TLR2 Looks at Lipoproteins

Toll-like receptors (TLRs) have evolved to recognize lipoproteins from diverse species of bacteria. In this issue of Immunity, Kang et al., 2009Kang J.Y. Nan X. Jin M.S. Youn S.-J. Ryu Y.H. Mah S. Han S.H. Lee H. Paik S.-G. Lee J.-O. Immunity. 2009; 31 (this issue): 873-884Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar provide insight into the mechanisms by which TLR2-TLR6 heterodimers recognize diacylated liporoteins, whereas TLR2-TLR1 heterodimers recognize triacylated lipopeptides. Toll-like receptors (TLRs) have evolved to recognize lipoproteins from diverse species of bacteria. In this issue of Immunity, Kang et al., 2009Kang J.Y. Nan X. Jin M.S. Youn S.-J. Ryu Y.H. Mah S. Han S.H. Lee H. Paik S.-G. Lee J.-O. Immunity. 2009; 31 (this issue): 873-884Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar provide insight into the mechanisms by which TLR2-TLR6 heterodimers recognize diacylated liporoteins, whereas TLR2-TLR1 heterodimers recognize triacylated lipopeptides. The posttranslational modification of proteins in bacteria, to form diacylated or triacylated lipoproteins, provides a mechanism by which proteins are directed, transported, and anchored into the bacterial cell membranes and outer envelopes. The localization of lipoproteins into either the inner or outer membranes of bacteria is pivotal to their function in a variety of cellular processes including structural integrity, transport, sensing, adhesion, and signal transduction. The host innate immune response, in contrast, has evolved to recognize these lipoproteins, which have distinct structures in diverse bacterial species. In this issue of Immunity, Kang et al., 2009Kang J.Y. Nan X. Jin M.S. Youn S.-J. Ryu Y.H. Mah S. Han S.H. Lee H. Paik S.-G. Lee J.-O. Immunity. 2009; 31 (this issue): 873-884Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar provide new insight into the mechanisms by which receptors of the innate immune system distinguish the fatty acid moieties of bacterial lipoproteins. Bacterial proteins are targeted for acylation via a conserved N-terminal signal sequence that terminates with a "lipobox," [LVI]-[AST]-[GA]-C, which is recognized by the three conserved enzymes responsible for the posttranslational lipid modification (Hutchings et al., 2009Hutchings M.I. Palmer T. Harrington D.J. Sutcliffe I.C. Trends Microbiol. 2009; 17: 13-21Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) (Figure 1). First, a prolipoprotein diacylglyceryltransferase (Lgt) transfers a diacylglyceride moiety from the phospholipid pool to the cysteine sulfhydryl group of the lipid box via a thioester linkage. Second, the signal peptide is cleaved just before the cysteine residue. These modifications result in the synthesis of diacylated lipoproteins in Gram-positive and Gram-negative bacteria, as well as in acid-fast bacteria including mycobacteria. The acylation of bacterial proteins facilitates anchoring in the bacterial cytoplasmic membrane. The third step, which occurs in Gram-negative and acid-fast bacteria, but is absent in Mycoplasma spp and some Gram-positive bacteria, involves an apolipoprotein N-acyltransferase (Lnt) that adds a third fatty acid to the amino group of the N-terminal cysteine. The triacylation of proteins in Gram-negative organisms facilitates transport of the lipoprotein to the outer membrane via the lipoprotein localization (Lol) pathway. The ability of the bacterial pathogens to produce two forms of lipoproteins, diacylated and triacylated, creates a diversity in biochemical patterns and hence a challenge for recognition by the innate immune system. Work from our lab (Brightbill et al., 1999Brightbill H.D. Libraty D.H. Krutzik S.R. Yang R.B. Belisle J.T. Bleharski J.R. Maitland M. Norgard M.V. Plevy S.E. Smale S.T. et al.Science. 1999; 285: 732-736Crossref PubMed Scopus (1370) Google Scholar) and others (Aliprantis et al., 1999Aliprantis A.O. Yang R.