Supplementary Material
Cloning, Expression and Purification of mCherry-TIR TLR
Recombinant TIR domain of hTLRs 1, 2 and 4 having a His-tag and mCherry on its N-terminal
were cloned in pET15b vector (Services of Top Gene Technologies, Pointe-Claire, Canada were
utilized for this part). It is pertinent to mention here that we found that tagless-, GST- and MBPtagged TIR domains prone to aggregation and formation of inclusion bodies. While exploring
options, we found that mCherry-tagged versions can be overexpressed and got retained in soluble
fraction. These chimeric proteins can be purified, concentrated and stored for longer durations.
Thus, mutants were created in the mCherry chimeric proteins. Seven mutants of hTLR4-TIR, four
for hTLR2-TIR and one for hTLR1-TIR were generated using QuikChange Site-Directed
Mutagenesis Kit (Stratagene, USA) and confirmed by DNA sequencing. The protein was initially
purified from a Ni-NTA column in buffer containing 20 mM HEPES (pH 8), 0.2 M NaCl, 5%
glycerol, 10 mM β-Mercaptoethanol, followed by anion-exchange chromatography under a linear
gradient of NaCl (50 mM to 1 M). The fraction containing native and mutant proteins were further
purified by gel filtration chromatography (Superdex200 column attached to AKTA Prime, GE
Healthcare, USA). Eluted proteins were eventually concentrated using membrane concentrators
(10 kDa cut-off; Millipore, Ireland). Purity of proteins was ascertained by 10% SDS-PAGE
containing molecular mass markers (Fermentas) also, a good correlation between expected and
observed values for the native and mutants in MALDI-TOF were seen. Concentration of the
purified protein was measured using UV absorption at A280/1mg/ml∼1.7 (U-2900 spectrophotometer,
Hitachi, Japan).
Circular Dichroism Experiments
CD studies were carried out with ~10 μM protein samples in 10 mM phosphate buffer (pH 8.0) on
a JASCO 810 spectrometer at 20C. A quartz cell with 1 mm path-length was used for the circular
dichroism experiments. Five scans ranging from 190 to 240 nm were taken for each protein
solution (hTLR4, 2, 1 and their mutants). Buffer-subtracted CD spectra was used to estimate the
secondary structural content in the protein using the K2D3 server (Perez-Iratxeta & AndradeNavarro 2008).
SWAXS Data Acquisition, Analysis and Modelling
The small/wide angle X-ray scattering (SWAXS) data was collected at the X9 beam line (National
Synchrotron Light Source, Brookhaven National Laboratory). 120 L of each protein in
concentration range of 1.2 – 4.1 mg/ml was used to collect scattering data by exposing samples for
120 seconds at ~15C. Using the Python script-based programs written by Dr. Lin Yang (X9 beam
line, NSLS), the images recorded on the two CCDs were scaled, merged, and circularly averaged,
and the buffer contribution was subtracted to obtain the scattering intensity (I) as a function of
momentum transfer vector, Q (𝑄 = [4𝜋𝑠𝑖𝑛𝜃]/𝜆) , where λ and θ represent the wavelength of Xray and the scattering angle, respectively. In this study, the SAXS and WAXS intensity profiles
were scaled and merged using the Q data between 0.12-0.2 Å-1. All the SAXS experiments
described in this study were carried out in triplicate and averaged. Similarity in migration pattern
of samples subjected to scattering experiments to those kept in lab confirmed lack of degradation
during transportation and data collection. Guinier analyses and Indirect Fourier transformation of
the SAXS data were carried out using PRIMUS software package (Konarev, Volkov, Sokolova,
Koch & Svergun 2003) and GNOM45 program (Svergun 1992), respectively to obtain RG and
Dmax values. The RG and Dmax are defined as the root-mean-square of all elemental volumes from
the centre-of-mass and maximum linear dimension of the scattering particle/protein molecule,
respectively. By employing dummy residues and constraints provided within the measured SAXS
I(Q) profile, the three-dimensional shapes of the two proteins were restored using the
1
GASBOR22IQ program (Svergun 1999). Ten models were generated without any predefined
shape or symmetry bias.
Supplementary Figure 1: A) This graph shows the distribution of energy profiles of the different
structures (5000 in total) generated during MD simulation of the TIR domain of hTLRs (example
hTLR1) For each TLR the total energy of the 5000 structures, adopted a single peak Boltzmann
distribution suggesting that ensemble of conformations simulated adopted similar global shape
(Ashish, Grover & Kishore 2000; Ashish & Kishore 2002). B) This graph shows the variation in
the computed radius of gyration (RG) values in the structural models of the TIR domains of ten
hTLRs as a function of the simulation time.
