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Originally published In Press as doi:10.1074/jbc.M414607200 on January 10, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11347-11351, March 25, 2005
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Crystal Structure of CD14 and Its Implications for Lipopolysaccharide Signaling*{diamondsuit}

Jung-In Kim{ddagger}§, Chang Jun Lee{ddagger}, Mi Sun Jin{ddagger}, Cherl-Ho Lee§, Sang-Gi Paik¶, Hayyoung Lee||**, and Jie-Oh Lee{ddagger}{ddagger}{ddagger}

From the {ddagger}Department of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea, §Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea, Department of Biology, School of Biosciences and Biotechnologies, and ||Institute of Biotechnology, Chungnam National University, Daejeon 305-701, Korea

Received for publication, December 27, 2004 , and in revised form, January 6, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Lipopolysaccharide, the endotoxin of Gram-negative bacteria, induces extensive immune responses that can lead to fatal septic shock syndrome. The core receptors recognizing lipopolysaccharide are CD14, TLR4, and MD-2. CD14 binds to lipopolysaccharide and presents it to the TLR4/MD-2 complex, which initiates intracellular signaling. In addition to lipopolysaccharide, CD14 is capable of recognizing a few other microbial and cellular products. Here, we present the first crystal structure of CD14 to 2.5 Å resolution. A large hydrophobic pocket was found on the NH2-terminal side of the horseshoe-like structure. Previously identified regions involved in lipopolysaccharide binding map to the rim and bottom of the pocket indicating that the pocket is the main component of the lipopolysaccharide-binding site. Mutations that interfere with lipopolysaccharide signaling but not with lipopolysaccharide binding are also clustered in a separate area near the pocket. Ligand diversity of CD14 could be explained by the generous size of the pocket, the considerable flexibility of the rim of the pocket, and the multiplicity of grooves available for ligand binding.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The innate immune response is the first line of defense against microbial infection (1). It uses a group of receptors to distinguish non-self-microbial products from host molecules by recognizing conserved structural patterns in the foreign molecules. Lipopolysaccharide (LPS)1 is an outer membrane glycolipid of Gram-negative bacteria and a well known inducer of the innate immune response (2, 3). It is composed of a hydrophobic lipid A component and the hydrophilic polysaccharides of the core and O-antigen. The lipid A portion represents part of the conserved molecular pattern of LPS and is responsible for most of the LPS-induced biological responses. Engagement of LPS on the host cell initiates strong pro-inflammatory responses that stimulate host defenses but can also lead to a pathological condition, septic syndrome, if the inflammatory responses are amplified and uncontrolled (4). Unfortunately, there is as yet no effective treatment for septic syndrome, which is one of the most common causes of death in intensive care units.

The core receptors recognizing LPS are CD14, Toll-like receptor 4 (TLR4), and MD-2 (5, 6). Binding of LPS to CD14 is enhanced by the serum LBP (LPS-binding protein) (7), which is acutely induced by infection. Since CD14 does not have an intracellular signaling domain, transfer of LPS to another receptor component, the TLR4/MD-2 complex, is required for downstream signaling. TLR4 has a cytoplasmic signaling domain that recruits MyD88, IRAK1/IRAK4, and TRIF to activate the transcription factors AP-1, NF-{kappa}B, and IRF3 (5). Mouse strains that have mutations of the TLR4 gene locus are hyporesponsive to LPS (8–10). MD-2 is associated with the extracellular domain of TLR4 and is required for LPS binding by TLR4.

