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CD14 functions as a key pattern recognition receptor for a diverse array of gram-negative and grampositive cell-wall components in the host innate immune response by binding to pathogen-associated molecular patterns (PAMPs) at partially overlapping binding site(s). To determine the potential contribution of CD14 residues in this pattern recognition, we have examined using solution NMR spectroscopy the binding of three different endotoxin ligands, Lipopolysaccharide, lipoteichoic acid and a PGN-derived compound, muramyl dipeptide to a 15 N isotopically labeled 152-residue Nterminal fragment of sCD14 expressed in Pichia pastoris. Mapping of NMR spectral changes upon addition of ligands revealed that the pattern of residues affected by binding of each ligand is partially similar and partially different. This first direct structural observation of the ability of specific residue combinations of CD14 to differentially affect endotoxin binding may help explain the broad specificity of CD14 in ligand recognition and provide a structural basis for pattern recognition. Another interesting finding from the observed spectral changes is that the mode of binding may be dynamically modulated and could provide a mechanism for binding endotoxins with structural diversity through a common binding site. KeywordsNMR; human CD14 structure; CD14-endotoxin complex; endotoxin; pattern recognition; pichia pastoris expression; isotopic labeling; cell-based assay; lipopolysaccharide; ReLPS; LTA; BODIPY Clinical manifestation of sepsis typically involves a complex interaction between the host immune system and major bacterial cell wall constituents known as endotoxins. The cluster differentiation antigen, CD14, has been shown to be a key high-affinity cellular receptor for these bacterial endotoxins and plays an important role in endotoxin-induced activation of innate immune cells [1]. In humans, CD14 is constitutively expressed on cell surfaces of monocytes/ macrophages as a 55 kDa membrane-bound protein and also exists in a soluble form (sCD14), in serum and bodily fluids in concentrations of 2-6 μg/ml [2,3]. Recognition and binding of different microbial endotoxins to both mCD14 and sCD14 initiates a signaling cascade mediated by Toll-like receptors (TLRs) that promotes the synthesis and secretion of multiple host-derived inflammatory mediators [4], overactivation of which is ultimately responsible for the deleterious effects of sepsis. Understanding mechanism of endotoxin recognition is