B
Radius of gyration (RG)
A
Frequency
500
400
300
200
100
0
k
1.0
-48
k
1.5
-48
k
2.0
-48
k
2.5
-48
k
3.0
-48
Energy(KJ/mol)
k
3.5
-48
0
20
40
60
Time (ns)
80
100
Supplementary Figure 2: The flowchart describes the steps followed for the generation of residue
interaction network used in this study.
2
Supplementary Figure 3: Multiple sequence alignment of the TIR domain in human TLRs:
Arrows and cylinders represent β-sheet and α-helix respectively. Residues highlighted in yellow
represent total conservation of residues in that column while those with conserved and semiconserved substitution are shown in cyan. Residues involved in “cross-talk” are shown in red and
those forming the hubs (H1-H4) are shown in fonts with underline. The residues in TLRs 1, 2 and
4 used for mutational studies, are shown in strikethrough fonts.
β1
sp|Q15399|hTLR1
sp|O60603|hTLR2
sp|O15455|hTLR3
sp|O00206|hTLR4
sp|O60602|hTLR5
sp|Q9Y2C9|hTLR6
sp|Q9NYK1|hTLR7
sp|Q9NR97|hTLR8
sp|Q9NR96|hTLR9
sp|Q9BXR5|hTLR10
β2
α1
BB Loop
LEELQRNLQFHAFISYSGHD---SFWVKNELLPNLE---KEG--MQICLHERNFVPGKSI
-KAPSRNICYDAFVSYSERD---AYWVENLMVQELE---NFNPPFKLCLHKRDFIPGKWI
IDRQTEQFEYAAYIIHAYKD---KDWVWEHFSSMEK----EDQSLKFCLEERDFEAGVFE
------ENIYDAFVIYSSQD---EDWVRNELVKNLE---EGVPPFQLCLHYRDFIPGVAI
QGTEPDMYKYDAYLCFSSKD---FTWVQNALLKHLDTQYSDQNRFNLCFEERDFVPGENR
LEELQRNLQFHAFISYSEHD---SAWVKSELVPYLE---KED--IQICLHERNFVPGKSI
--LISPDCCYDAFIVYDTKDPAVTEWVLAELVAKLEDP-REKHFN-LCLEERDWLPGQPV
--LSTSQTFYDAYISYDTKDASVTDWVINELRYHLEES-RDKNVL-LCLEERDWDPGLAI
SGRDEDALPYDAFVVFDKTQSAVADWVYNELRGQLEEC-RGRWALRLCLEERDWLPGKTL
QEQLKRNVRFHAFISYSEHD---SLWVKNELIPNLE---KEDGSILICLYESYFDPGKSI
H2
H1
β3
α2
sp|Q15399|hTLR1
sp|O60603|hTLR2
sp|O15455|hTLR3
sp|O00206|hTLR4
sp|O60602|hTLR5
sp|Q9Y2C9|hTLR6
sp|Q9NYK1|hTLR7
sp|Q9NR97|hTLR8
sp|Q9NR96|hTLR9
sp|Q9BXR5|hTLR10
679
685
799
718
740
684
939
946
919
678
α3
β4
DD
VENIITC-IEKSYKSIFVLSPNFVQSEWCH-YELYFAHHNLFHEGSNSLILILLEPIPQY 737
IDNIIDS-IEKSHKTVFVLSENFVKSEWCK-YELDFSHFRLFDENNDAAILILLEPIEKK 743
LEAIVNS-IKRSRKIIFVITHHLLKDPLCKRFKVHHAVQQAIEQNLDSIILVFLEEIPDY 858
AANIIHEGFHKSRKVIVVVSQHFIQSRWCI-FEYEIAQTWQFLSSRAGIIFIVLQKVEK- 776
IANIQDA-IWNSRKIVCLVSRHFLRDGWCL-EAFSYAQGRCLSDLNSALIMVVVGSLSQY 798
VENIINC-IEKSYKSIFVLSPNFVQSEWCH-YELYFAHHNLFHEGSNNLILILLEPIPQN 742
