Crystal Structures of Human MD-2 and Its Complex with Antiendotoxic Lipid IVa

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Science  15 Jun 2007:
Vol. 316, Issue 5831, pp. 1632-1634
DOI: 10.1126/science.1139111


Endotoxic lipopolysaccharide (LPS) with potent immunostimulatory activity is recognized by the receptor complex of MD-2 and Toll-like receptor 4. Crystal structures of human MD-2 and its complex with the antiendotoxic tetra-acylated lipid A core of LPS have been determined at 2.0 and 2.2 angstrom resolutions, respectively. MD-2 shows a deep hydrophobic cavity sandwiched by two β sheets, in which four acyl chains of the ligand are fully confined. The phosphorylated glucosamine moieties are located at the entrance to the cavity. These structures suggest that MD-2 plays a principal role in endotoxin recognition and provide a basis for antiseptic drug development.

Innate immunity is the first line of defense against microbial infections (1). Defense responses are activated when microbial components are recognized by a variety of pathogen sensors, including the Toll family of receptors, Nod-like receptors, and double-stranded RNA sensors (2). Among microbial components, lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is a potent stimulant of immune responses, and a small difference in LPS structure has a great influence on host responses against Gram-negative bacteria (3). Excessive responses to the endotoxic LPS frequently result in severe sepsis, a rapidly progressing inflammatory disease with up to 29% mortality, leading to more than 215,000 annual deaths in the United States alone (4). Toll-like receptor 4 (TLR4) and MD-2 form a complex, and both have been implicated in LPS recognition because mice lacking either molecule are hyporesponsive to LPS (5, 6).

TLR4 is a type I transmembrane protein consisting of extracellular leucine-rich repeats and a cytoplasmic signaling domain similar to the type I interleukin-1 (IL-1) receptor (1). MD-2 is a 160–amino acid glycoprotein with a16–amino acid secretion signal at the N terminus (7) and represents a class of MD-2–related lipid recognition (ML) proteins that also include mite allergen proteins (8). MD-2 forms a stable complex with TLR4 on the cell surface (9), and MD-2 alone as well as in the complex directly binds to LPS with nanomolar affinity (10). On the other hand, it has also been reported that TLR4 recognizes and binds to LPS (11, 12).

Lipid A, the primary immunostimulatory core of LPS, is diverse in several species (13), and these variations are discriminated by the TLR4–MD-2 complex as endotoxic or as antiendotoxic (14). Lipid A of the Escherichia coli type (Fig. 1) acts as a potent agonist in human macrophage cells and in mouse cells. However, its precursor lipid IVa (15), the tetra-acylated form of lipid A, acts as an antagonist in human cells but as an agonist in mouse cells (16). Despite identification of the complex as the LPS receptor, no information is available on the structures of liganded pathogen sensors. Here, we report crystal structures of human MD-2 and its complex with lipid IVa. The structures suggest that MD-2 plays a principal role in LPS recognition.

Fig. 1.

Chemical structure of lipid IVa. Lipid A has additional XA3′ and XA4′ acyl chains.

We expressed human MD-2 in methyltropic yeast Pichia pastoris, as described (17). Polysaccharide moieties of MD-2 were trimmed off by endoglycosidase treatment, which leaves a single N-acetylglucosamine (NAG) at each glycosylation site. Monomeric MD-2 was crystallized into space group P41212 with a = 53.1 Å, c = 111.5 Å, and one MD-2 molecule contained in the asymmetric unit. The structure of the native MD-2 crystal was determined at 2.0 Å resolution by multiple isomorphous replacement. A cocrystal with the lipid IVa complex was obtained from a mixture of MD-2 and lipid IVa and is nearly isomorphous to the native crystal. The structure of the complex was refined at 2.2 Å resolution. Details of the crystallographic analyses are given in (17).

Residues from Glu17 to Asn160 are defined in the structure with two N-linked NAGs at Asn26 and Asn114. MD-2 is folded into a single domain consisting of two β sheets in the immunoglobulin fold conserved among the ML proteins (8): One sheet consists of three antiparallel β strands, and the other consists of six antiparallel strands (Fig. 2). Between these sheets is a large and deep hydrophobic cavity. It has a volume of 1710 Å3 with approximate dimensions of 15 Å by 8 Å by 10 Å. The β6 and β7 strands line the entrance to the cavity. The cavity makes the span of the two sheets much wider than that in the previously predicted model of MD-2 (18). Three disulfide bridges are located between Cys25 and Cys51, between Cys37 and Cys148, and between Cys95 and Cys105, in contrast to the predicted bridges between Cys25 and Cys148 and between Cys37 and Cys51 (18). The sole free Cys133 is located deep in the cavity and seems not to be involved in the oligomerization that has previously been reported (19).

