Human Toll-Like Receptor 4
Created by Jeremy Hines
Toll-like receptors are a set of set of signaling proteins of paramount importance in the immune system. Sensing the presence of infection, these multifaceted receptors are each specific for different sets of microbial components which allows a variety of protective mechanisms [13]. Human Toll-Like Receptor 4 is a homodimer positioned in the plasma membrane of macrophages, dendritic cells, mast cells, and eosinophils. In an immune related response, TLR4 (PDB ID: 3FXI) recognizes ligands composed of lipopolysaccharide in Gram negative bacteria [13]. The pathogen sensor domain is horseshoe shaped and consists of a leucine rich repeat of 20-29 amino acid residues. The cytoplasmic signaling domain, known as the Toll-interleukin receptor domain, mediates transcription of genes encoding inflammatory cytokines which aide in limiting the spread of infection. Specifically, LPS is bound on macrophages by a co-receptor protein to TLR4, called CD14. Another associated protein called MD2 forms a complex with CD14 and LPS before the intracellular domain is activated [13]. MD-2 is a 25-kDa co-receptor which physically associates with TLR4, binds the lipid moiety of LPS through the central hydrophobic pocket, accommodating up to five acyl chains. Therefore in theory, CD-14 catalyzes the LPS-bound MD2 complex to interact with TLR4; this results in the dimerization of TLR4 chains A and B to initiate the signaling cascade [10]. TLR4 has a molecular weight of 95680.13 daltons, composed of 839 amino acids, and a theoretical pI of 5.88 [7].
As a type-1 transmembrane protein, the TLR4 secondary structure is comprised of two identical chains
(A and B). Each chain contains 605 residues composed as follows: 9 % helical (
13 helices, 57 residues) and 20 % beta sheet (41 strands, 124 residues) [9]. The associated protein MD2 (lymphocyte antigen 96) consists of 1 alpha helix and 12 beta strands [9]. The quaternary structure of the
TLR4-MD2 complex details the
MD-2 protein fitting inside the crescent of the TLR4 protein serving as a bridge between the TLR4 dimers, in which both function to bind LPS of Gram-negative bacteria [12]. MD-2 binding to TLR4 is dependent on
Cys95 and Cys105 residues forming an intermolecular
disulfide bond, as well as hydrophilic charged residues within the vicinity including Lys91, Asd100, Tyr102, and Arg90 [17]. In respect to MD-2 interaction with LPS, Kobayashi et al. have demonstrated a critical subset of amino acids within the region from
Phe119 to Lys132 of MD-2 allow for interaction between LPS and the TLR4-MD2 complex. LPS associates with a large hydrophobic pocket in MD-2, with five of the six lipid chains of LPS buried inside the pocket and the remaining chain exposed to the surface of MD-2 [14]. In this manner, the lipid-MD2 component can form a hydrophobic interaction with the conserved phenylalanines of TLR4. Specifically, F126 loop of MD-2 undergoes localized conformational changes and supports this core hydrophobic interface by making hydrophilic interactions with TLR4 [14]. The negatively charged phosphate residues of the lipid A moiety can stabilize the TLR4-MD2 complex through interaction with associated positively charged residues within the vicinity. To reiterate, the primary interface between TLR4 and MD2 is established before LPS binding, afterwhich LPS induced dimerization between TLR4 chains A and B with associated MD2 complex occurs [14] See Figure 4. Experimentally, Replacement of Val-82, Met-85, and Leu-87 with polar amino acids reduced receptor activation, and subsequently lead to a loss of LPS transfer from CD14 to MD-2 [15]. Resman et al. have also identified a pair of conserved hydrophobic residues, Phe440 and Phe463, in the leucine rich repeats 16 and 17 of TLR4 extracellular domain, critical for activation of TLR4 by LPS. Replacement of Phe-440 or Phe-463 with alanine eliminated LPS sensitivity, but replacement of Phe-440 with another large, hydrophobic residue, tryptophan, substantially preserved TLR4 signaling activity. Surprisingly, replacement of
Leu-444 on leucine-rich repeat 16 with alanine increased cell activation by LPS, an insight to functionally specific and dependent residual characteristics of TLR4 [15] . Fujumoto et al. have demonstrated through genetic alterations that the N-terminal region of TLR-4 is essential for association with MD-2, and therefore recognition of the ligand and subsequent intracellular signaling. Deletions within this terminal region reduced interaction potential with MD2 and therefore LPS signaling capacity [4].
Toll-Like Receptor 4's extracellular domain can bind and recognize many different bacterial components of LPS including n-acetyl-d-glucosamine (Nag) pocket, lauric acid, 3-hydroxy-tetradecanoic acid, and L-glycero-D-Manno-Heptopyranose in addition to microbe-derived ligands, TLR4 recognizes various endogenous ligands including heat shock proteins HSP60, HSP70 and gp96, Alpha-defensin, Tamm-Horsfall protein, biglycan , fibrinogen, surfactant protein, low-molecular weight oligosaccharide fragments of hyaluronan, fibronectin, and heparansulfate [10]. Hence, the TLRmediated immune response can be activated in the absence of foreign microbes. Prosthetic groups of TLR4 include magnesium and phosphate ions, which serve to arbitrate receptor functionality. Important post-translational modifications defining functionality of TLR4 include glycosylation of Asn residues and di-sulfide bond formation between Cys residues [7].