LENLSQS-IQLSKKTVFVMTDKYAKTENFK-IAFYLSHQRLMDEKVDVIILIFLEKPFQ- 996
IDNLMQS-INQSKKTVFVLTKKYAKSWNFK-TAFYLALQRLMDENMDVIIFILLEPVLQ-1003
FENLWAS-VYGSRKTLFVLAHTDRVSGLLR-ASFLLAQQRLLEDRKDVVVLVILSPDGR- 976
SENIVSF-IEKSYKSIFVLSPNFVQNEWCH-YEFYFAHHNLFHENSDHIILILLEPIPFY 736
H3
α4
β5
H4
α5
Loop
sp|Q15399|hTLR1
sp|O60603|hTLR2
sp|O15455|hTLR3
sp|O00206|hTLR4
sp|O60602|hTLR5
sp|Q9Y2C9|hTLR6
sp|Q9NYK1|hTLR7
sp|Q9NR97|hTLR8
sp|Q9NR96|hTLR9
sp|Q9BXR5|hTLR10
SIPSSYHKLKSLMARRTYLEWPKEKSKRGLFWANLRAAINIKLTEQAKK----------- 786
AIPQRFCKLRKIMNTKTYLEWPMDEAQREGFWVNLRAAIKS------------------- 784
KLNHALCLRRGMFKSHCILNWPVQKERIGAFRHKLQVALGSKNSVH-------------- 904
TLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEA 836
Q-LMKHQSIRGFVQKQQYLRWPEDLQDVGWFLHKLSQQILKKEKEKKKDNNIPLQTVATI 857
SIPNKYHKLKALMTQRTYLQWPKEKSKRGLFWANIRAAFNMKLTLVTENNDVKS------ 796
--KSKFLQLRKRLCGSSVLEWPTNPQAHPYFWQCLKNALATDNHVAYSQVFKETV-----1049
--HSQYLRLRQRICKSSILQWPDNPKAEGLFWQTLRNVVLTENDSRYNNMYVDSIKQY--1059
--RSRYVRLRQRLCRQSVLLWPHQPSGQRSFWAQLGMALT--------------------1014
CIPTRYHKLKALLEKKAYLEWPKDRRKCGLFWANLRAAINVNVLATREMYELQTFTELNE 796
3
Supplementary Figure 4: MSA of TIR domain sequences from human to C. elegans. Conserved residues
are highlighted in yellow and those involved in networking are shown in red fonts.
sp|Q15399|TLR1_HUMAN
sp|O60603|TLR2_HUMAN
sp|O15455|TLR3_HUMAN
sp|O00206|TLR4_HUMAN
sp|O60602|TLR5_HUMAN
sp|Q9Y2C9|TLR6_HUMAN
sp|Q9NYK1|TLR7_HUMAN
sp|Q9NR97|TLR8_HUMAN
sp|Q9NR96|TLR9_HUMAN
sp|Q9BXR5|TLR10_HUMAN
sp|Q9EPQ1|TLR1_MOUSE
sp|Q9QUN7|TLR2_MOUSE
sp|Q99MB1|TLR3_MOUSE
sp|Q9QUK6|TLR4_MOUSE
sp|Q9JLF7|TLR5_MOUSE
sp|Q9EPW9|TLR6_MOUSE
sp|P58681|TLR7_MOUSE
sp|P58682|TLR8_MOUSE
sp|Q9EQU3|TLR9_MOUSE
sp|Q6R5P0|TLR11_MOUSE
sp|Q6QNU9|TLR12_MOUSE
sp|Q6R5N8|TLR13_MOUSE
tr|Q5WA51|Q5WA51_CHICK
sp|Q9DD78|TLR21_CHICK
tr|Q7ZTG5|Q7ZTG5_CHICK
tr|Q5GR02|Q5GR02_CHICK
tr|Q5ZJD0|Q5ZJD0_CHICK
tr|E0AEW6|E0AEW6_CHICK
tr|A5YBP4|A5YBP4_CHICK
tr|A4UXC9|A4UXC9_XENLA
tr|F7C313|F7C313_XENTR
tr|F6YQH2|F6YQH2_XENTR
tr|Q5U5B1|Q5U5B1_XENLA
tr|F6ZAC8|F6ZAC8_XENTR
tr|F6QHI4|F6QHI4_XENTR
tr|B2GUH5|B2GUH5_XENTR
tr|F6U8S1|F6U8S1_XENTR
tr|F7EDI8|F7EDI8_XENTR
tr|B3DIW3|B3DIW3_DANRE
tr|Q6TS42|Q6TS42_DANRE
tr|Q6IWL5|Q6IWL5_DANRE
tr|B3U3W0|B3U3W0_DANRE
tr|Q6TS41|Q6TS41_DANRE
tr|B3DIN1|B3DIN1_DANRE
