The 1.4 Å Crystal Structure of the Human Oxidized Low Density Lipoprotein Receptor Lox-1

The lectin-like oxidized low density lipoprotein receptor-1 (Lox-1) mediates the recognition and internalization of oxidatively modified low density lipoprotein by vascular endothelial cells. This interaction results in a number of pro-atherogenic cellular responses that probably play a significant role in the pathology of atherosclerosis. The 1.4 Å crystal structure of the extracellular C-type lectin-like domain of human Lox-1 reveals a heart-shaped homodimer with a ridge of six basic amino acids extending diagonally across the apolar top of Lox-1, a central hydrophobic tunnel that extends through the entire molecule, and an electrostatically neutral patch of 12 charged residues that resides next to the tunnel at each opening. Based on the arrangement of critical binding residues on the Lox-1 structure, we propose a binding mode for the recognition of modified low density lipoprotein and other Lox-1 ligands. The lectin-like oxidized low density lipoprotein receptor-1 (Lox-1) mediates the recognition and internalization of oxidatively modified low density lipoprotein by vascular endothelial cells. This interaction results in a number of pro-atherogenic cellular responses that probably play a significant role in the pathology of atherosclerosis. The 1.4 Å crystal structure of the extracellular C-type lectin-like domain of human Lox-1 reveals a heart-shaped homodimer with a ridge of six basic amino acids extending diagonally across the apolar top of Lox-1, a central hydrophobic tunnel that extends through the entire molecule, and an electrostatically neutral patch of 12 charged residues that resides next to the tunnel at each opening. Based on the arrangement of critical binding residues on the Lox-1 structure, we propose a binding mode for the recognition of modified low density lipoprotein and other Lox-1 ligands. Atherosclerosis is understood to be a disease of chronic vascular inflammation resulting from the interaction of oxidatively modified low density lipoprotein (oxLDL) 1The abbreviations used are: oxLDL, oxidatively modified low density lipoprotein; LDL, low density lipoprotein; CTLD, C-type lectin-like domain; Lox-1, lectin-like oxidized low-density lipoprotein receptor-1. 1The abbreviations used are: oxLDL, oxidatively modified low density lipoprotein; LDL, low density lipoprotein; CTLD, C-type lectin-like domain; Lox-1, lectin-like oxidized low-density lipoprotein receptor-1. with macrophages, lymphocytes, and various cellular components of artery walls, including vascular endothelial cells (1.Glass C.K. Witztum J.L. Cell. 2001; 104: 503-516Abstract Full Text Full Text PDF PubMed Scopus (2576) Google Scholar, 2.Libby P. Nature. 2002; 420: 868-874Crossref PubMed Scopus (6841) Google Scholar). oxLDL causes vascular endothelial cell activation and dysfunction, resulting in pro-inflammatory responses, pro-oxidative conditions, and apoptosis, all of which are pro-atherogenic. The lectin-like oxidized low-density lipoprotein receptor-1 (Lox-1) has been characterized as the primary receptor for oxLDL on the surface of vascular endothelial cells and is up-regulated in atherosclerotic lesions (3.Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Aiba Y. Tanaka T. Miwa S. Katsura Y. Kita T. Masaki T. Nature. 1997; 386: 73-77Crossref PubMed Scopus (1150) Google Scholar, 4.Kataoka H. Kume N. Miyamoto S. Minami M. Moriwaki H. Murase T. Sawamura T. Masaki T. Hashimoto N. Kita T. Circulation. 