TLR Molecule Architecture
Toll-like receptors are large transmembrane proteins whose structure comprises three key regions:
- Extracellular (LRR domain). Functions as an "antenna" that recognizes foreign ligands. It contains 19 to 25 leucine-rich repeat regions (Leucine-rich repeats). Spatially, this domain is folded into a horseshoe shape: its outer part consists of $\alpha$-helices, and its inner part, which directly contacts the ligand, consists of $\beta$-sheets.
- Transmembrane segment. Responsible for anchoring the receptor in the lipid bilayer and determining precisely where the receptor will function—on the cell membrane or on the membrane of intracellular granules.
- Intracellular (TIR domain). The signaling part of the receptor (Toll/IL-1 receptor and resistance domain). It represents a central $\beta$-sheet surrounded by five $\alpha$-helices. It is responsible for transmitting the activation signal inside the cell via interaction with adapter molecules.
Classification and Receptor Localization
The intracellular location of TLRs strictly depends on which specific structures of the pathogen they are programmed to recognize.
Surface receptors (on the cell membrane) Scan the extracellular space for components of bacterial and fungal cell walls and flagella.
- TLR-1, TLR-2, TLR-6: Recognize lipid-containing structures. They form pairs (heterodimers) for precise recognition. The TLR-1/TLR-2 combination responds to triacyl lipopeptides of Gram-positive bacteria, while TLR-2/TLR-6 responds to diacyl lipopeptides.
- TLR-4: Specializes in the lipopolysaccharide (LPS) of Gram-negative bacteria. Exception: this receptor can be present both on the membrane and in endolysosomes.
- TLR-5: Responds to flagellin (the flagellar protein of Salmonella and other motile bacteria).
- TLR-11: Recognizes profilin from uropathogenic strains of *Escherichia coli*.
Intracellular receptors (in endosomes/lysosomes) Located on the membranes of the endoplasmic reticulum and translocate to phagolysosomes to recognize foreign nucleic acids.
- TLR-3: Responds to double-stranded viral RNA.
- TLR-7 and TLR-8: Recognize single-stranded viral RNA.
- TLR-9: Targets bacterial and viral DNA rich in unmethylated CpG motifs.
Activation Mechanism: From Monomer to Complex
At rest, receptors exist as single molecules — monomers. As soon as a ligand binds to the receptor, dimerization occurs. Most often, homodimers are formed (two identical molecules, e.g., in TLR-3).
For successful target capture, receptors often require helpers (coreceptors). The most striking example is the multi-step recognition of bacterial LPS by the TLR-4 receptor:
- LPS is released from the bacterial cell wall.
- In the blood, it binds to the serum protein LBP.
- This complex attaches to the membrane molecule CD14.
- The LPS component (lipid A) embeds into the hydrophobic part of the MD-2 molecule.
- Only after this does the MD-2 molecule (along with LPS) perfectly "fit" into the inner horseshoe of the TLR-4 receptor.
Thus, TLR-4 itself does not directly recognize the bacterial toxin, but rather the complex structure together with MD-2.
Why Are Nucleic Acid TLRs Hidden Inside?
The TLR-3, 7, 8, and 9 receptors have their recognition regions directed toward the interior of cytoplasmic granules. When a cell engulfs a pathogen via endocytosis, receptors are mobilized from the endoplasmic reticulum to the phagolysosome.
Inside the phagolysosome, microorganisms are broken down, and their genetic material becomes accessible to TLRs. This hidden localization is a defensive mechanism. It prevents receptors from contacting the organism's own nucleic acids, protecting us against autoimmune reactions. Self DNA and RNA can enter phagolysosomes only during massive pathological cell apoptosis.
Briefly: answers to topical questions
Which adapter proteins interact with the intracellular TIR domain of Toll-like receptors?
Several specialized adapter proteins interact with the intracellular TIR domain of Toll-like receptors to ensure signal transduction.
- MyD88 — the primary adapter protein used by all receptors except TLR-3.
- TIRAP (Mal) — participates in signal transduction together with MyD88.
- TRIF — an adapter protein involved in the MyD88-independent pathway (used by TLR-3 and TLR-4).
- TRAM — an accessory adapter for TLR-4 in the MyD88-independent pathway.
- Tollip — part of the molecular complex to which the signal from the TIR domain is relayed.