tr|B3DJW3|B3DJW3_DANRE
tr|B3DKG5|B3DKG5_DANRE
tr|B3DKB1|B3DKB1_DANRE
tr|B0S591|B0S591_DANRE
tr|B3DJL6|B3DJL6_DANRE
tr|Q9NBK6|Q9NBK6_DROME
tr|Q9VLE6|Q9VLE6_DROME
tr|Q9NBK8|Q9NBK8_DROME
tr|Q9NBK9|Q9NBK9_DROME
tr|Q7KIN0|Q7KIN0_DROME
tr|Q9V477|Q9V477_DROME
tr|Q9VPH1|Q9VPH1_DROME
sp|P08953|TOLL_DROME
tr|Q9N5Z3|Q9N5Z3_CAEEL
tr|Q5I9V9|Q5I9V9_CAEEL
tr|Q5I9W0|Q5I9W0_CAEEL
tr|Q5I9W1|Q5I9W1_CAEEL
------LEELQRNLQFHAFISYSGHD---SFWVKNELLPNLEKEG----------K-APSRNICYDAFVSYSERD---AYWVENLMVQELENFNPP--------IDRQTEQFEYAAYIIHAYKD---KDWVWEHFSSMEKEDQS---------------ENIYDAFVIYSSQD---EDWVRNELVKNLEEGVPP--------QGTEPDMYKYDAYLCFSSKD---FTWVQNALLKHLDTQYSDQNR
------LEELQRNLQFHAFISYSEHD---SAWVKSELVPYLEKED------------LISPDCCYDAFIVYDTKDPAVTEWVLAELVAKLED-PREKH--------LSTSQTFYDAYISYDTKDASVTDWVINELRYHLEE-SRDKN------SGRDEDALPYDAFVVFDKTQSAVADWVYNELRGQLEECRGRWA------QEQLKRNVRFHAFISYSEHD---SLWVKNELIPNLEKEDGS--------LEELQRNLQFHAFVSYSGHD---SAWVKNELLPNLEKDD----------K-APCRDVCYDAFVSYSEQD---SHWVENLMVQQLENSDPP--------IDTQAEQFEYTAYIIHAHKD---RDWVWEHFSPMEEQDQS---------------ESIYDAFVIYSSQN---EDWVRNELVKNLEEGVPR--------WSLEPGAYRYDAYFCFSSKD---FEWAQNALLKHLDAHYSSRNR
------LEELQRNLQFHAFVSYSEHD---SAWVKNELLPNLEKDD------------LQSMESCYDAFIVYDTKNSAVTEWVLQELVAKLED-PREKH--------SSTSQTFYDAYISYDTKDASVTDWVINELRYHLEE-SEDKS------SRRSAQALPYDAFVVFDKAQSAVADWVYNELRVRLEERRGRRA-------RLRG-QFNYDVFISYCEED---QAWVLEELVPVLEKAPPEGEG
-------GDKGKRFLFDVFVSHCRQD---QGWVIEELLPALEGFLPAGLG
--------KTEKKFLYDAFVSFSATD---EAWVYKELVPALEQGSQTT-------PEERETALQFHAFISYSERD---SLWVKNELIPNLEKGEGC--------R-APTKDICYDAFVSYSEND---SNWVENIMVQQLEQACPP--------------GDIYDAFVIHSSKD---QEWVMKELVEPLEEGKPP--------PAADTSEYMYDAYLCYSKND---FEWVQNSLLKHLDSQYFDKNR
--------IPLPDACYDAFIAYDNTDLAVNEWVMTELVEKLED-QKARQ------YKKRPENKPFDAFISYSEHD---ADWTKEHLLKKLETDG----------PEERETALQFHAFISYSERD---SLWVKNELIPNLEKGEGC------------------------------------------------C--------K-NCDREICYNGFVSYSERD---SEWVENMMVPKLENAVPP--------IDPRKRNFDYDAYIIHAQND---VSWVENHLIPLEKNAGSK--------QEEAADTCKYDAYLCYSGKD---FQWVQDAFLQNLDTQYSDRNR