-B. Mark M.R. Suggett S. Devaux B. Radolf J.D. Klimpel G.R. Godowski P. Zychlinsky A. Science. 1999; 285: 736-739Crossref PubMed Scopus (1236) Google Scholar) led to the discovery that the human innate immune system recognizes bacterial lipoproteins via Toll-like receptor 2 (TLR2). Further investigation revealed that TLR2 is able to recognize the bacterial lipoproteins that are either diacylated or triacylated by forming heterodimers with TLR6 or TLR1, respectively (Takeuchi et al., 2002Takeuchi O. Sato S. Horiuchi T. Hoshino K. Takeda K. Dong Z. Modlin R.L. Akira S. J. Immunol. 2002; 169: 10-14PubMed Google Scholar). In this issue of Immunity, Kang et al. describe the crystal structure of the TLR2-TLR6-diacylated lipopeptide complex (Kang et al., 2009Kang J.Y. Nan X. Jin M.S. Youn S.-J. Ryu Y.H. Mah S. Han S.H. Lee H. Paik S.-G. Lee J.-O. Immunity. 2009; 31 (this issue): 873-884Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar), which together with previous papers on the TLR2-TLR1-triacylated lipopeptide complex (Jin et al., 2007Jin M.S. Kim S.E. Heo J.Y. Lee M.E. Kim H.M. Paik S.G. Lee H. Lee J.O. Cell. 2007; 130: 1071-1082Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar), provides new insight into the mechanism by which TLR2 looks at lipoproteins. Specifically, the ability of TLR2 to heterodimerize with either TLR6 or TLR1 results in the formation of distinct binding pockets which facilitate the recognition of diacylated and triacylated lipopeptides, respectively. A key feature of TLRs is the leucine-rich repeat (LRR) domains, which contribute to recognition of microbial ligands. The major driving force for TLR2 binding of lipopeptides is a strong but relatively nonspecific hydrophobic interaction between ester-bound fatty acids and the internal pocket formed by hydrophobic residues from the LRR9-12 modules. There is a minimal requirement of diacylated lipopeptides for TLR2 recognition, and subsequent induction of a robust response requires that the lipopeptide have two acyl chains of at least 12 carbons each (eight for mouse TLR2) (Buwitt-Beckmann et al., 2006Buwitt-Beckmann U. Heine H. Wiesmuller K.H. Jung G. Brock R. Akira S. Ulmer A.J. J. Biol. Chem. 2006; 281: 9049-9057Crossref PubMed Scopus (197) Google Scholar). TLR2-TLR1 recognition of triacylated lipopeptides and heterodimer formation requires the interaction between a fatty acid of at least eight carbons in length that is amide linked on the N-terminal cysteine of the lipopeptide and a hydrophobic channel present in the LRR module of TLR1 (Figure 1). For TLR6, this hydrophobic channel is blocked by two phenylalanine residues, F343 and F365 (Figure 1). A mutant TLR6, in which F343 and F365 were replaced with less bulky methionine and lysine amino acids found in TLR1, was able to form a complex with TLR2 and triacylated lipopeptides and transduce an immunological response. Unlike the TLR2-TLR1 heterodimer formation, in which fatty acid moieties were bound to each TLR in the complex, Kang et al., 2009Kang J.Y. Nan X. Jin M.S. Youn S.-J. Ryu Y.H. Mah S. Han S.H. Lee H. Paik S.-G. Lee J.-O. Immunity. 2009; 31 (this issue): 873-884Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar demonstrate that TLR2-TLR6 heterodimer formation depends on hydrogen bonds between glycerol and the peptide backbone of the ligand and the LRR11 loops of TLRs. These hydrogen bonds simultaneously interact with the ligand and bridge TLR2 and TLR6, but also fix the conformation of the hydrophobic residues in the center of the dimerization interface. Therefore, the entire lipopeptide—the fatty acids and the peptide head group—is responsible for the specificity of the interaction in the formation of TLR2-TLR6 heterodimers (Buwitt-Beckmann et al., 2006Buwitt-Beckmann U. Heine H. Wiesmuller K.H. Jung G. Brock R. Akira S. Ulmer A.J. J. Biol. Chem. 2006; 281: 9049-9057Crossref PubMed Scopus (197) Google Scholar). Kang et al., 2009Kang J.Y. Nan X. Jin M.S. Youn S.