1999; 99: 3110-3117Crossref PubMed Scopus (395) Google Scholar). It is also expressed to a lower extent in macrophages, smooth muscle cells, and dendritic cells. Upon recognition of oxLDL, Lox-1 is observed to initiate oxLDL internalization and degradation as well as the induction of a variety of pro-atherogenic cellular responses including a reduction of nitric oxide (NO) release (5.Cominacini L. Rigoni A. Pasini A.F. Garbin U. Davoli A. Campagnola M. Pastorino A.M. Lo Cascio V. Sawamura T. J. Biol. Chem. 2001; 276: 13750-13755Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), secretion of monocyte chemoattractant protein-1 (MCP-1) (6.Li D. Mehta J.L. Circulation. 2000; 101: 2889-2895Crossref PubMed Scopus (368) Google Scholar), production of reactive oxygen species (7.Cominacini L. Pasini A.F. Garbin U. Davoli A. Tosetti M.L. Campagnola M. Rigoni A. Pastorino A.M. Lo Cascio V. Sawamura T. J. Biol. Chem. 2000; 275: 12633-12638Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar), expression of matrix metalloproteinases-1 and -3 (8.Li D. Liu L. Chen H. Sawamura T. Ranganathan S. Mehta J.L. Circulation. 2003; 107: 612-617Crossref PubMed Scopus (203) Google Scholar), monocyte adhesion (6.Li D. Mehta J.L. Circulation. 2000; 101: 2889-2895Crossref PubMed Scopus (368) Google Scholar), and apoptosis (9.Li D. Mehta J.L. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1116-1122Crossref PubMed Scopus (313) Google Scholar). In addition, Lox-1 expression is up-regulated by various elicitors of vascular stress, including oxLDL (9.Li D. Mehta J.L. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1116-1122Crossref PubMed Scopus (313) Google Scholar), reactive oxygen species (10.Nagase M. Ando K. Nagase T. Kaname S. Sawamura T. Fujita T. Biochem. Biophys. Res. Commun. 2001; 281: 720-725Crossref PubMed Scopus (116) Google Scholar) and fluid shear stress (11.Murase T. Kume N. Korenaga R. Ando J. Sawamura T. Masaki T. Kita T. Circ. Res. 1998; 83: 328-333Crossref PubMed Scopus (187) Google Scholar), suggesting that Lox-1 may help amplify oxLDL-induced vascular dysfunction. Moreover, Lox-1 is a multifunctional receptor involved in several other cellular events. In addition to binding oxLDL, Lox-1 is reported to be a dendritic cell receptor for the 70-kDa heat shock protein involved in antigen cross-presentation to naive T cells (12.Delneste Y. Magistrelli G. Gauchat J. Haeuw J. Aubry J. Nakamura K. Kawakami-Honda N. Goetsch L. Sawamura T. Bonnefoy J. Jeannin P. Immunity. 2002; 17: 353-362Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar) and a receptor for advanced glycation end products (13.Jono T. Miyazaki A. Nagai R. Sawamura T. Kitamura T. Horiuchi S. FEBS Lett. 2002; 511: 170-174Crossref PubMed Scopus (114) Google Scholar), monocytes (14.Hayashida K. Kume N. Minami M. Kita T. FEBS Lett. 2002; 511: 133-138Crossref PubMed Scopus (42) Google Scholar), apoptotic cells (15.Oka K. Sawamura T. Kikuta K. Itokawa S. Kume N. Kita T. Masaki T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9535-9540Crossref PubMed Scopus (338) Google Scholar), and both Gram-negative and Gram-positive bacteria (16.Shimaoka T. Kume N. Minami M. Hayashida K. Sawamura T. Kita T. Yonehara S. J. Immunol. 2001; 166: 5108-5114Crossref PubMed Scopus (149) Google Scholar). Lox-1 is a member of the scavenger receptor family, a structurally diverse group of cell surface receptors of the innate immune system that recognize modified lipoproteins. It is a disulfide-linked homodimeric type II transmembrane protein with a short 34-residue cytoplasmic region, a single transmembrane region, and an extracellular region consisting of an 80-residue domain predicted to be a coiled coil followed by a 130-residue C-terminal C-type lectin-like domain (CTLD) (3.Sawamura T. Kume N. Aoyama T. Moriwaki H. Hoshikawa H. Aiba Y. Tanaka T. Miwa S. Katsura Y. Kita T. Masaki T. Nature. 