------YQSLQRDFDFNAFISYSEHD---ASWVKNIFLPSIERSNDC----------FP--KCCYDALIMYDTKDSAVSDWVFNDLVNILEK-QGNKM--------FP--KCCYDALIMYDTKDSAVSDWVFNDLVNILEK-QGNKM--------VCKSKCLYDVFITYDNKDPNVSDWIFNELCQHLED-KGDKH------LHDKST-EHFDAFIAFNTKNSSVRDWVYNELLVQLES-PERGG------EEDVER-LHFHAFVSYSQKN---AGWVKSQFLPKLEGDCG---------VGRLPEELRYDAFVSYSQHD---AEWVEEILVAELEDTQPS--------AKVTEGRFQFDAYIIHAGED---KSWVERSLLSLEDKDLN---------------ECSYDAFVIFSSYD---EAWVMNELMENLENGVPP--------------ECSYDAFVIFSSYD---EAWVMNELMENLENGVPP--------EEPDPDRFLYDVYLCFSSKD---MKWVERALLKRLDSQFSEHNT
------ANGNPTDTQYDAFVVFDTSNKAVRDWIYKEMLVRLEN-RGRWR------GLGQGVDYAFHAFISYSHSD---ADWVRNHLLPCLENAKPP---------MKEKQQYRYDAFVSYSGKD---EHWVIEELLPNLEQRGPPF-------LRDQEEKYNYDAFVSYNSAD---EDWVMEQLLPNLEGSS-----------NQSGQTFQYDAFISYNTLD---EAWVMEELIPKLEGEQG----------RELDKDKRFDAFLAFTHKDE----ALLEEFVDRLERGRPR---------RELDEDKKYDAFLSFTHKDE----DLIEEFVDRLENGRHK--------EEELDKDKTYDAFISYSHKD----EELISKLLPKLESGPHP--------VDQLDKERPNDAYFAYSLQDEH---FVNQILAQTLEN-DIG--------E---ESEKLYDAVLLHSAKDSE---FVCQHLAAQLETGRPP--------VDKNEREKLFDAFVSYSSKDEL---FVNEELAPMLEMGEHR--VNKFTNISQRDPSAVYDIFISYCQND---RTWVLNELLPNVEETGDVS-------EEDLDKDKKFDAFISYSHKD---QSFIEDYLVPQLEHGPQK--------SPLPVPLLSYHAFVSYSKKD---EKMVIDQLCRPLEDED----------SLLKNIQAAKHFILVLTPNS-------LDRLLNDDNCEDWVHKE
------SLLKNIQAAKHFILVLTPNS-------LDRLLNDDNCEDWVHKE
------SLLKNIQAAKHFILVLTPNS-------LDRLLNDDNCEDWVHKE
663
669
783
702
724
668
923
930
903
662
666
669
784
700
725
668
924
903
903
805
792
863
676
678
716
727
933
733
676
1
662
779
741
660
928
928
914
903
669
670
784
632
703
737
927
681
117
823
822
673
1002
671
1140
1126
1104
780
887
1082
447
842
789
4
sp|Q15399|TLR1_HUMAN
sp|O60603|TLR2_HUMAN
sp|O15455|TLR3_HUMAN
sp|O00206|TLR4_HUMAN
sp|O60602|TLR5_HUMAN
sp|Q9Y2C9|TLR6_HUMAN
sp|Q9NYK1|TLR7_HUMAN
sp|Q9NR97|TLR8_HUMAN
sp|Q9NR96|TLR9_HUMAN
sp|Q9BXR5|TLR10_HUMAN
sp|Q9EPQ1|TLR1_MOUSE
sp|Q9QUN7|TLR2_MOUSE
sp|Q99MB1|TLR3_MOUSE
sp|Q9QUK6|TLR4_MOUSE
sp|Q9JLF7|TLR5_MOUSE
sp|Q9EPW9|TLR6_MOUSE
sp|P58681|TLR7_MOUSE
sp|P58682|TLR8_MOUSE
sp|Q9EQU3|TLR9_MOUSE
sp|Q6R5P0|TLR11_MOUSE
sp|Q6QNU9|TLR12_MOUSE
sp|Q6R5N8|TLR13_MOUSE
tr|Q5WA51|Q5WA51_CHICK
sp|Q9DD78|TLR21_CHICK
tr|Q7ZTG5|Q7ZTG5_CHICK
tr|Q5GR02|Q5GR02_CHICK
tr|Q5ZJD0|Q5ZJD0_CHICK
tr|E0AEW6|E0AEW6_CHICK
tr|A5YBP4|A5YBP4_CHICK
tr|A4UXC9|A4UXC9_XENLA
tr|F7C313|F7C313_XENTR
tr|F6YQH2|F6YQH2_XENTR
tr|Q5U5B1|Q5U5B1_XENLA
tr|F6ZAC8|F6ZAC8_XENTR
tr|F6QHI4|F6QHI4_XENTR
tr|B2GUH5|B2GUH5_XENTR
tr|F6U8S1|F6U8S1_XENTR
