Protocol to reveal the binding partner of secreted housekeeping enzymes in Listeria monocytogenes via in silico prediction to in vivo validation.

Numerous interacting protein partners exist without recognized interactive domains, necessitating a standardized methodology to decipher more in-depth interaction profiles. Here, we present a protocol to reveal the binding partner of a secreted housekeeping enzyme, alcohol acetaldehyde dehydrogenase (Listeria adhesion protein), in Listeria monocytogenes through in silico modeling and in vivo experiments. We describe steps for target protein modeling, biophysical profiling, ClusPro docking optimization, protein variant modeling, and docking comparison. We then provide detailed procedures for in vitro and in vivo protein binding validation. For complete details on the use and execution of this protocol, please refer to Liu et al.1.

Cell-surface anchoring of Listeria adhesion protein on L. monocytogenes is fastened by internalin B for pathogenesis.

Listeria adhesion protein (LAP) is a secreted acetaldehyde alcohol dehydrogenase (AdhE) that anchors to an unknown molecule on the Listeria monocytogenes (Lm) surface, which is critical for its intestinal epithelium crossing. In the present work, immunoprecipitation and mass spectrometry identify internalin B (InlB) as the primary ligand of LAP (KD ∼ 42 nM). InlB-deleted and naturally InlB-deficient Lm strains show reduced LAP-InlB interaction and LAP-mediated pathology in the murine intestine and brain invasion. InlB-overexpressing non-pathogenic Listeria innocua also displays LAP-InlB interplay. In silico predictions reveal that a pocket region in the C-terminal domain of tetrameric LAP is the binding site for InlB. LAP variants containing mutations in negatively charged (E523S, E621S) amino acids in the C terminus confirm altered binding conformations and weaker affinity for InlB. InlB transforms the housekeeping enzyme, AdhE (LAP), into a moonlighting pathogenic factor by fastening on the cell surface.