CD14 is expressed on the surface of myelomonocytic cells as a glycosylphosphatidylinositol-linked glycoprotein or in soluble form in the serum (2). The crucial role of CD14 in LPS signaling has been confirmed with knock-out mice; CD14-deficient mice are highly resistant to septic shock initiated by injection of either LPS or live bacteria (11). The CD14 pathway has been suggested as a therapeutic target because anti-CD14 monoclonal antibodies gave significant protection against septic shock in animal models (12, 13). In addition to the LPS of Gram-negative bacteria, CD14 can bind other microbial products such as peptidoglycan (PGN), lipoteichoic acid, lipoarabinomannan, and lipoproteins (14, 15). Therefore, it has broad ligand specificity and functions as a pattern recognition receptor by recognizing structural motifs in various microbial products (16). The molecular mechanism of ligand binding and transfer by CD14 has been intensively investigated by mutagenesis and epitope mapping of blocking antibodies. However, the structural basis of LPS binding and transfer between receptors remains to be clarified. As a first step to addressing these questions, we undertook structural studies of CD14.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Expression and Purification of Soluble Recombinant CD14 —A cDNA of mouse CD14 (residues 5–313) was amplified by PCR using sense primer, 5'-actacggatcccgagccctgcgagctagacgaggaa-3', and antisense primer, 5'-atccaggatccgcagagtccaaaaagggattt-3'. The PCR products were digested with BamHI and cloned into a modified pAcGP67A baculovirus vector (Pharmingen) that contains the Fc domain of human immunoglobulin G1 and a thrombin site between the NotI and BglII restriction sites. To obtain the recombinant virus, the cloned plasmid was co-transfected with Baculogold (Pharmingen) into SF9 insect cells as described in the manufacturer's protocol. To produce the protein, Hi-5 insect cells (Invitrogen) cultured in SF900II serum-free medium (Invitrogen) were incubated for 3 days with the recombinant virus, and the secreted CD14-Fc fusion protein was purified by protein-A-Sepharose (Peptron, Daejeon) affinity chromatography. The Fc tag of the fusion protein was removed by thrombin digestion at 4 °C. This cloning strategy generates recombinant CD14 with an additional ADPGYLLEFRSGRLVPR sequence at its COOH terminus and an ADP sequence at its NH2 terminus. The cleaved CD14 protein was further purified by Hitrap Q anion exchange chromatography and Superdex-200 gel filtration chromatography (AP Biotech). CD14-containing fractions were pooled and concentrated to 30 mg/ml for crystallization.

Crystallization and Data Collection—Crystals of CD14 were grown at 22 °C using the hanging-drop vapor diffusion method, by mixing 1 µl of the protein solution and 1 µl of crystallization buffer containing 100 mM sodium HEPES (pH 7.5), 1.9 M Li2SO4 and 5mM NiCl2. The crystals belong to space group P212121 with unit cell dimensions, a = 70.5 Å, b = 117.7 Å and c = 102.4 Å. Diffraction data were collected with the F2 beam line of the Cornell High Energy Synchrotron Source (MacCHESS) and the BL41XU beam line of Spring 8, using crystals flash-frozen at -170 °C in the crystallization buffer supplemented with 30% glycerol. The diffraction data were processed with DENZO and SCALEPACK packages (Table I) (17).


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  		TABLE I
Statistics from the crystallographic analysis

 
Structure Determination and Refinement—The crystal structure of CD14 was determined by the multiple isomorphous replacement technique (Table I). Initial phases were calculated using the program MLPHARE (18) with data obtained from crystals soaked in 5 mM thimerosal for 8 h, 5 mM lead acetate for 1 week, or 5 mM potassium osmate (VI) for 18 h. The phases were improved by 2-fold non-crystallographic symmetry averaging and solvent flattening, using the program DM (18). The initial CD14 model was refined with the CNS program (19) and rebuilt manually using the O program (20). Atoms related by 2-fold non-crystallographic symmetry were tightly restrained throughout the refinement (weight = 300 kcal/mol-1 Å2) except for the last modeling and refinement cycle. The refined atomic model contains two CD14 molecules corresponding to residues 5–313 and 200 water molecules. Three N-glycosylation sites with the clear electron densities of two to three carbohydrate residues are present (Fig. 1A). All eight cysteines of CD14 are involved in the formation of disulfide bridges (Fig. 1A). After refinement, strong and continuous electron densities were found inside the NH2-terminal pocket. These are probably derived from a mixture of endogenous lipid like molecules co-purified with CD14 (21).