tr|F7EDI8|F7EDI8_XENTR
tr|B3DIW3|B3DIW3_DANRE
tr|Q6TS42|Q6TS42_DANRE
tr|Q6IWL5|Q6IWL5_DANRE
tr|B3U3W0|B3U3W0_DANRE
tr|Q6TS41|Q6TS41_DANRE
tr|B3DIN1|B3DIN1_DANRE
tr|B3DJW3|B3DJW3_DANRE
tr|B3DKG5|B3DKG5_DANRE
tr|B3DKB1|B3DKB1_DANRE
tr|B0S591|B0S591_DANRE
tr|B3DJL6|B3DJL6_DANRE
tr|Q9NBK6|Q9NBK6_DROME
tr|Q9VLE6|Q9VLE6_DROME
tr|Q9NBK8|Q9NBK8_DROME
tr|Q9NBK9|Q9NBK9_DROME
tr|Q7KIN0|Q7KIN0_DROME
tr|Q9V477|Q9V477_DROME
tr|Q9VPH1|Q9VPH1_DROME
sp|P08953|TOLL_DROME
tr|Q9N5Z3|Q9N5Z3_CAEEL
tr|Q5I9V9|Q5I9V9_CAEEL
tr|Q5I9W0|Q5I9W0_CAEEL
tr|Q5I9W1|Q5I9W1_CAEEL
BB loop
MQICLHERN---FVPGKSIVENIITC-IEKSYKSIFVLSPNFVQSE---W
FKLCLHKRD---FIPGKWIIDNIIDS-IEKSHKTVFVLSENFVKSE---W
LKFCLEERD---FEAGVFELEAIVNS-IKRSRKIIFVITHHLLKDP--LC
FQLCLHYRD---FIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSR---W
FNLCFEERD---FVPGENRIANIQDA-IWNSRKIVCLVSRHFLRDG--WC
IQICLHERN---FVPGKSIVENIINC-IEKSYKSIFVLSPNFVQSE---W
FNLCLEERD---WLPGQPVLENLSQS-IQLSKKTVFVMTDKYAKT---EN
VLLCLEERD---WDPGLAIIDNLMQS-INQSKKTVFVLTKKYAKS---WN
LRLCLEERD---WLPGKTLFENLWAS-VYGSRKTLFVLAHTDRVS---GL
ILICLYESY---FDPGKSISENIVSF-IEKSYKSIFVLSPNFVQNE---W
IQICLHERN---FVPGKSIVENIINF-IEKSYKSIFVLSPHFIQSE---W
FKLCLHKRD---FVPGKWIIDNIIDS-IEKSHKTVFVLSENFVRSE---W
LKFCLEERD---FEAGVLGLEAIVNS-IKRSRKIIFVITHHLLKDP--LC
FHLCLHYRD---FIPGVAIAANIIQEGFHKSRKVIVVVSRHFIQSR---W
LRLCFEERD---FIPGENHISNIQAA-VWGSRKTVCLVSRHFLKDG--WC
IRVCLHERN---FVPGKSIVENIINF-IEKSYKAIFVLSPHFIQSE---W
FNLCLEERD---WLPGQPVLENLSQS-IQLSKKTVFVMTQKYAKT---ES
VLLCLEERD---WDPGLPIIDNLMQS-INQSKKTIFVLTKKYAKS---WN
LRLCLEDRD---WLPGQTLFENLWAS-IYGSRKTLFVLAHTDRVS---GL
LRLCLPARD---FGIGNDRMESMIAS-MGKSRATLCVLTGQALASP---W
LRLCLPERD---FEPGKDVVDNVVDS-MLSSRTTLCVLSGQALCNP---R
FKLCLHQRD---FEPGIDIFENIQNA-INTSRKTLCVVSNHYLHSE---W
IQLCQHERN---FIPGKSIVENIINC-IEKSYKSIFVLSPNFVQSE---W
FRLCLHKRD---FVPGKWIVDNIIDS-IEKSHKTLFVLSEHFVQSE---W
FQLCLYFRD---FLPGVPIVTNIIQEGFLSSRNVIAVISADFLESK---W
FTLCFEERD---FLPGEEHINNIRDA-IWKSRKTICVVTRQFLKDG--WC
FNLCLEERD---WLPGQPVFDNLSQS-IQLSKKTIFVLTNKYIKS---GT
FKICYHERD---FKPGHPVLGNIFYC-IENSHKDLFVLSPSFVNSC---W
IQLCQHERN---FIPGKSIVENIINC-IEKSYKSIFVLSPNFVQSE---W
IRICQHERN---FVPGKSIIENIINC-IDKSYKSIFILSPNFVQSE---W
MKLCLHKRD---FVPGKWIIDNIIDA-MEKSYKTVFVLSEHFVRSE---W
LQFCFEERD---LEAGTSTLSAFVDC-INRSRKTIFVVTRNLLNDS--WC
