Domain Interactions in a Chimeric Dual-domain Lysin Lead to Broad Bactericidal Activity.

Phage-derived lysins represent a novel, non-traditional therapeutic agent against multidrug-resistant bacteria. However, engineering lysins with broad host range remain poorly addressed. Previously, we reported that the chimeric lysin ClyR, which harbors the CHAP catalytic domain of PlyC lysin (PlyCAC) and the SH3b cell-wall binding domain of PlySs2 lysin (PlySb), exhibits an expanded host range. However, the mechanism by which ClyR exhibits expanded bactericidal activity is still not fully understood. Since the structure of PlyCAC is publicly available (PDB code: 4F88), here, we first solve the crystal structure of PlySb using X-ray diffraction, and then use various biophysical methods, such as X-ray diffraction, cross-linking coupled mass spectrometry (CXMS), and small-angle X-ray scattering (SAXS) to analyze the potential full-length structures of ClyR. Our results demonstrate that ClyR exhibits a dynamic conformation supported by multiple inter-domain interactions between the two constituent domains. Mutagenesis and biochemical analysis further support the notion that these inter-domain interactions may modulate the bactericidal activity of ClyR. Altogether, our findings provide novel insights into the action mechanism of ClyR and a better understanding of how inter-domain interactions influence the host range of chimeric lysins.