MD-2 is required for disulfide HMGB1-dependent TLR4 signaling.

Innate immune receptors for pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) orchestrate inflammatory responses to infection and injury. Secreted by activated immune cells or passively released by damaged cells, HMGB1 is subjected to redox modification that distinctly influences its extracellular functions. Previously, it was unknown how the TLR4 signalosome distinguished between HMGB1 isoforms. Here we demonstrate that the extracellular TLR4 adaptor, myeloid differentiation factor 2 (MD-2), binds specifically to the cytokine-inducing disulfide isoform of HMGB1, to the exclusion of other isoforms. Using MD-2-deficient mice, as well as MD-2 silencing in macrophages, we show a requirement for HMGB1-dependent TLR4 signaling. By screening HMGB1 peptide libraries, we identified a tetramer (FSSE, designated P5779) as a specific MD-2 antagonist preventing MD-2-HMGB1 interaction and TLR4 signaling. P5779 does not interfere with lipopolysaccharide-induced cytokine/chemokine production, thus preserving PAMP-mediated TLR4-MD-2 responses. Furthermore, P5779 can protect mice against hepatic ischemia/reperfusion injury, chemical toxicity, and sepsis. These findings reveal a novel mechanism by which innate systems selectively recognize specific HMGB1 isoforms. The results may direct toward strategies aimed at attenuating DAMP-mediated inflammation while preserving antimicrobial immune responsiveness.

Genome-wide expression profiling and mutagenesis studies reveal that lipopolysaccharide responsiveness appears to be absolutely dependent on TLR4 and MD-2 expression and is dependent upon intermolecular ionic interactions.

Lipid A (a hexaacylated 1,4' bisphosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. In this article, we define the role of TLR4 and MD-2 in LPS signaling by using genome-wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulation with peptidoglycan-free LPS and synthetic Escherichia coli lipid A. Of the 1396 genes significantly induced or repressed by any one of the treatments in the wild-type macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin and that both are required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. According to our murine TLR4/MD-2-activation model, the two phosphates on lipid A were predicted to interact extensively with the two positively charged patches on mouse TLR4. When either positive patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were nearly abrogated. However, the MyD88-dependent and -independent pathways were impaired to the same extent, indicating that the adjuvant activity of monophosphorylated lipid A most likely arises from its decreased potential to induce an active receptor complex and not more downstream signaling events. Hence, we concluded that ionic interactions between lipid A and TLR4 are essential for optimal LPS receptor activation.

MD-2 residues tyrosine 42, arginine 69, aspartic acid 122, and leucine 125 provide species specificity for lipid IVA.

Lipopolysaccharide (LPS) activates the innate immune response through the Toll-like receptor 4 (TLR4).MD-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 murine-like 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).

MD-2-mediated ionic interactions between lipid A and TLR4 are essential for receptor activation.

Lipopolysaccharide (LPS) activates innate immune responses through TLR4.MD-2. LPS binds to the MD-2 hydrophobic pocket and bridges the dimerization of two TLR4.MD-2 complexes to activate intracellular signaling. However, exactly how lipid A, the endotoxic moiety of LPS, activates myeloid lineage cells remains unknown. Lipid IV(A), a tetra-acylated lipid A precursor, has been used widely as a model for lipid A activation. For unknown reasons, lipid IV(A) activates proinflammatory responses in rodent cells but inhibits the activity of LPS in human cells. Using stable TLR4-expressing cell lines and purified monomeric MD-2, as well as MD-2-deficient bone marrow-derived macrophages, we found that both mouse TLR4 and mouse MD-2 are required for lipid IV(A) activation. Computational studies suggested that unique ionic interactions exist between lipid IV(A) and TLR4 at the dimerization interface in the mouse complex only. The negatively charged 4'-phosphate on lipid IV(A) interacts with two positively charged residues on the opposing mouse, but not human, TLR4 (Lys(367) and Arg(434)) at the dimerization interface. When replaced with their negatively charged human counterparts Glu(369) and Gln(436), mouse TLR4 was no longer responsive to lipid IV(A). In contrast, human TLR4 gained lipid IV(A) responsiveness when ionic interactions were enabled by charge reversal at the dimerization interface, defining the basis of lipid IV(A) species specificity. Thus, using lipid IV(A) as a selective lipid A agonist, we successfully decoupled and coupled two sequential events required for intracellular signaling: receptor engagement and dimerization, underscoring the functional role of ionic interactions in receptor activation.

