Bacterial biofilms on medical devices and implants pose a serious health challenge, and the interaction between bacterial surface proteins and abiotic surfaces is an important initial step towards bacterial colonization and biofilm development. The autolysin (AtlE) surface protein of S. epidermidis functions in cell wall homeostasis by catalyzing the hydrolysis of peptidoglycan (PGN) subunits. AtlE is also known to play a direct role in biofilm formation. The amidase domain (Ami) of AtlE is a 26 kDa zinc-dependent metalloenzyme and is primarily responsible for the amide bond cleavage between N acetyl muramic acid and L alanine of the PGN. Ami is thought to play a role in binding to surfaces, but the structural basis of this role is unclear. Here, we have characterized the structure, function, and interaction of the Ami domain with a serum-coated abiotic surface using solution NMR spectroscopy. We find that the RDC-based solution structure of Ami is similar to the previously-determined crystal structure. The ¹⁵N-relaxation rates (R₁, R₂, and hetNOE) indicate motions on various NMR time scales for the active site residues. The Zn²⁺ binding to the Ami domain is characterized by high affinity (K_D ~ 30 nM) and induces a local conformational change around the active site as determined by solution NMR. In contrast, the substrate (muramyl tripeptide) showed a much weaker affinity (K_D ~ 60 μM); however, this peptide induces a line broadening effect on the active site residues upon binding. We find that Ami binds to serum-coated surfaces, and upon binding, it significantly reduces staphylococcal biofilms on those surfaces. Surface binding was further investigated using serum-coated nanoparticles, which allowed a structural characterization using NMR relaxation measurements. These experiments revealed a moderate affinity of Ami for serum coated surfaces with enhanced transverse relaxation rates (R_2,cpmg). Taken together, these results reveal the structural and functional properties of the Ami domain required for normal cell growth, and they also demonstrate its role in biofilm formation.

Rahul Yadav

Mississippi State University

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