FHFCFEERD---FVPGEDHIVNIRDA-IWNSKKTICVVTKQFLKDG--WC
IRICQHERN---FIPGKSIIENIINC-IEKSYKSIFILSPNFVQSE---W
LNLCLEERD---FLAGQPFLDNLSES-IQISRKTVFVLTRKYVKK---GH
LNLCLEERD---FLAGQPFLDNLSES-IQISRKTVFVLTRKYVKK---GH
MYLCLEERD---WEPGKAIIDNLAHS-INQSNKTLFVLTKKYVKS---GK
FTLCLEERD---WIAGRSSIENLYDS-IYRSKKTIFIITREWFN---CGL
LRMCHHERD---FIPGKTVVQNILRC-IEQSRRCVFVLSSHFVQSE---W
FSLCLHKRD---FRPGRWIVDNIIDS-IEKSYRTLFVLSEHFVSSE---W
--FFYEQRD---STPGHSRLKTIVDN-MVHSRKITFVITEMLLKDP--WC
IQLCLHMRD---FQAGKSIASNIIDEGIMGSRKIIVVVSQHFIASA---W
IQLCLHMRD---FQAGKSIASNIIDEGIMGSRKIIVVVSQHFIDSS---W
LRCCFEERD---FIPGEDHLTNMRSA-IQNSRKTICVVSEHFLKDG--WC
FQLCLEERD---WIPGVSCIENLHKS-VYSSRKTVFVLTSPGGYSDASGI
YRLCIHERD---FIPGKWIIDNIIEN-IENSRKVIFVLSHNFVNSE---W
LRLCLHSRD---FQLGHDIVENITDS-IYASRRTLCLVSRNYLNSN---W
FRLCLHHRD---FELGRDIVDNIVAA-VYGSRKTICVVSQSFLRSE---W
WRLCLHHRD---FEPGRPIIDNIVDG-IYSSRKTICLITRNYLKSN---W
FQLCFYLRD---WLAGESIPDCIGQS-IKDSRRIIVLMTENFMNST---W
FRLCFYLRD---WLVGESIPDCINQS-VKGSRRIIILMTKNFLKST---W
FRLCLHDRD---WLVGDCIPEQIVRT-VDDSKRVIIVLSQHFIDSV---W
YRLCLHYRD---VNINAYITDALIEA-AESAKQFVLVLSKNFLYNE---W
LRVCLQHRD---LAHDATH-YQLLEA-TRVSRRVVILLTRNFLQTE---W
YKLCLHQRD---FPVGGYLPETIVQA-IDSSRRTIMVVSENFIKSE---W
--ICLHERD---FQIGVTILDNIISC-MDRSYSLMLIISSKFLLSH---W
FQLCVHERD---WLVGGHIPENIMRS-VADSRRTIIVLSQNFIKSE---W
YQLCLLHRDGPTYCSNLHAISDELIAQMDSSQCLILVLTKHFLENE---W
LKCAFEHQK---NIIPIFDTAFEFPTKEDQIPNDIRMITKYNGVKWVHDY
LKCAFEHQK---NIIPIFDTAFEFPTKEDQIPNDIRMITKYNGVKWVHDY
LKCAFEHQK---NIIPIFDTAFEFPTKEDQIPNDIRMITKYNGVKWVHDY
:::
706
712
827
746
768
711
966
973
946
705
709
712
828
744
769
711
967
946
946
848
835
906
719
721
760
771
976
776
719
44
705
823
785
703
971
971
957
946
712
713
826
676
747
781
973
724
160
866
865
716
1045
714
1183
1168
1147
821
930
1129
494
889
836
5
sp|Q15399|TLR1_HUMAN
sp|O60603|TLR2_HUMAN
sp|O15455|TLR3_HUMAN
sp|O00206|TLR4_HUMAN
sp|O60602|TLR5_HUMAN
sp|Q9Y2C9|TLR6_HUMAN
sp|Q9NYK1|TLR7_HUMAN
sp|Q9NR97|TLR8_HUMAN
sp|Q9NR96|TLR9_HUMAN
sp|Q9BXR5|TLR10_HUMAN
sp|Q9EPQ1|TLR1_MOUSE
sp|Q9QUN7|TLR2_MOUSE
sp|Q99MB1|TLR3_MOUSE
sp|Q9QUK6|TLR4_MOUSE
sp|Q9JLF7|TLR5_MOUSE
sp|Q9EPW9|TLR6_MOUSE
sp|P58681|TLR7_MOUSE
sp|P58682|TLR8_MOUSE
sp|Q9EQU3|TLR9_MOUSE
sp|Q6R5P0|TLR11_MOUSE
sp|Q6QNU9|TLR12_MOUSE
sp|Q6R5N8|TLR13_MOUSE
tr|Q5WA51|Q5WA51_CHICK
sp|Q9DD78|TLR21_CHICK