Heterogeneity and dynamics in villin headpiece crystal structures.

The villin headpiece domain (HP67) is the C-terminal F-actin-binding motif that confers F-actin-bundling activity to villin, a component of the actin bundles that support the brush-border microvilli. It has been investigated extensively by both experimental and theoretical measurements. Our laboratory, for example, has determined both its NMR and its crystal structures. This study presents the structures of HP67 and its pH-stabilized mutant (H41Y) in a different crystal form and space group. For both constructs, two molecules are found in each asymmetric unit in the new space group P6(1). While one of the two structures (Mol A) is structurally similar to our previously determined structure (Mol X), the other (Mol B) has significant deviations, especially in the N-terminal subdomain, where lattice contacts do not appear to contribute to the difference. In addition, the structurally most different crystal structure, Mol B, is actually closer to the averaged NMR structure. Harmonic motions, as suggested by the B-factor profiles, differ between these crystal structures; crystal structures from the same space group share a similar pattern. Thus, heterogeneity and dynamics are observed in different crystal structures of the same protein even for a protein as small as villin headpiece.

Crystal structure of a pH-stabilized mutant of villin headpiece.

Villin-type headpiece domains are compact F-actin-binding motifs that have been used extensively as a model system to investigate protein folding by both experimental and computational methods. Villin headpiece (HP67) harbors a highly helical, thermostable, and autonomously folding subdomain in the C terminus (HP35), and because of this feature, HP67 is usually considered to be composed of a N- and C-terminal subdomain. Unlike the C-terminal subdomain, the N-terminal subdomain consists mainly of loops and turns, and the folding is dependent upon the presence of the C-terminal subdomain. The pH sensitivity of this subdomain is thought to arise from, at least partially, protonation of H41 buried in the hydrophobic core. Substitution of this histidine with tyrosine, another permissive residue at this position for naturally occurring sequences, increases not only the pH stability of HP67 but also the thermal stability and the cooperativity of thermal unfolding over a wide pH range (0.9-7.5). The crystal structures of wild-type HP67 and the H41Y mutant, determined under the same conditions, indicate that the H41Y substitution causes only localized rearrangement around the mutated residue. The F-actin-binding motif remains essentially the same after the mutation, accounting for the negligible effect of the mutation on F-actin affinity. The hydrogen bond formed between the imidazole ring of H41 and the backbone carbonyl of E14 of HP67 is eliminated by the H41Y mutation, which renders the extreme N terminus of H41Y more mobile; the hydrogen bond formed between the imidazole ring of H41 and the backbone nitrogen of D34 is replaced with that between the hydroxyl group of Y41 and the backbone nitrogen of D34 after the H41Y substitution. The increased hydrophobicity of tyrosine compensates for the loss of hydrogen bonds in the extreme N terminus and accounts for the increased stability and cooperativity of the H41Y mutant.

The differential impact of disulfide bonds and N-linked glycosylation on the stability and function of CD14.

Innate immunity is the first line defense against invading pathogens. During Gram-negative bacterial infection, the Toll-like 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(6)-Cys(17) and Cys(15)-Cys(32)) being indispensable, the third and fourth (Cys(168)-Cys(198) and Cys(222)-Cys(253)) being important, and the last (Cys(287)-Cys(333)) being dispensable. A functional role is observed for the first disulfide bond because the C6A substitution severely reduces the ability of CD14 to confer lipopolysaccharide responsiveness to U373 cells. Two of the four predicted glycosylation sites, asparagines 132 and 263, are actually involved in N-linked glycosylation, resulting in heterogeneity in CD14 molecular weight. Furthermore, glycosylation at Asn(132) plays a role in CD14 trafficking and upstream and/or downstream ligand interactions. When mapped onto the crystal structure of mouse CD14, the first two disulfide bonds and Asn(132) are in close proximity to the initial beta strands of the leucine rich repeat domain. Thus, disulfide bonds and N-linked glycosylation in the initial beta sheets of the inner concave surface of CD14 are crucial for structure and function.

High-resolution crystal structures of villin headpiece and mutants with reduced F-actin binding activity.

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.