1997; 386: 73-77Crossref PubMed Scopus (1150) Google Scholar, 17.Xie Q. Matsunaga S. Niimi S. Ogawa S. Tokuyasu K. Sakakibara Y. Machida S. DNA Cell Biol. 2004; 23: 111-117Crossref PubMed Scopus (47) Google Scholar). Deletion analysis has localized oxLDL recognition to the highly conserved (61–83% sequence identity) CTLD of Lox-1 (18.Chen M. Inoue K. Narumiya S. Masaki T. Sawamura T. FEBS Lett. 2001; 499: 215-219Crossref PubMed Scopus (45) Google Scholar). The human Lox-1 CTLD shares its highest sequence identity (35–42%) with the leukocyte-expressed immunoreceptors dectin-1, NKG2D, DC-SIGN, and DC-SIGNR. Like NKG2D, Lox-1 does not have any of the conserved calcium binding residues observed in classic CTLDs, such as mannose binding protein, and is not known to bind carbohydrate. The oxidative modification of low density lipoprotein (LDL) results in an increased net negative charge and potential conformational rearrangements of ApoB100 domains (19.Esterbauer H. Ramos P. Rev. Physiol. Biochem. Pharmacol. 1995; 127: 31-64Crossref Google Scholar, 20.Parasassi T. Bittolo-Bon G. Brunelli R. Cazzolato G. Krasnowska E.K. Mei G. Sevanian A. Ursini F. Free Radic. Biol. Med. 2001; 31: 82-89Crossref PubMed Scopus (61) Google Scholar). Although several positively charged Lox-1 residues are known to play a role in the recognition of modified LDL (18.Chen M. Inoue K. Narumiya S. Masaki T. Sawamura T. FEBS Lett. 2001; 499: 215-219Crossref PubMed Scopus (45) Google Scholar, 21.Chen M. Narumiya S. Masaki T. Sawamura T. Biochem. J. 2001; 355: 289-296Crossref PubMed Scopus (80) Google Scholar, 22.Shi X. Niimi S. Ohtani T. Machida S. J. Cell Sci. 2001; 114: 1273-1282Crossref PubMed Google Scholar), the lack of a three-dimensional structure for Lox-1 has hindered our understanding of the binding mode(s) employed by Lox-1. A detailed understanding of this interaction could be of significant medical interest, because antagonists could potentially mitigate the progression of atherosclerosis. In an effort to begin elucidating the molecular recognition mechanisms of Lox-1, we have determined the crystal structure of the human Lox-1 CTLD in two crystal forms refined to 1.4 and 3.0 Å respectively. Plasmid Construction—cDNA encoding human Lox-1 was amplified by PCR from a pDNR-LIB plasmid (Open Biosystems) to create the constructs Lox1R136 and Lox1A142 (residues 136–273 and 142–273, respectively). These constructs were respectively subcloned into a pET15b expression vector (Novagen) downstream of a histidine tag and a thrombin cleavage site. The cDNA for leaderless Escherichia coli chaperone/disulfide isomerase DsbC was amplified by PCR from a pET40 plasmid (Novagen) and subcloned downstream of each Lox-1 construct. A separate ribosome binding site was added just upstream of DsbC to facilitate translation. Plasmids pET15b-Lox1R136-DsbC and pET15b-Lox1A142-DsbC were transformed into Origami B (DE3) E. coli (Novagen). Human Lox-1 R136 and A142 were expressed using 0.4 mm isopropyl β-d-thiogalactoside in E. coli cells grown in LB medium containing kanamycin, tetracycline, and carbenicillin at 23 °C, with 250 rpm agitation, for 20 h. Purification and Thrombin Digestion—Each of the human Lox-1 fragments were purified by passage through a nickel-nitrilotriacetic acid column followed by a cation exchange column. In brief, cells were harvested by centrifugation and resuspended in sonication buffer (50 mm NaH2PO4, pH 8.0, 300 mm NaCl, and 15 mm imidazole), sonicated on an ice-water bath, and centrifuged for 25 min at 22,000 × g. Supernatant was applied to a nickel-nitrilotriacetic acid column (Qiagen). The column was then washed with a linear gradient from 20 mm to 250 mm imidazole. The elutions were pooled and concentrated with a 10-kDa cutoff ultrafiltration unit (Amicon), and the buffer was changed to phosphate-buffered saline for thrombin digestion. A ratio of 0.1 unit of thrombin to 1 mg of Lox-1 was applied to the sample and