There are two signaling pathways in response to TLR4 binding to microbial components. One pathway is activated by TIRAP adaptor protein (Toll Interleukin 1 receptor-domain-containing adaptor protein) and MyD88, which leads to proliferation and secretion of pro-inflammatory cytokines [1]. The second pathway of action includes TRIF (TIR-domain-containing adaptor protein inducing interferon-Beta) and TRAM (TRIF-related adaptor molecule) which leads to the production of type 1 interferons [1].
Shu-Ling Fu et al. have discovered the glycoprotein Dioscorind from Dioscorea alata, to be a medicinal herb-derived activator of TLR4. Dioscorin ultimately triggered TLR4 signaling and subsequent production of cytokines in macrophages as well as immune related signaling patterns through proteins such as NF-kB [3]. The secondary structure of Dioscorin A has prominent alpha helix configuration, while that of Dioscorin B shows anti-parallel beta sheet configurations [11]. The prominence of phenylalanine, tryptophan, and tyrosine in these proteins may provide insight to natural triggers of TLR4 mediated responses.
A newly discovered inhibitor of the TLR4 signaling pathway involves the body's own negative feedback response. A homolog to TLR4, a type 1 transmembrane protein known as RP105, contains similar extracellular leucine rich repeats and conserved juxtamembrane cysteine residues [2]. In correspondence to TLR4-MD2 dependence for LPS recognition and signaling, RP105 relies on MD-1, the homologue of MD-2. Divanovic et al. recently demonstrated the negative inhibition potential of RP105-MD1 against TLR4-MD2 signaling in human embryonic kidney cells. Coimmunoprecipitation techniques have led to the theory that contact between MD2 and MD1 proteins in a heterodimerization fashion, with inhibition of TLR following [2]. It has been hypothesized by Divanovic et al. that RP105:MD-1 interacts directly with the TLR4 complex, hampering its capability to interact with a microbial ligand. Such discoveries provide insight into the fundamentally conserved structure of Toll-Like Receptors, their function as progressed through time, and nature's attempt in feedback inhibition to prevent mechanisms involved in overextended control.
TLR4's chain A shows an 81% similarity similarity to TLR3's chain A (PBD ID: 1ZIW) which has 11 helices and 46 beta sheet strands [9]. TLR3 also plays a key role in activating immune system responses by binding to double stranded RNA of viruses, and contains a similar 23-leucine rich repeat to TLR4 (Choe et al. 2005). TLR4's chain A shows an 81% similarity to TLR2 Variable Lymphocyte Receptor B (PBD ID: 2Z81) which has 14 helices and 36 beta sheet strands [9]. TLR2 shows a similar function in the innate immune response by recognizing microbial lipoproteins and lipopeptides, containing extensive leucine repeats, hydrogen bonding, and hydrophobic interactions [8]. In a similar manner to Fujimoto et. Al's discovery of amino-terminal functionality, a report by Mitsuzawa et. Al demonstrates deletion of amino terminal residues 40-63 in TLR2 abolishes the receptor's function for peptidoglycan [4]. Torben et al. have discovered that TLR1, TLR2, and TLR3 have a tertiary structure dominated by leucine rich repeats in the shape of a horseshoe, with the MD-2 complex binding at the concave face of the N-terminus and central domains of TLR4 [18]. Protein Blast Analysis of TLR4's A chain identified conserved sequences within the Toll-Like Receptor Superfamily. These sequences are related mainly by crucial Leucine rich repeats that are essential to the protein's functionality and ligand binding capabilities. TLR3 (PDB ID: 1ZIW) showed a query coverage of 92% and an E-value of 3E-19. G protein coupled receptor 4 also showed a leucine rich repeat similarity, with an E value of 5E-16 and query coverage of 92%. Dali Server 3D structural analysis was used to identify tertiary structure similarities. In comparison to TLR4, Toll-Like Receptor 3 showed a root mean square deviation of 5.0 and a Z score of 24.2. Toll-like Receptor 2 showed an RMSD of 4.4 Å and a Z score of 22.9. Reticulon 4 Receptor (1P8T) showed a RMSD of 3.1 Å and a Z score of 16.4. The lower the RMSD, the smaller the averagedistance between the backbones of superimposed proteins. All three structures in comparison to TLR4 show significant Z scores, above 2.0, possibly representing conserved sequences through the receptor’s evolution [5].
TLR4 serves as a crucial signaling mechanism within an organism by initiatingproponents of the innate immunity. The importance of such influences is due tothe immediacy of the effects. Nonspecific immune responses allow othercomponents in the body to ward off infection and recovery, while adapting inways which retain memory specific responses for future interaction. Rapid signaling cascades to deter infection and commence secretion of cytokines, influencing a variety of defensive mechanisms, has been conserved in manyorganisms throughout evolution. The leucine rich repeats in Toll-Like Receptorproteins among other important conserved residues is a key insight into the mechanism of TLR signaling.