Fig. 2.

Stereo ribbon model of human MD-2 in complex with lipid IVa. The N terminus is drawn in blue and the C terminus in red. The β strands are indicated with their labels, and some amino acid residue numbers are shown. Bound lipid IVa and NAGs as well as cysteine residues are drawn as ball-and-stick models. The two β sheets are inclined toward each other by about 45°.

In the native structure, unexpected electron densities were observed in the cavity; these were attributed to bound lipidic molecules that presumably copurified with MD-2 (fig. S1A). Three myristic acid molecules were built into the structure (fig. S2). In the structure of the lipid IVa complex, electron densities in the cavity (fig. S1B) were assigned to the different parts of lipid IVa (Fig. 1): two glucosamine, two phosphate, and four fatty acid chains. The glycosylation sites of both Asn26 and Asn114 are distant from the cavity region, indicating that the glycosylation plays a role not in ligand binding, but (presumably) in the secretion and protection of MD-2.

Root mean square (RMS) positional deviations between the superposed native and complexed structures are 0.3 Å for the main-chain atoms and 0.7 Å for all the protein atoms. Thus, the MD-2 structure is not significantly altered upon lipid IVa binding. A major difference is in the side-chain conformation of Lys122 located at the entrance to the cavity: The side chain is shifted toward the second glucosamine moiety XG2 of lipid IVa, with RMS deviations of 0.3 Å for the main-chain atoms and 2.8 Å for the side-chain atoms. Overall B factors for the native and complexed structures are 39 Å2 and 37 Å2, respectively, and a slight decrease (3.3 Å2) in the averaged B factor is noticed for β7 upon complexation.

Details of the interactions between lipid IVa and MD-2 are shownin Fig. 3 and fig. S3. The phosphate and sugar groups are aligned in parallel with β7 in the order XP1, XG1, XG2, and XP2, with an XP1-XP2 distance of 12.5 Å, which explains how the peptide fragment from Phe119 to Lys132 can bind to LPS (20). Residues Phe119 to Gly123 are important for the LPS recognition, and these residues, with the exception of Lys122, are conserved in all the species of MD-2 (21). Three hydrogen bonds to lipid IVa are noticed: Ser120 N to XA1 O1′ (distance of 2.87 Å), Lys122 N to XA3 O1′ (3.07 Å), and Ser120 O to XA3 O3′ (2.66 Å). Water atoms mediating lipid IVa and MD-2 are located at the cavity entrance (Fig. 3). Among a total of 18 lysine and arginine residues of human MD-2, which is highly basic with an isoelectric point value of 8.7, only Lys122 and Arg90 are located in the vicinities of the entrance, and their side chains cover XG2 and XP2. These interactions tether the hydrophilic moiety of lipid IVa to the cavity. Hydrophobic and electrostatic surface potentials in the vicinities of the entrance indicate that the entrance is positively charged and the inside of the cavity is highly hydrophobic (Fig. 4). None of the phosphate groups of lipid IVa, which are reported to be essential to the activation of immune responses (13), are involved in direct hydrogen bonds to MD-2 atoms. The lysine and arginine residues mainly contribute to the attraction of negatively charged lipid IVa.

Fig. 3.

Binding interface to lipid IVa. (A) Ribbon representation of the lipid IVa complex, as viewed from a 40° rotation with respect to Fig. 2. The representation is similarly drawn as in Fig. 2 so as to show that the entrance to the MD-2 cavity is lined by the β6b and β7 strands. (B) Stereo close-up view of the binding interface. Amino acid residues located in the vicinities of the entrance are drawn as ball-and-stick models with their residue labels. The structure of the lipid IVa moiety is similarly drawn in darker gray, O atoms in red, N in blue, C in gray, and P in pink. Water O atoms involved in hydrogen bonds (broken lines) are also depicted: W1 between Gly123 N and XG2 O3, as well as a group of W2, W3, W4, W5, and W6, in which W2 is hydrogen-bonded to Glu92 Oϵ1, W2 to W3, and W3 to XG2 N2.

Fig. 4.

Binding pocket and surface properties of MD-2. MD-2 is viewed from a 90° rotation with respect to Fig. 2, and residues of interest are indicated. (A) Protein surface showing hydrophobic and hydrophilic properties. The lipid IVa structure is removed from the complexed structure. Green and red represent hydrophobicity and hydrophilicity, respectively, and the extent is indicated by color darkness. (B) Electrostatic potential surface. Positive and negative potentials are shown in blue and red, respectively. Bound lipid IVa is drawn as a ball-and-stick representation: O in red, N in blue, C in yellow, and P in green.