incubated on room temperature for 2 h. Thrombin-digested protein was applied to a nickel-nitrilotriacetic acid column, and the flow-through was loaded on a HiPrep S 16/10 column (Amersham Biosciences) equilibrated with 25 mm HEPES, pH 7.5. Protein was eluted with a linear gradient from 100 mm to 500 mm NaCl. Fractions containing protein were concentrated to 20 mg/ml with an ultrafiltration unit (Amicon). Covalent dimerization of the Arg-136 fragment was confirmed by SDS-PAGE and mass spectrometry under reducing and nonreducing conditions. Crystallization—Crystals of Lox-1 were grown by vapor diffusion using 2 μl of 20 mg/ml protein and an equal volume of precipitant and were fully grown within 24 h. Lox-1R136 crystals grew from two different crystallization conditions yielding the same monoclinic spacegroup (C2) and identical cell dimensions (a = 71.0 Å, b = 49.1 Å, c = 76.3 Å, β = 98.5°). In the first condition, crystals were grown at 4 °C using a precipitant solution containing 0.1 m Bicine, pH 9.0, 5% dioxane, and 10% polyethylene glycol 10,000 (w/v). For the second condition (referred to as “dioxane free” in the text), the same protein was crystallized at room temperature in a precipitant solution containing 0.2 m ammonium acetate, 0.1 m sodium acetate, pH 4.6, and 30% polyethylene glycol 3000. Crystals of Lox1A142 grew in a trigonal space group (P3121, a = b = 97.7 Å, c = 215.1 Å, γ = 120°) by the addition of 0.1 m ammonium acetate, 0.1 m Bis-Tris, pH 5.5, and 17% polyethylene glycol 10,000 at room temperature. Data Collection and Structure were to containing an 30% A from the first crystal condition of human Lox-1 R136 with 1.4 Å was on a at the at the Data from the monoclinic crystals of Lox-1 R136 and trigonal crystals of were with a and an Data were with 1997; 276: Scopus Google Scholar). The structure of Lox-1 Arg-136 was by the molecular using 4 D. PubMed Scopus Google Scholar). A was by the CTLD of and into a single using T. J. N. Res. 2003; 31: PubMed Scopus Google Scholar). was in using a an and with of P. J. M. T. D. 1998; PubMed Scopus Google Scholar). were were using The structure of human was also determined by molecular with A of the refined Lox-1 Arg-136 A with was used as the The were refined with using and and of the Lox-1 were using S. M. A. PubMed Scopus Google Scholar). Data and for all crystals are in were using L. The Scholar). and surface was using 1997; PubMed Scopus Google Scholar) and 4 D. PubMed Scopus Google Scholar), respectively. The volume was determined using with a Å Biol. PubMed Scopus Google R136 R136 = b = c = = γ = β = = b = c = = γ = β = = b = c = = β = γ = to for the highest to for the highest to for the highest = is the of of with of to for the highest to for the highest is with of 5% of the as the at the of of in to for the highest = is the of of with is with of 5% of the as the at the of in a six Lox-1 CTLD were from and with Res. PubMed Scopus Google Scholar). of the human Lox-1 CTLD fragments were with the E. coli chaperone/disulfide isomerase DsbC in the of cells, a E. coli with a and This expression to mg of Lox-1 of the for from the of A CTLD fragment (residues was crystallized in the monoclinic spacegroup with in the Data were with and 5% A second Lox-1 fragment two residues the (residues crystallized in a trigonal spacegroup with in the unit and to 3.0 crystal forms were using molecular and and the in this to the monoclinic with Human Lox-1 forms a heart-shaped homodimer with a tunnel through the of the a and from the the Lox-1 has a CTLD consisting of two and by two and a and The is by conserved and A at in human Lox-1, forms an the at the of the In of structure, Lox-1 is highly to a of and in of Å for 100 low sequence with Lox-1 of for several The conformational in Lox-1 are observed in