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  		FIG. 1.
Secondary structure assignment and overall structure of mouse CD14. A, alignment of mouse, human, rabbit, and bovine CD14. Residues deleted in the crystallized CD14 are not shown. Secondary structure assignments are shown schematically above the sequences. N-Glycosylation sites identified in the crystal structure are marked with an asterisk. Cysteines involved in disulfide bridges are linked by broken yellow lines. Residues on the rim of the NH2-terminal pocket are written in yellow, residues on the walls and base of the main pocket are written in orange, and residues on the sub-pocket are written in purple. LPS-binding regions identified by previous mutagenesis experiments (23) are labeled R1–R4 and enclosed within blue boxes. Residues important for LPS signaling (36–38) are enclosed within green boxes and labeled T1–T3. B, overall structure of the CD14 dimer. Two monomers of CD14 in the crystal are colored in gray and cyan. Disulfide bridges are shown in orange. The position of the NH2-terminal pocket is indicated by an arrow.

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Structure Determination and Overall Structure of CD14 — We have determined the crystal structure of mouse CD14 to 2.5 Å resolution. The carboxyl-terminal 32 residues not needed for ligand binding or for signaling were deleted by cloning to improve the ability of the protein to crystallize (Fig. 1A) (22). The monomeric subunit of CD14 contains thirteen {beta} strands, and 11 of them, from {beta}3 to {beta}13, overlap with conserved leucine-rich repeats (LRRs) (Fig. 1). The concave surface of the horseshoe-shaped structure consists of a large {beta}-sheet of 11 parallel and two antiparallel beta strands. The convex surface of CD14 contains both helices and loops, in no regular pattern. As a result, it is rough rather than smooth and contains several grooves and pockets that are crucial for ligand binding (discussed in the following paragraphs). CD14 forms a dimer in the crystallographic asymmetric unit as well as in solution (Fig. 1B and data not shown). Dimerization in the crystal is mediated by residues in {beta}13 and in the loop between {beta}12 and {beta}13. Parallel {beta}-sheets from the two monomers interact in an antiparallel fashion and form a large and continuous {beta}-sheet encompassing the entire CD14 dimer. Surprisingly, the total number of LRR motifs in the CD14 dimer is close to that in TLR4, and therefore the overall shape of the two functionally related proteins is likely to be very similar.

The NH2-terminal Hydrophobic Pocket—The most striking feature of the structure of CD14 is the NH2-terminal pocket. The pocket is located on the side of the horseshoe near the NH2 terminus, and it is entirely hydrophobic except for the rim (Figs. 1B and 2A). The main pocket contains a smaller sub-pocket at the bottom. This sub-pocket is formed by hydrophobic residues from {beta}4–6, {alpha}4, {alpha}5, and connecting loops (Figs. 1A and 2B). It is narrow and deep with dimensions 4.5 Å wide, 9.6 Å long, and 8 Å deep. The bottom and walls of the main pocket are lined with residues from {beta}1–3, {alpha}1–4, and their connecting loops (Fig. 2C). The main pocket is both wide and deep with dimensions, 8 Å wide, 13 Å long, and 10 Å deep. Overall, the pocket including the sub-pocket has a total volume of 820 Å3 and hence is large enough to accommodate at least part of the lipid chains of LPS. The residues on the hydrophilic rim of the main pocket are highly flexible (Fig. 2D). The average temperature factor of the rim residues is 79.9 Å2, significantly higher than 43.4 Å2 of the complete protein. Furthermore, some of the rim residues, Pro22, Lys23, Val54, and Asp55, have different conformations in the two subunits of the crystal asymmetric unit.



