Yang et al. show that a disulfide isoform of HMGB1, with a role in TLR4 signaling, physically interacts with and binds MD-2. MD-2 deficiency in macrophage cell lines or in primary mouse macrophages stimulated with HMGB1 implicates MD-2 in TLR4 signaling. They also identify an HGMB1 peptide inhibitor, P5779, which when administered in vivo can protect mice from acetaminophen-induced hepatoxicity, ischemia/reperfusion injury, and sepsis.

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Villin-type headpiece domains are approximately 70 amino acid modular motifs found at the C terminus of a variety of actin cytoskeleton-associated proteins. The headpiece domain of villin, a protein found in the actin bundles of the brush border epithelium, is of interest both as a compact F-actin binding domain and as a model folded protein. We have determined the high-resolution crystal structures of chicken villin headpiece (HP67) at 1.4 A resolution as well as two mutants, R37A and W64Y, at 1.45 and 1.5 A resolution, respectively. Replacement of R37 causes a 5-fold reduction in F-actin binding affinity in sedimentation assays. Replacement of W64 results in a much more drastic reduction in F-actin binding affinity without significant changes in headpiece structure or stability. The detailed comparison of these crystal structures with each other and to our previously determined NMR structures of HP67 and the 35-residue autonomously folding subdomain in villin headpiece, HP35, provides the details of the headpiece fold and further defines the F-actin binding site of villin-type headpiece domains.

Lipopolysaccharide (LPS) activates the innate immune response through the Toll-like receptor 4 (TLR4)⅐MD-2 complex. A synthetic lipid A precursor, lipid IV A , induces an innate immune response in mice but not in humans. Both TLR4 and MD-2 are required for the agonist activity of lipid IV A in mice, with TLR4 interacting through specific surface charges at the dimerization interface. In this study, we used site-directed mutagenesis to identify the MD-2 residues that determine lipid IV A species specificity. A single mutation of murine MD-2 at the hydrophobic pocket entrance, E122K, substantially reduced the response to lipid IV A . Combining the murine MD-2 E122K with the murine TLR4 K367E/S386K/R434Q mutations completely abolished the response to lipid IV A , effectively converting the murine cellular response to a human-like response. In human cells, however, simultaneous mutations of K122E, K125L, Y41F, and R69G on human MD-2 were required to promote a response to lipid IV A . Combining the human MD-2 quadruple mutations with the human TLR4 E369K/Q436R mutations completely converted the human MD-2/human TLR4 receptor to a murinelike receptor. Because MD-2 residues 122 and 125 reside at the dimerization interface near the pocket entrance, surface charge differences here directly affect receptor dimerization. In comparison, residues 42 and 69 reside at the MD-2/TLR4 interaction surface opposite the dimerization interface. Surface charge differences there likely affect the binding angle and/or rigidity between MD-2 and TLR4, exerting an indirect influence on receptor dimerization and activation. Thus, surface charge differences at the two MD-2/TLR4 interfaces determine the species-specific activation of lipid IV A .Lipopolysaccharide (LPS), also known as endotoxin, activates the innate immune response during Gram-negative bacterial infection. Lipid A, the active component of LPS, anchors LPS to the outer leaflet of the outer membrane of Gram-negative bacteria. When bacteria divide or die, LPS released into local tissue or the circulation can trigger innate immune recognition (1). Lipid A from most species is highly proinflammatory, although certain lipid A molecules and precursors lack stimulatory power. The synthetic precursor of Escherichia coli lipid A, tetraacylated lipid IV A (2) (compound 406), is an agonist in murine cells and a partial agonist in equine cells but is an antagonist in human cells (3).The Toll-like receptor 4 (TLR4) 2 and MD-2 complex constitute the essential components of a receptor system for LPS (4, 5). CD14, initially believed to be the LPS receptor, is an enhancing component of the LPS receptor (6). The lack of a transmembrane or an intracellular signaling domain led to speculation about an additional LPS receptor component that actually transduces the LPS signal. TLR4 was identified as the signaling component of the LPS receptor, based on positional cloning in mice that fail to respond to LPS (4, 7) and on the finding that TLR4 knock-out cells did not respond to LPS (8). The co-recept...

Innate immunity is the first line defense against invading pathogens. During Gram-negative bacterial infection, the Tolllike receptor 4 and MD-2 complex recognize lipopolysaccharide present in the bacterial cell wall. This recognition can be enhanced 100 -1000-fold by CD14. However, the beneficial role provided by CD14 becomes detrimental in the context of sepsis and septic shock. An understanding of how CD14 functions will therefore benefit treatments targeted at both immune suppression and immune enhancement. In the present study, we use site-directed mutagenesis to address the role of disulfide bonds and N-linked glycosylation on CD14. A differential impact is observed for the five disulfide bonds on CD14 folding, with the first two (Cys CD14 is myeloid-specific leucine rich repeat (LRR) 3 protein expressed abundantly on mature monocytes and macrophages (1-3) and at low levels on some neutrophils (4). CD14 is dubbed a pattern recognition receptor because it recognizes multiple pathogen-related molecules (reviewed in Ref. 5) and some common phospholipids (6 -8). The best studied and the most biologically relevant ligand is lipopolysacharide (LPS, also known as endotoxin) from the Gram-negative bacterial cell wall during infection (3). By facilitating the monomeric dissociation of LPS and downstream interaction with the ultimate LPS receptor, the Toll-like receptor 4 (TLR4), and myeloid differentiation protein 2 (MD-2) complex (9, 10), CD14 heightens the sensitivity of the immune system to infection (3,5,(11)(12)(13)(14). This protective role of CD14, however, becomes devastating in the context of septic shock because it exaggerates an already overactive response. Corroborative evidence comes from the fact that CD14 knock-out mice are LPS-insensitive and resistant to septic shock (15, 16), whereas transgenic mice expressing human CD14 are hypersensitive to LPS stimulation (17). An understanding of how CD14 functions would therefore benefit treatment targeted not only at enhancing innate immunity but also at ameliorating septic shock (18, 19).The underlying mechanism by which CD14 facilitates LPS recognition remains obscure. It remains unknown whether a conformational change in CD14 is involved or whether CD14 merely acts as a shuttle between LPS and the TLR4⅐MD-2 complex (13). It remains unknown whether a ternary complex is formed between LPS, CD14 and TLR4⅐MD-2, and if so, whether CD14 interacts directly with MD-2 or TLR4 in the transferring process. It is unlikely that CD14 would participate directly in the signaling cascade, because direct interactions can be observed between lipid A and TLR4⅐MD-2 complex (K d Ϸ 3 nM) (14). With CD14, LPS can be recognized at extremely low concentrations (0.01-1 ng/ml), ϳ2-3 orders of magnitude lower than that without (5,20).Biochemical studies have identified residues on CD14 essential for LPS binding. By deletion mutagenesis, Juan et al. (21) established that residues 57-64 are important for LPS binding. This region is also protected from limited proteolysis after incub...