What endogenous ligands (DAMPs) can Toll-like receptors recognize upon tissue damage?
Upon tissue damage, Toll-like receptors can recognize various endogenous ligands (DAMPs), including alarmins and nucleic acids.
- Heat shock proteins (HSP20, HSP40, HSP60, HSP70, HSP90, Hsp96) — interact with TLR-2 and TLR-4; Hsp96 is listed among endogenous ligands of TLR-2, while Hsp60 and Hsp70 are listed for TLR-4.
- HMGB1 protein — listed among endogenous ligands of TLR-4; HMGB1 is also part of the chromatin-containing immune complexes mentioned for TLR-9.
- Extracellular matrix components — fibronectin, fibrinogen, and hyaluronic acid are listed among DAMPs associated with TLR-4.
- Nucleic acids — auto-RNA and ribonucleoproteins are recognized by TLR-3, TLR-7, and TLR-8; auto-DNA, chromatin, and chromatin-containing immune complexes are recognized by TLR-9.
- Other molecules — lipoproteins are listed among endogenous ligands of TLR-2/TLR-1, and $\beta$-defensins among endogenous ligands of TLR-4.
Which transcription factors are activated in the cell during signal transduction from Toll-like receptors?
Four main transcription factors are activated in the cell during signal transduction from Toll-like receptors.
- NF-$\kappa$B — a key factor responsible for the expression of pro-inflammatory genes.
- AP-1 (Activation protein 1) — participates in the activation of numerous genes responsible for the development of inflammation and the formation of adaptive immune responses.
- IRF3 (Interferon-regulatory factor 3) — specific predominantly for the induction of interferon-$\beta$ genes (IFN$\beta$).
- IRF7 (Interferon-responding factor 7) — specific predominantly for the induction of interferon-$\alpha$ genes (IFN$\alpha$).
Additionally, the IRF5 factor participates in the induction of type I interferon genes upon TLR activation.
On which immune system cells are Toll-like receptors expressed?
Toll-like receptors are expressed on a wide range of immune system cells, with the highest expression observed on myeloid cells.
- Monocytes and macrophages — express TLRs 1–10.
- Dendritic cells — myeloid, plasmacytoid, and lymphoid subpopulations bear various combinations of TLRs.
- Granulocytes — neutrophils and eosinophils.
- Mast cells (mastocytes) — express TLR-2, TLR-3, and TLR-4.
- Lymphocytes — T cells, B cells, natural killer cells (NK cells), and natural regulatory T cells.
Which cytokines are synthesized by myeloid cells as a result of Toll-like receptor activation?
Upon Toll-like receptor activation, myeloid cells synthesize various groups of cytokines.
- Proinflammatory cytokines — tumor necrosis factor alpha (TNFα), interleukin-1 (IL-1, IL-1β), and interleukin-6 (IL-6).
- Interleukin-12 (IL-12) — a link between innate and adaptive immunity, produced by macrophages and dendritic cells.
- Type I interferons — interferon alpha (IFNα) and interferon beta (IFNβ), synthesized upon activation of intracellular TLRs (e.g., by plasmacytoid dendritic cells).
- Chemokines — their synthesis is induced by the direct action of pathogens via TLRs.
Surface TLRs (1, 2, 4, 5, 6, 11) "sense" microbes from the outside — cell walls and flagella. Intracellular TLRs (3, 7, 8, 9) "read" the genetic code (DNA and RNA) inside phagolysosomes.
Class and exam questions
What state are TLRs in before encountering a pathogen?
In resting cells, receptors are present as inactive monomers. They assemble into dimers only upon ligand binding.
Is TLR-4 capable of independently recognizing bacterial LPS?
No. Signal transduction requires a multimolecular complex including the CD14 and MD-2 coreceptors. In fact, TLR-4 interacts with the MD-2 molecule bound to LPS.
Can TLRs react to the body's own tissues?
Yes, certain receptors (e.g., TLR-2, 4) can recognize DAMPs — damage-associated molecular patterns or heat shock proteins released upon host cell damage.
What else to study on this topic
- Changes in the TLR expression profile under the influence of cytokines
- Structure and functions of the TIR domain in signal transduction
- Specificity of TLR-1/2 and TLR-2/6 heterodimer formation
- Scavenger receptors and their role in the clearance of apoptotic cells
- C-type lectin receptors of myeloid cells
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