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  		FIG. 2.
Structure of the NH2-terminal pocket. A, surface representation of the NH2-terminal pocket. The molecular surface of the pocket is shown in mesh. The hydrophobic and hydrophilic surfaces are colored yellow and green, respectively. Kyte and Doolittle's scale (39) was used for the calculation of hydrophobicity. Stereo views of the sub-pocket (B), wall and base (C), and the rim of the main pocket (D) are shown. The viewing orientation is approximately the same for all figures. The side chains of the pocket residues are shown in cyan. The {alpha}2 helix is omitted for clarity in C.

 
The Pocket Area Is Responsible for LPS Binding—The-binding sites for LPS in CD14 have been intensively studied by mutagenesis and by epitope mapping of antibodies that block LPS binding, and four regions have been identified within the NH2-terminal 65 residues of CD14 (23). Deletion or missense mutations of these regions significantly reduce LPS binding or responsiveness (23–29). We found that all of these regions are clustered around the pocket; region 1 is located close to the wall and region 3 is at the bottom of the pocket, whereas regions 2 and 4 are located on the rim of the pocket (Fig. 3A). Residues from the turn between the {beta}1 and {beta}2 strands constitute region 1. Region 2 is the loop between the {beta}2 strand and the {alpha}1 helix. Monoclonal antibodies that recognize this area inhibited LPS binding (23, 25, 30). Region 3 consists of residues from the {beta}3 strand. Region 3 is the most frequent target of LPS blocking antibodies. At least nine monoclonal antibodies that recognize the {beta}2 and {beta}3 strands reduce binding of LPS by soluble CD14 (23, 25, 30). Region 4 includes residues from the loop connecting {alpha}2 and {alpha}3 helices. This area is labile to proteolysis in the absence of bound LPS but becomes resistant when LPS is bound (31). Furthermore, the antibodies MEM18, CRIS-6, and 6C8 that block LPS binding bind to the same region (23–25). Collectively, these mutagenesis and epitope mapping data strongly suggest that the NH2-terminal pocket is the principal component of the LPS-binding site in CD14.



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  		FIG. 3.
Regions involved in LPS binding and signaling. A, the molecular surface of CD14 is depicted. The four LPS-binding regions, R1–R4, identified by previous mutagenesis experiments are colored in cyan (Fig. 1A). The view is rotated clockwise along the horizontal axis by 60 degrees from that of Fig. 1B. B, regions that interfere with LPS transfer are shown in green and labeled T1–T3 (Fig. 1A). The positions of the secondary structures are indicated. G1 and G2 indicate grooves that may play roles in LPS and/or PGN binding (see "Results and Discussion").

 
Model of LPS Bound to CD14 —LPS is a large amphipathic molecule with a molecular weight ranging from a few thousand to more than 10 thousand (3). It has typically five to seven fatty acid groups with 12–14 carbon atoms attached to the phosphorylated diglucosamine backbone. A long carbohydrate chain of the conserved core and the highly variable O-antigen branches out from the glucosamine backbone. Based on our structural findings and previous biochemical investigations, we propose that the lipid portion of LPS binds to the NH2-terminal pocket. This proposal is based on the following reasoning: 1) as summarized in the previous paragraph, the epitopes of antibodies and mutations that block LPS binding map around the NH2-terminal pocket; and 2) the pocket is the only hydrophobic surface large enough to accommodate lipid portion of LPS.

It is unlikely that binding of LPS induces a global structural change of CD14, since many residues making up the hydrophobic pocket are in conserved LRR motifs, and the overall shape of LRR proteins displays limited variability (32). Besides, it has been reported that binding of LPS induces only minor changes in the tryptophan fluorescence and CD spectrum of CD14 (31). However, LPS binding can induce local structural changes especially within the highly mobile {alpha}2 and {alpha}3 helices and the connecting loops (31).