Four fatty acid chains of lipid IVa are all deeply confined in the cavity. The XA1 chain is in an extended linear conformation and is stuck deeply into the cavity: Three of its four sides are surrounded by hydrophobic MD-2 side chains. The XA2 chain is also in the cavity and lined up with XA1. The XA3 and XA4 chains are curved, and the regions of XA3 C10′-11′ and XA4 C11′-13′ atoms are loosely packed in the cavity. The tip end of XA4 folds back toward the XG2 moiety, and that of XA3 hangs over XA4. The fatty acid chains in the cavity are packed next to each other through van der Waals contacts, as exemplified in the lipid molecules bound to the GM2 activator protein (22). The packed and confined fatty acid structures are distinct from the extended structures of the fatty acid chains of LPS associated with the membrane-embedded region of the FhuA ferrichrome ion receptor (23). The cavity of MD-2 is divided into four sites on the basis of their interactions to the fatty acid chains: L1 through L4 respectively correspond to XA1 through XA4 (table S3). In the L1, L2, and L3 sites of the native structure, the fatty acid molecules assigned as myristic acids exist in nearly identical configurations to those of XA1, XA2, and XA3, respectively (figs. S2 and S3). The averaged B factor for the lipid IVa molecule is 46 Å2; comparable values are obtained for XA1 (33 Å2), XA2 (46 Å2), and XA3 (41 Å2), with a larger one for XA4 (53 Å2). These suggest that the L1, L2, and L3 sites have higher affinities to fatty acid chains. The surface area accommodating lipid IVa is very wide, 890 Å2. This large value is comparable to that of ligands bound to the antibodies (24) and explains the nanomolar affinity of MD-2 toward LPS (10).

The MD-2 residues essential to the interaction with TLR4 are reported to be Arg90, Lys91, Asp100, Tyr102, Cys95, and Cys105 in the absence of the ligands (25). A synthetic peptide from Cys95 to Cys105, in the oxidized form, exhibits a decrease in LPS-induced activation (18) and is supposed to compete with MD-2 through the interaction with TLR4. These residues are located at the cavity entrance (Fig. 4).

The structure of CD14, which transfers LPS to MD-2, also has a hydrophobic cavity of dimensions nearly equal to those of MD-2 (26); hence, it is presumed to recognize acyl chains of LPS. The only differences between the structures of the antagonist lipid IVa and of the agonist lipid A are two additional acyl chains, XA3′ and XA4′ (Fig. 1). The MD-2 cavity likely could not accommodate more than four acyl chains. When the additional XA3′ chain ester-linked to the XA3 O3′ atom is directed toward the inside of the cavity, the hydrogen bonds of the XA3 O3′ atom to both Ser120 O and to XA1 O1′ are disrupted, and hence the XA3 and XA4 portions are rearranged. This rearrangement would displace some portions of XA3 and XA4 toward the region near Val82, Leu87, and Phe126, which is reported to affect ligand-stimulated TLR4 clustering (27).

Binding sites other than L1 through L4 for the additional acyl chains or conformational changes enlarging the cavity are conceivable for lipid A. The additional lipid A acyl chains displaced from the hydrophobic cavity might be involved in activation upon MD-2 complexation with TLR4, and they may induce the reported oligomerization of TLR4 (28). This activation scheme is consistent with the increased MD-2 affinity to lipid A upon association with TLR4 (29). Recombinant human MD-2 in which Ser57, Leu61, and Lys122 are replaced with the corresponding mouse residues (Thr57, Val61, and Glu122) is reported to be activated by lipid IVa and lipid A (30). The hydroxy Oγ atom of Ser57 in the β4 strand is hydrogen-bonded to Glu53 N, Leu61 is located deep in the cavity, and Lys122 is on the surface of the cavity entrance. The former two replacements would bring subtle changes in the construction of the cavity, and the replacement with the glutamate side chain would change the electrostatic properties of the cavity entrance.

We hypothesize that the MD-2 structure unaltered by lipid IVa binding might be essential to antagonistic properties in human cells. The complexed structure that confines most of lipid IVa suggests that MD-2 plays a principal role in recognizing LPS. Moreover, it provides a basis for structure-based development of antiseptic drugs that might be effective in preventing endotoxin shock.

Supporting Online Material

Materials and Methods

Figs. S1 to S3

Tables S1 to S3

References and Notes

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