Listeria adhesion protein (LAP) is a housekeeping bifunctional enzyme consisting of N-terminal acetaldehyde dehydrogenase (ALDH) and C-terminal alcohol dehydrogenase (ADH). It aids Listeria monocytogenes in crossing the epithelial barrier through a paracellular route by interacting with its host receptor, heat shock protein 60 (Hsp60). To gain insight into the binding interaction between LAP and Hsp60, LAP subdomain(s) participating in the Hsp60 interaction were investigated.

Listeria adhesion protein (Lap), an alcohol acetaldehyde dehydrogenase (lmo1634) promotes bacterial paracellular translocation through epithelial cell junctions during gastrointestinal phase of infection. Secreted Lap is critical for pathogenesis and is mediated by SecA2 system; however, if strain dependent variation in Lap secretion would affect L. monocytogenes paracellular translocation through epithelial barrier is unknown.

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.

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.