the unique ability to direct sec-mediated export and membrane insertion and to support release into the periplasm from the bilayer, the TMD of Lyz_P1 was designated a signal anchor release (SAR) domain. More recently, other SAR endolysins have been identified and characterized (15, 16). In fact, bioinformatic analysis suggests that most members of the T4 lysozyme family, recognizable by the Glu-8aa-(Asp/Cys)-5aa-Thr catalytic triad (Fig. ​(Fig.1A),1A), are SAR endolysins (43 of 58 entries in the GenBank protein database) (16).Open in a separate windowFIG. 1.Sequence alignments. (A) Alignment of T4E, Lyz_P1, and Lyz₁₀₃. Catalytic residues are highlighted in blue, Cys residues in pink, and SAR domains in yellow. Locations of Leu substitutions are indicated below the corresponding Gly residue (green). Inactivating/caging disulfides for Lyz_P1 and Lyz₁₀₃, respectively, are shown as arrows connected with a black line above the participating Cys residues. Disulfides resulting from isomerization are shown below the participating Cys residues as arrows connected with a brown line. (B) Alignments of Lyz_P1→103 and Lyz_103→P1 conversion mutants. Catalytic residues are highlighted in blue, Cys residues in pink, and SAR domains in yellow. Locations of Cys substitutions are indicated below the corresponding residue.Since the lyz_P1 gene is expressed well before progeny P1 phage have been assembled, there must be a mechanism to ensure that the membrane-tethered form of the protein is kept enzymatically inactive so that premature lysis is avoided. A key to the regulation of Lyz_P1 is the fact that the P1 enzyme has a catalytic cysteine residue, Cys₅₁ (Fig. ​(Fig.1A),1A), in the central position of the catalytic triad, in contrast to E and most of its orthologs, which have an Asp residue in this position (20). Genetic, biochemical, and structural analysis of Lyz_P1 demonstrated that the membrane-tethered form is inactive for two reasons: first, the entire catalytic domain is misfolded, so the active-site cleft is completely missing, and second, the catalytic Cys₅₁ is covalently occupied in a disulfide bond with another Cys at position 44. This led to a model for activation in which a thiol (Cys₁₃) present in the SAR domain becomes unmasked upon membrane release and triggers a disulfide bond isomerization, liberating the thiol of the catalytic Cys₅₁. This model was confirmed by crystal structures showing the alternative disulfide linkages in the inactive and active forms of Lyz_P1 (19).Lyz_P1 became the prototype of a class of SAR endolysins recognizable by the Asp→Cys substitution in the catalytic triad and the presence of activating Cys in the N-terminal SAR domain. However, most SAR endolysins belong to a second major class, represented by R₂₁, the endolysin of the lambdoid phage 21 (20). These enzymes have the canonical Glu-8aa-Asp-5aa-Thr catalytic triad and no Cys residue in the SAR domain. Instead, genetic and structural analysis revealed that in the inactive form, the catalytic domain has nearly the correct fold, except for a displacement of the active-site Glu, but is subject to steric hindrance by the proximity of the bilayer in which the SAR domain is embedded. In the soluble, active form, the SAR domain of R₂₁ has refolded into the main body of the enzyme, providing a floor to the active site and repositioning the catalytic glutamate to its proper place (16). Thus, the R₂₁ regulatory scheme is markedly different from that of Lyz_P1, where the released SAR domain provides only the free thiol for the disulfide bond rearrangement and makes few contacts with the enzyme itself.Here, we examine the regulation of the endolysin Lyz₁₀₃ of the Erwinia amylovora phage ERA103 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EF160123","term_id":"121621816","term_text":"EF160123"}}EF160123), which seems to have characteristics of both of these major classes: it has a Cys residue in an N-terminal hydrophobic sequence but retains the canonical Asp residue in the catalytic triad. The results are discussed in terms of a model for SAR-dependent disulfide bond isomerization distinct from that of Lyz_P1 and its homologs but which nevertheless confers a covalent constraint on premature activation of the muralytic activity.