The long carbohydrate chain of LPS is hydrophilic and negatively charged and must have its own binding site, as previous research has shown that LPS that has been enzymatically delipidized retains some affinity for CD14 (33). Previous studies of the binding of PGN to CD14 in vitro provide clues to the binding site of the carbohydrate portion of LPS, although the biological importance of PGN binding to CD14/TLR2 is under debate (34, 35). The hydrophobic NH2-terminal pocket of CD14 is unlikely to be involved in PGN binding, since PGN is a completely hydrophilic molecule. However, the LPS- and PGN-binding sites must overlap, at least in part, because PGN competes with LPS for binding to CD14 (30). The binding site of PGN appears to be shifted to the COOH-terminal side of the pocket, since deletion mutants of regions 1 and 2 that have a profound effect on LPS binding have only minor effects on PGN binding (Figs. 1A and 3A) (28). On the other hand, deletion of region 4 reduces binding of LPS as well as of PGN, and region 4 is on the COOH-terminal side of the pocket. Furthermore, Dziarski et al. (30) reported that a monoclonal antibody, Leu_M3, specifically reduced the affinity for peptidoglycan without affecting that for LPS. The epitope of this antibody maps to the upper side of the G2 groove formed by the {alpha}5 helix and loops on the far COOH-terminal side of the pocket (Fig. 3A). Collectively, the LPS-binding site of CD14 appears to extend further beyond the NH2-terminal pocket and includes grooves in the neighboring area.

The structural characteristics of the binding site may explain the broad ligand specificity of CD14. Although the hydrophobic bottom and walls of the pocket are rigid, the generous size of the pocket may allow structural variation in the hydrophobic portion of the ligand. Structural diversity in the hydrophilic part of the ligands could be explained by the considerable flexibility of the hydrophilic rim combined with the multiplicity of grooves available for ligand binding.

Regions Responsible for LPS Signaling—To initiate signaling, LPS bound to CD14 should be transferred to the TLR4/MD-2 complex on the cell membrane. Several laboratories have reported CD14 mutants that have only minor defects in LPS binding but have virtually no signaling activity (36–38). They are alanine mutations of Glu7–Asp10, Asp9–Phe13, or Leu91–Glu101 in human CD14 or Pro151–Leu153 in mouse CD14. These regions are labeled T1–T3 in Figs. 1A and 3B. It is noteworthy that they are clustered on the same side of CD14, although most of them are far apart in the primary sequence (Figs. 1A and 3B). The sequences of Glu7–Asp10 and Asp9–Phe13 overlap with region 1 of the LPS blocking mutations. Therefore region 1 appears to play a role in both LPS binding and transfer because some mutations in this area block LPS binding and others LPS transfer. The sequences Leu91–Glu101 and Pro151–Leu153 are the lower parts of the two grooves, G1 and G2. As shown in Fig. 3B, all these mutations are located in the same area near the NH2-terminal pocket. These data suggest that the area close to the pocket plays an important role in the transfer of LPS from CD14 to the TLR4/MD-2 complex.

In conclusion, we present here the first crystal structure of CD14. The structure provides evidence that different regions around the pocket contribute to LPS binding and signaling. Our structural studies provide new insights into the mechanism by which it recognizes LPS and may help in developing therapeutic agents to counteract septic shock syndrome.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1WWL) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by Functional Proteomics Program M102KM010017 from the Ministry of Science of Korea and by Korea Research Foundation Grant KRF-2000-005-D00004). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{diamondsuit} This article was selected as a Paper of the Week. Back

** To whom correspondence may be addressed. Tel.: 82-42-821-7533; Fax: 82-42-822-9690; E-mail: hlee{at}cnu.ac.kr. {ddagger}{ddagger} To whom correspondence may be addressed. Tel.: 82-42-869-2839; Fax: 82-42-869-5839; E-mail: jieoh.lee{at}kaist.ac.kr.

1 The abbreviations used are: LPS, lipopolysaccharide; TLR, toll-like receptor; PGN, peptidoglycan; LRR, leucine-rich repeat. Back


   ACKNOWLEDGMENTS
 
We thank the staff of the Cornell High Energy Synchrotron Source (MacCHESS), the BL41XU beam line of Spring 8, and the 6B beam line of Pohang Accelerator Laboratory for help with data collection.



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 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

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