TLR Xray structures

1
TLR X-ray structures

• Harma Brondijk
• Bio-informatics course 2009

2
TIR domain structures

• Protein-protein interaction surfaces
• Oligomerizationn interface
• Interaction surface(s) with TIR domains of
  adapter molecules
• MAL/MyD88
• TRAM/TRIF
• SARM

3
TIR-domain structures

• TLR-TIR domains
• hTLR1 2.90 Å 1fyv (2000)
• hTLR2 3.00 Å 1fyw (2000)
• hTLR2-P681H 2.80 Å 1fyx (2000)
• hTLR2-C713S 3.20 Å 1o77 (2002)
• hTLR10 2.20 Å 2j67 (2006)
• Other TIR-domains
• hMyD88 (NMR) 2js7/2z5v (2008)
• IL1RAPL 2.30 Å 1t3g (2005)

4
TIR domains similar structure, flexible regions
hTLR1
hTLR2
hTLR2_P681H
hTLR10
IL1RAPL
hTLR2_C713S
5
TIR domains similar structure, flexible regions
BB-loop
6
Dimer interfaces which is the true interface?
hTLR1 - 1 molecule in the asymetric unit -
protein-protein contacts -gt two possible dimer
interfaces
hTLR1 807 Å2, 2 saltbridges
hTLR1 725 Å2, 2 disulfide
7
Dimer interfaces which is the true interface?
hTLR1
hTLR10
hTLR2
hTLR2-P681H
hTLR2-C713S
8
TLR1-TLR2 TIR domain docking putative dimer
GAUTAM et al. JBC 2006
9
hTLR10 TIR dimer most likely real signaling
dimer
Molecule B
Molecule A
Nyman et al. JBC 2008
10
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 -gt Interaction surfaces are relatively
higly conserved
Xu et al. Nature 2000
11
Modelling Information driven docking programs
-gt Putative TLR4-Mal/TRAM interactions
Miguel et al. PLoS ONE 2007
12
LRR domain structures

• Issues
• Overall structure
• Interaction with ligands (agonist/antagonist)
• Ligand induced dimerization?
• Conformational changes
• Interfaces
• Interactions with co-factors

13
LRR-domain structures

• TLR ectodomains
• hTLR1/hTLR2 dimer PAM3CSK4 (TLR1 aa 25-475/TLR2
  aa 27-506)
• 2.10 Å 2z7x (2007)
• hTLR2 aa 1-284 1.80 Å 2z80 (2007)
• mTLR2 aa 27-506 1.80 Å/2.60 Å 2z81/2z82 (2007)
• hTLR3 2.10 Å 1ziw (2005)
• hTLR3 2.40 Å 1aoz (2005)
• mTLR3 2.66 Å 3cig (2008)
• mTLR3 dsRNA 3.41 Å 3ciy (2008)
• hTLR4 aa27-228 1.70 Å 2z62 (2007)
• hTLR4 aa 27-527 2.00 Å 2z63 (2007)
• hTLR4/MD2/Eritoran 2.70 Å 2z65 (2007)
• - hTLR4/MD2/LPS 3.10 Å 3FXI (2009)
• mTLR4/MD2 2.84 Å 2z64 (2007)
• CD14 2.50 Å 1wwl (2005)

14
TLR ecto-domains consist ofleucine-rich-repeats
hTLR3
hTLR3
Choe et al. Science 2005 Bell et al. PNAS 2005
Curvature may vary along the ectodomain
TLR1/2/4 divided in 3 distinct regions
15
LRR domain architecture

• LRR
• 20-30 residues (extensions possible)
• defining motif
• LxxLxLxxNxL
• LLeu/Val/Ile/Phe
• NAsn/Thr/Ser/Cys
• Repeat -gt curved solenoid structure
• Concave side continuous parallel ß-sheet
• Convex side variable
• Cavity of solenoid structure filled with
  hydrophobic residues

16
The Leucine-rich repeat structure diversity
rules!
Always ß-sheet on concave side, convex side
varaible
Polyproline II helix
a-helix
Extensions possible on convex and lateral sides
(examples from TLR3 structure)
310-helix
2 Polyproline II helices
Other combinations of helices and strands
17
Ligand binding TLRs recognize chemically diverse
compounds
18
How do TLRs recognize/bind their ligands?
How does ligand binding induce TLR
oligomerization?
19
mTLR3-dsRNA complex
2 TLR3 molecules bind adjacently to the dsRNA
C-termini 25 Å apart
Liu et al. Science, 2008
20
dsRNA-mTRL3 interactions
N
C

• 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

N
C
Liu et al. Science, 2008
21
mTRL3-mTLR3 interactions

• Direct TLR3-TLR3 contacts near C-terminal
  interaction site explains concerted binding

C
N
N
C
Liu et al. Science, 2008
22
hTLR1-hTLR2 Pam3CSK4 complex
C-termini lt42 Å apart
Jin et al. Cell 2007
23
hTLR1-hTLR2 Pam3CSK4 complex
Ligand binds to both TLR1 and TLR2 -gt
heterodimerization
Top view
Question How does the hTLR2-hTLR6 dimer form??
(ligand lacks the 3rd lipid chain)
Jin et al. Cell 2007
24
mTLR4-mMD2 complex
MD2 binds on the edge of the central and
N-terminal region, at the lateral side of the
molecule
Kim et al. Cell 2007
25
hTLR4/VLR-hMD2 Eritoran
Kim et al. Cell 2007
26
hTLR4-MD2-LPS homodimer
Park et al. Nature 2009
27
Dimerization through LPS and MD2 interactions
MD2
TLR4
MD2
TLR4
28
What makes LPS an agonist?
Lipid Iva and Eritoran have fewer lipid tails -gt
bind deeper in the MD2 binding pocket -gt
Phosphates not available for interactions
Park et al. Nature 2009
29
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
30
Central theme ligand interactions induce
dimerization -gt C-termini in close approximation
Park et al. Nature 2009
31
Thanks for your attention
32
Dimerization through LPS and MD2 interactions
33
TLR4-MD2-LPS dimerization interactions
34
TIR domain plasticity
TLR2-TLR2 comparison
TLR1-TLR2 comparison
Xu et al. Nature 2000
Tao et al. BBRC 2002