-J. Ryu Y.H. Mah S. Han S.H. Lee H. Paik S.-G. Lee J.-O. Immunity. 2009; 31 (this issue): 873-884Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar provide compelling evidence that the side chains of the first two amino acids of diacylated lipopeptides have substantial interactions with the TLRs. The amino-terminal cysteine binds to the "sulfur site" formed by the hydrophobic F325, L328, F349, L350, and P325 residues of TLR2 and the L318 residue of TLR6. An amino acid with a small R group side chain such as serine is preferred as the second residue, so that it can fit into the narrow neck area of the ligand-binding pocket generated by the LRR11 loops of TLR2 and TLR6 and form a hydrogen bond with the F325 on the backbone of TLR2. Although studies with defined lipopeptides indicate that the amino acids directly adjacent to the diacylglycerol can affect their potency in activating TLR2-dependent responses, it must be recognized that during natural infections, it is lipoproteins with long polypeptide chains and not lipopeptides that activate the TLR2-TLR1 and TLR2-TLR6 dimers. Some studies indicate that native lipoproteins are more potent inducers of TLR2 responses than synthetic lipopeptides, suggesting the possibility that downstream amino acids of certain lipoproteins interact with the extensive LRR domains of the TLRs. The ability of cell-surface TLR2 to form heterodimers with TLR1 and TLR6 is not the only mechanism by which the innate immune system recognizes the biochemical diversity between Gram-positive and Gram-negative bacteria. The closely related cytosolic receptors NOD1 and NOD2 distinguish structures in bacterial cell walls, specifically peptidoglycan motifs. An important difference between Gram-positive and Gram-negative peptidoglycan is the nature of the third amino acid in the peptidoglycan stem peptide motif. A diaminopimelic acid (DAP) is found in the peptidoglycan motif of most Gram-negative bacteria, such that the naturally occurring peptidoglycan degradation product sensed by NOD1 is GlcNAc-MurNAc-L-Ala- D-Glu-meso-DAP (GM-triDAP) (Girardin et al., 2003Girardin S.E. Boneca I.G. Viala J. Chamaillard M. Labigne A. Thomas G. Philpott D.J. Sansonetti P.J. J. Biol. Chem. 2003; 278: 8869-8872Crossref PubMed Scopus (1797) Google Scholar). The recognition of these products by NOD1 is dependent on the presence of an exposed meso-DAP, an amino acid absent from eukaryotes, representing a very simple signature of bacterial origin. In contrast, the minimal motif recognized by NOD2 is the muramyl dipeptide MurNAc-L-Ala-D-isoGln (MDP), present in Gram-positive and Gram-negative and mycobacterial peptidoglycans (Girardin et al., 2003Girardin S.E. Boneca I.G. Viala J. Chamaillard M. Labigne A. Thomas G. Philpott D.J. Sansonetti P.J. J. Biol. Chem. 2003; 278: 8869-8872Crossref PubMed Scopus (1797) Google Scholar). However, NOD2 also recognize a muramyl tripeptide in which the third amino acid position is occupied by one of the amino acids specific to Gram-positive peptidoglycan (L-lysine or L-ornithine) (Girardin et al., 2003Girardin S.E. Boneca I.G. Viala J. Chamaillard M. Labigne A. Thomas G. Philpott D.J. Sansonetti P.J. J. Biol. Chem. 2003; 278: 8869-8872Crossref PubMed Scopus (1797) Google Scholar). The distinct locations of TLRs and NLRs in cellular compartments and/or cell subsets provide an important mechanism of regulating innate immune responses and the outcome of an infection. Furthermore, simultaneous activation of TLRs by lipoproteins and NOD proteins by peptidoglycan results in a synergistic antibacterial response. Although TLR2-TLR6 versus TLR2-TLR1 and NOD1 versus NOD2 allow the innate immune system to recognize biochemical differences between Gram-positive and Gram-negative bacteria, several other innate receptors recognize conserved motifs, e.g., DNA by TLR9 and RNA by RIG-I helicase. The capacity of the innate