the and the The second of the Lox-1 has two different in the A and B in the of which any of the in any other known CTLD B crystal at suggesting an from crystal In addition, both the C-terminal end of and the are from the in other These to an for Lox-1 Lox-1 of the within the Lox-1 homodimer is to that observed in the cell receptors NKG2D, and in that the the and the C-terminal of the each other with two are observed in the human Lox-1 to the NKG2D, and the Lox-1 are from each other by Å in the of the two in the to the of the This is in by an of the C-terminal to the by two to residues to other a the not to each other to a across the as in other CTLD A is observed in both the and that is in the to Lox-1, and a across the is as in this forms a 20 Å tunnel through the of the a and This is a not observed in any other CTLD The tunnel is by the and the that the from each The is of residues and results in a surface of The Lox-1 arrangement is by including the a in A and a conserved to Lox-1 that forms an of the hydrophobic just the two conserved and and involved in an with a of protein These in a of just the tunnel The at the are to be conserved across species for the at Lox-1 have at this Lox-1 tunnel is Å in for a by the of and that the of the tunnel to a of 4 Å In addition, the at end of the tunnel to to the a into A. B has on its of the tunnel because the The amino acid that the of the tunnel are and highly conserved and and are the residues that a potential through It is that is a histidine in the other known Lox-1 other residues are to through to potential tunnel The tunnel are with and A of the Å trigonal crystal of Lox-1 with the monoclinic crystal reveals tunnel The tunnel in the monoclinic is not observed in any of the in the trigonal a the and is observed in the of the trigonal These are by different for the and In addition, the in the trigonal crystal have with resulting in tunnel Lox-1 structure was observed to have a within the tunnel oxygen is to the of The other oxygen is through two to the of and the of and The the of and A crystal structure of Lox-1 in the same space group reveals conformational to Lox-1 to the does not the of the does bind with an apolar Lox-1 tunnel to a molecule, a acid a to The Lox-1 tunnel may recognize the of that have of are in oxLDL a acid of a a resulting in a short to its S. Witztum J.L. D. J. Res. 2000; Full Text Full Text PDF PubMed Google Scholar, Y. A. N. M. S. T. K. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). of may also potential ligands. The scavenger receptor was to recognize that a at the E. R. Y. M. L. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, short products such as and ApoB100 with that may bind the Lox-1 tunnel as does K. Free Radic. Biol. Med. 2000; PubMed Scopus Google Scholar). Lox-1 recognition of ApoB100 is with a that Lox-1 ApoB100 from oxLDL H. Kume N. Sawamura T. Aoyama T. Hoshikawa H. H. E. Masaki T. Kita T. Arterioscler. Thromb. Vasc. Biol. 1998; PubMed Scopus Google Scholar). Moreover, are on the of apoptotic cells A. S. P. Witztum J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar) and advanced glycation end K. Free Radic. Biol. Med. 2000; PubMed Scopus Google Scholar), the that the tunnel could in the recognition of Lox-1 ligands. for the tunnel ApoB100 fragments to of the are with each consisting of a surface and basic respectively that are to be to a each six to a region to as the to each tunnel A the just each tunnel and is the of the other known Lox-1 the human Lox-1 structure that the number of and including that to the same The electrostatically neutral of this highly charged patch the potential for with the observed on the surface of LDL, such as for top of Lox-1 the and is and has a basic A ridge of six and from each and two diagonally