TLR X-ray structures Harma Brondijk Bio-informatics course 2009

TIR domain structures Protein-protein interaction surfaces: 1.Oligomerizationn interface 2.Interaction surface(s) with TIR domains of adapter molecules:  MAL/MyD88  TRAM/TRIF  SARM

TIR-domain structures TLR-TIR domains: –hTLR12.90 Å1fyv(2000) –hTLR23.00 Å1fyw(2000) –hTLR2-P681H2.80 Å 1fyx(2000) –hTLR2-C713S3.20 Å1o77(2002) –hTLR Å2j67(2006) Other TIR-domains –hMyD88 (NMR)2js7/2z5v(2008) –IL1RAPL2.30 Å 1t3g(2005)

TIR domains: similar structure, flexible regions hTLR1 hTLR10 IL1RAPL hTLR2 hTLR2_P681H hTLR2_C713S

TIR domains: similar structure, flexible regions BB-loop

Dimer interfaces: which is the ‘true’ interface? hTLR1 725 Å 2, 2 disulfide hTLR1 807 Å 2, 2 saltbridges hTLR1: - 1 molecule in the asymetric unit - protein-protein contacts -> two possible dimer interfaces

Dimer interfaces: which is the ‘true’ interface? hTLR1 hTLR10 hTLR2 hTLR2-P681H hTLR2-C713S

TLR1-TLR2 TIR domain docking: putative dimer GAUTAM et al. JBC 2006

hTLR10 TIR dimer: most likely ‘real’ signaling dimer Nyman et al. JBC 2008 Molecule B Molecule A

Conserved surface patches: possible interaction surfaces In general surface residues are much less conserved than core residues Interaction surfaces need to change concerted in both interaction partners -> Interaction surfaces are relatively higly conserved Xu et al. Nature 2000

Modelling + Information driven docking programs -> Putative TLR4-Mal/TRAM interactions Miguel et al. PLoS ONE 2007

LRR domain structures Issues: 1.Overall structure 2.Interaction with ligands (agonist/antagonist) 3.Ligand induced dimerization?  Conformational changes  Interfaces 4.Interactions with co-factors

LRR-domain structures TLR ectodomains: –hTLR1/hTLR2 dimer + PAM3CSK4 (TLR1 aa /TLR2 aa ) 2.10 Å2z7x(2007) –hTLR2 aa Å2z80(2007) –mTLR2 aa Å/2.60 Å 2z81/2z82(2007) –hTLR32.10 Å1ziw(2005) –hTLR32.40 Å1aoz(2005) –mTLR32.66 Å3cig(2008) –mTLR3 + dsRNA3.41 Å3ciy(2008) –hTLR4 aa Å2z62(2007) –hTLR4 aa Å2z63(2007) –hTLR4/MD2/Eritoran2.70 Å2z65(2007) - hTLR4/MD2/LPS3.10 Å3FXI(2009) –mTLR4/MD22.84 Å2z64(2007) –CD Å1wwl(2005)

TLR ecto-domains consist of leucine-rich-repeats Choe et al. Science 2005 Bell et al. PNAS 2005 hTLR3 convex concave lateral Curvature may vary along the ectodomain: TLR1/2/4: divided in 3 distinct regions

LRR domain architecture LRR: –20-30 residues (extensions possible) –defining motif: LxxLxLxxNxL L=Leu/Val/Ile/Phe N=Asn/Thr/Ser/Cys Repeat -> curved solenoid structure –Concave side: continuous parallel ß-sheet –Convex side: variable Cavity of solenoid structure filled with hydrophobic residues

The Leucine-rich repeat structure: diversity rules! α-helix Always ß-sheet on concave side, convex side varaible: Polyproline II helix helix Other combinations of helices and strands 2 Polyproline II helices Extensions possible on convex and lateral sides (examples from TLR3 structure)

Ligand binding: TLRs recognize chemically diverse compounds Flagellin (TLR5) (TLR3) Pam2CSK4 (TLR6/TLR2 heterodimer)

How do TLR’s recognize/bind their ligands? How does ligand binding induce TLR oligomerization?

mTLR3-dsRNA complex Liu et al. Science, 2008 C-termini 25 Å apart 2 TLR3 molecules bind adjacently to the dsRNA

Two interaction sites close to N and C terminus Interactions with sugar-phosphate backbones only explains lack of sequence specificity Histidine involvement explains pH dependence Liu et al. Science, 2008 dsRNA-mTRL3 interactions N N C C

mTRL3-mTLR3 interactions Direct TLR3-TLR3 contacts near C- terminal interaction site explains concerted binding Liu et al. Science, 2008 N N C C

hTLR1-hTLR2 Pam 3 CSK 4 complex Jin et al. Cell 2007 C-termini <42 Å apart

hTLR1-hTLR2 Pam 3 CSK 4 complex Jin et al. Cell 2007 Top view Ligand binds to both TLR1 and TLR2 -> heterodimerization Question: How does the hTLR2-hTLR6 dimer form?? (ligand lacks the 3 rd lipid chain)

mTLR4-mMD2 complex Kim et al. Cell 2007 MD2 binds on the edge of the central and N-terminal region, at the lateral side of the molecule

hTLR4/VLR-hMD2 Eritoran Kim et al. Cell 2007

hTLR4-MD2-LPS homodimer Park et al. Nature 2009

Dimerization through LPS and MD2 interactions TLR4 TLR4 * MD2 * MD2

What makes LPS an agonist? Lipid Iva and Eritoran have fewer lipid tails -> bind deeper in the MD2 binding pocket -> Phosphates not available for interactions Park et al. Nature 2009

TLR4 dimerization appears to induce a conformational change Central domains of TLR1/2/4 less rigid compared to standard LRR structures; needed to accommodate conformational changes??? Park et al. Nature 2009

Central theme: ligand interactions induce dimerization -> C-termini in close approximation Park et al. Nature 2009

Thanks for your attention

Dimerization through LPS and MD2 interactions

TLR4-MD2-LPS dimerization interactions

TIR domain plasticity Xu et al. Nature 2000 TLR1-TLR2 comparison Tao et al. BBRC 2002 TLR2-TLR2 comparison