immune system to distinguish biochemical differences between Gram-positive and Gram-negative bacteria does not appear to result in distinct functional responses. Activation of TLR2-TLR1 and TLR2-TLR6 induces innate immune pathways required for host defense against microbial pathogens including cytokine release and dendritic cell and macrophage differentiation (Krutzik et al., 2005Krutzik S.R. Tan B. Li H. Ochoa M.T. Liu P.T. Sharfstein S.E. Graeber T.G. Sieling P.A. Liu Y.J. Rea T.H. et al.Nat. Med. 2005; 11: 653-660Crossref PubMed Scopus (317) Google Scholar), as well as microbicidal activity (Liu et al., 2006Liu P.T. Stenger S. Li H. Wenzel L. Tan B.H. Krutzik S.R. Ochoa M.T. Schauber J. Wu K. Meinken C. et al.Science. 2006; 311: 1770-1773Crossref PubMed Scopus (2739) Google Scholar) (Figure 1). Therefore, the function of the different TLR2 heterodimers is to enable recognition of bacteria with different cell envelopes, but that once triggered, the immunologic mechanisms appear to activate the same pathways. The elegant recognition mechanisms by which TLRs can distinguish lipoproteins provides a new impetus to search for differences in the subsequent innate immune response. The ability of TLR2 to mediate the recognition and immune response to various nonpeptide ligands, including lipoteichoic acid, is still controversial and thought by some to be the result of contamination of preparations with lipoproteins, as extensively discussed by Kang et al. in the Supplemental Data (Kang et al., 2009Kang J.Y. Nan X. Jin M.S. Youn S.-J. Ryu Y.H. Mah S. Han S.H. Lee H. Paik S.-G. Lee J.-O. Immunity. 2009; 31 (this issue): 873-884Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). Nevertheless, Kang et al. provide evidence that lipoteichoic acid from Streptococcus pneumoniae binds to TLR2, with the lipid chains inserted into the TLR2 hydrophobic pocket. They also provide evidence that the polar head groups of TLR2 ligands are critical for heterodimer formation. These studies indicate that such nonpeptide ligands can bind to TLR2 but do not resolve the issue as to whether they, under physiologic conditions, activate cellular responses. Clearly, more research into this area is warranted to define the universe of natural TLR2 ligands that can trigger host innate immune responses. In summary, the innate immune system is encoded to distinguish biochemical differences between Gram-positive and Gram-negative bacteria. In particular, the laboratory of Jie-Oh Lee has provided clear evidence that TLR2 has 20/20 vision for lipoproteins, the heterodimerization of TLR2-TLR1 and TLR2-TLR6 heterodimers forms different lipid-binding pockets that distinguish triacylated from diacylated lipopeptides (Jin et al., 2007Jin M.S. Kim S.E. Heo J.Y. Lee M.E. Kim H.M. Paik S.G. Lee H. Lee J.O. Cell. 2007; 130: 1071-1082Abstract Full Text Full Text PDF PubMed Scopus (917) Google Scholar, Kang et al., 2009Kang J.Y. Nan X. Jin M.S. Youn S.-J. Ryu Y.H. Mah S. Han S.H. Lee H. Paik S.-G. Lee J.-O. Immunity. 2009; 31 (this issue): 873-884Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar). Although TLR2 has perfect vision to distinguish distinct lipoproteins, the subsequent immune responses appear to be identical carbon copies. Recognition of Lipopeptide Patterns by Toll-like Receptor 2-Toll-like Receptor 6 HeterodimerKang et al.ImmunityNovember 19, 2009In BriefToll-like receptor 2 (TLR2) initiates potent immune responses by recognizing diacylated and triacylated lipopeptides. Its ligand specificity is controlled by whether it heterodimerizes with TLR1 or TLR6. We have determined the crystal structures of TLR2-TLR6-diacylated lipopeptide, TLR2-lipoteichoic acid, and TLR2-PE-DTPA complexes. PE-DTPA, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid, is a synthetic phospholipid derivative. Two major factors contribute to the ligand specificity of TLR2-TLR1 or TLR2-TLR6 heterodimers. Full-Text PDF Open Archive