across the entire and at each end of the six and are conserved as basic residues in other species of Lox-1 of Lox-1 to the crystal structure reveals that each Lox-1 species has two to basic residues at each end and two to basic residues to six the basic ridge in addition to the basic of the is the top and of both human and Lox-1 have of conserved basic residues as a significant role in recognition of LDL oxLDL, respectively (18.Chen M. Inoue K. Narumiya S. Masaki T. Sawamura T. FEBS Lett. 2001; 499: 215-219Crossref PubMed Scopus (45) Google Scholar, 21.Chen M. Narumiya S. Masaki T. Sawamura T. Biochem. J. 2001; 355: 289-296Crossref PubMed Scopus (80) Google Scholar, 22.Shi X. Niimi S. Ohtani T. Machida S. J. Cell Sci. 2001; 114: 1273-1282Crossref PubMed Google Scholar). The Lox-1 crystal structure reveals that the first residues to the end and the second including residues and to the basic ridge and basic residues to the surface region as 1 in and Lox-1, and and in and Lox-1, respectively. The and which at the of the patch the and and of two to residues within any of are observed to a reduction of Lox-1 binding to modified LDL (18.Chen M. Inoue K. Narumiya S. Masaki T. Sawamura T. FEBS Lett. 2001; 499: 215-219Crossref PubMed Scopus (45) Google Scholar, 21.Chen M. Narumiya S. Masaki T. Sawamura T. Biochem. J. 2001; 355: 289-296Crossref PubMed Scopus (80) Google Scholar, 22.Shi X. Niimi S. Ohtani T. Machida S. J. Cell Sci. 2001; 114: 1273-1282Crossref PubMed Google Scholar). This a binding that two residues from each of the basic modified LDL has a negative charge because of the of and histidine from ApoB100 and the addition of such as (19.Esterbauer H. Ramos P. Rev. Physiol. Biochem. Pharmacol. 1995; 127: 31-64Crossref Google Scholar, K. Free Radic. Biol. Med. 2000; PubMed Scopus Google Scholar, D. J. 1999; PubMed Scopus Google Scholar). is that critical basic residues on Lox-1 1 and with modified ApoB100 on It is that advanced glycation end modification of a negative as does the of on apoptotic cells and the of within the of various of and acids on the of Gram-positive bacteria K. Free Radic. Biol. Med. 2000; PubMed Scopus Google Scholar, PubMed Google Scholar). not be to that the conserved basic of Lox-1 also play a role in the recognition of other Lox-1 ligands. of the also a hydrophobic patch of 15 highly conserved residues an and It is that a of the conserved to within the hydrophobic patch next to and the first basic has been observed to recognition of LDL by human Lox-1 X. Ogawa S. T. Machida S. Cell. Biol. Res. Commun. 2001; PubMed Scopus Google Scholar). In of was to have on LDL binding X. Niimi S. Ohtani T. Machida S. J. Cell Sci. 2001; 114: 1273-1282Crossref PubMed Google Scholar). for on the of the conserved basic on the three-dimensional structure of human Lox-1 we propose that entire of the with modified LDL to all basic Although each basic is from the Lox-1 probably with modified of ApoB100 that the LDL recognition mode an entire of the Lox-1 binding in a two charged ApoB100 domains on the surface of modified the binding mode is for several This mode potentially to basic to with oxLDL at not just the tunnel and of the patch are such that could potentially in the recognition of oxLDL the tunnel may bind ApoB100 In addition, the and may facilitate interaction with the surface of the role of the tunnel and the patch in recognition to be by the of the Lox-1 to recognize a variety of charged such as oxLDL, with on This type of interaction a of binding on Lox-1 that could to and several receptors to bind with such as apoptotic cells could Lox-1 in a localized region of the cell such as oxLDL may bind both of Lox-1 a of on the cell could potentially in a cellular for and at the at the J. for mass spectrometry and for critical on the