Solution NMR studies provide structural basis for endotoxin pattern recognition by the innate immune receptor CD14

Abstract

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

The Crystal Structure of Human Soluble CD14 Reveals a Bent Solenoid with a Hydrophobic Amino-Terminal Pocket

“…2A and 2B). Similar to MD-2, previous circular dichroism, tryptophan fluorescence, and NMR studies suggest that LPS binding does not induce large structural changes in human CD14 (50, 80). Given these volume constraints and pocket rigidity, it is unlikely that CD14 can accommodate all the acyl chains of LPS.…”

“…For example, previous work has shown that mutation of E47, which falls near the front of the rim in α1, to either lysine or arginine can block binding of P. gingivalis LPS (85). Solution NMR spectroscopy analyzing soluble CD14 aa 20-171 bound to the Re chemotype of LPS did not have structural assignments available upon publication, but does show high average temperature factor evidence for hydrophilic residues at the rim suggesting local flexibility to accommodate LPS binding (50). Further work to develop a ligand bound CD14 structure would help to clarify the roles of both the hydrophobic binding pocket and the hydrophilic rim residues required for ligand binding.…”

“…Additionally, the mechanisms involved in delivery of these ligands to multiple Toll-like receptor complexes are also unresolved. To address this gap in knowledge, many groups have purified various recombinant forms of soluble CD14 using bacteria, yeast, insect, and human cellular expression systems (12, 41-51), often with the goal of structure determination (49, 50). Currently, only the unliganded crystal structure of mouse CD14, purified from SF9 insect cells, is known (52).…”

Chimeric Anti-CD14 IGG2/4 Hybrid Antibodies for Therapeutic Intervention in Pig and Human Models of Inflammation

“…However, as they block CD14 function, it is likely that they bind to either the N-terminal LPS-binding pocket of CD14 or parts of the LPS-signaling motif. Importantly, the hydrophobic binding pocket can also accommodate other acylated endogenous and exogenous ligands of CD14 and TLRs (49)(50)(51). Therefore anti-CD14 Abs, like r18D11 and rMil2, may affect pattern recognition signaling upon a wide range of threats, being more efficient than, for example, LPS mimics.…”

Membrane CD14, but not soluble CD14, is used by exoenzyme S fromP. aeruginosato signal proinflammatory cytokine production

“…Moreover, other serum molecules, such as LBP, play a role by extracting LPS from the bacterial cell wall, disrupting LPS aggregates through recognition of the lipid component of LPS, and enhancing binding of LPS to sCD14 and mCD14 [4 -6]. Furthermore, other lipid-based TLR agonists, such as the TLR2 agonists lipoteichoic acid and triacylated and diacylated lipopeptides, also use LBP and CD14 in a similar manner to LPS [7][8][9][10].…”