tr|Q7ZTG5|Q7ZTG5_CHICK
tr|Q5GR02|Q5GR02_CHICK
tr|Q5ZJD0|Q5ZJD0_CHICK
tr|E0AEW6|E0AEW6_CHICK
tr|A5YBP4|A5YBP4_CHICK
tr|A4UXC9|A4UXC9_XENLA
tr|F7C313|F7C313_XENTR
tr|F6YQH2|F6YQH2_XENTR
tr|Q5U5B1|Q5U5B1_XENLA
tr|F6QHI4|F6QHI4_XENTR
tr|B2GUH5|B2GUH5_XENTR
tr|F6U8S1|F6U8S1_XENTR
tr|F7EDI8|F7EDI8_XENTR
tr|B3DIW3|B3DIW3_DANRE
tr|Q6TS42|Q6TS42_DANRE
tr|Q6IWL5|Q6IWL5_DANRE
tr|B3U3W0|B3U3W0_DANRE
tr|Q6TS41|Q6TS41_DANRE
tr|B3DIN1|B3DIN1_DANRE
tr|B3DJW3|B3DJW3_DANRE
tr|B3DKG5|B3DKG5_DANRE
tr|B3DKB1|B3DKB1_DANRE
tr|B0S591|B0S591_DANRE
tr|B3DJL6|B3DJL6_DANRE
tr|Q9NBK6|Q9NBK6_DROME
tr|Q9VLE6|Q9VLE6_DROME
tr|Q9NBK8|Q9NBK8_DROME
tr|Q9NBK9|Q9NBK9_DROME
tr|Q7KIN0|Q7KIN0_DROME
tr|Q9V477|Q9V477_DROME
tr|Q9VPH1|Q9VPH1_DROME
sp|P08953|TOLL_DROME
tr|Q9N5Z3|Q9N5Z3_CAEEL
tr|Q5I9V9|Q5I9V9_CAEEL
tr|Q5I9W0|Q5I9W0_CAEEL
tr|Q5I9W1|Q5I9W1_CAEEL
DD loop
CHYELYFAHHNLFHE-GSNSLILILLEPIPQYSIPSSYHKLKSLMARRTY
CKYELDFSHFRLFDE-NNDAAILILLEPIEKKAIPQRFCKLRKIMNTKTY
KRFKVHHAVQQAIEQ-NLDSIILVFLEEIPDYKLNHALCLRRGMFKSHCI
CIFEYEIAQTWQFLS-SRAGIIFIVLQKVEKTLLRQQVELY-RLLSRNTY
LEAFS-YAQGRCLSD-LNSALIMVVVGSLSQYQLMKHQSIR-GFVQKQQY
CHYELYFAHHNLFHE-GSNNLILILLEPIPQNSIPNKYHKLKALMTQRTY
FKIAFYLSHQRLMDE-KVDVIILIFLEKPFQKSKFLQLRKR---LCGSSV
FKTAFYLALQRLMDE-NMDVIIFILLEPVLQHSQYLRLRQR---ICKSSI
LRASFLLAQQRLLED-RKDVVVLVILSPDGRRSRYVRLRQR---LCRQSV
CHYEFYFAHHNLFHE-NSDHIILILLEPIPFYCIPTRYHKLKALLEKKAY
CHYELYFAHHNLFHE-GSDNLILILLAPIPQYSIPTNYHKLKTLMSRRTY
CKYELDFSHFRLFDE-NNDAAILVLLEPIERKAIPQRFCKLRKIMNTKTY
RRFKVHHAVQQAIEQ-NLDSIILIFLQNIPDYKLNHALCLRRGMFKSHCI
CIFEYEIAQTWQFLS-SRSGIIFIVLEKVEKSLLRQQVELY-RLLSRNTY
LEAFR-YAQSRSLSD-LKSILIVVVVGSLSQYQLMRHETIR-GFLQKQQY
CHYELYFAHHNLFHE-GSDNLILILLEPILQNNIPSRYHKLRALMAQRTY
FKMAFYLSHQRLLDE-KVDVIILIFLEKPLQKSKFLQLRKR---LCRSSV
FKTAFYLALQRLMDE-NMDVIIFILLEPVLQYSQYLRLRQR---ICKSSI
LRTSFLLAQQRLLED-RKDVVVLVILRPDAHRSRYVRLRQR---LCRQSV
CNLELRLATYHLVARPGTTHLLLLFLEPLDRQRLHSYHRLS-RWLQKEDY
CRLELRLATSLLLAAPSPPVLLLVFLEPISRHQLPGYHRLA-RLLRRGDY
CRLEVQLASMKMFYE-HKDVIILIFLEEIPNYKLSSYHRLR-KLINKQTF
CHYELYFAHHRLFSE-NSNSLILILLEPIPSYVIPARYHKLKALMAKRTY
CKYELDFSHFRLFDE-NNDVAILILLEPIQSQAIPKRFCKLRKIMNTKTY
CSFEFDIARSWQLVE-GKAGIIMIILGEVDKTLLRQRLGLS-RYLRRNTY
VEAFN-FAQSRYFSD-LKEVLIMVVVGSLSQYQLMKHKPIR-IFLQRSRY
FKTTFYMAHQRLLDE-KIDVIILIFLEKVLQKSRYVQLRKR---LCRSSV
CQYELYFAEHRVLDE-NQDSLIMVVLEDLPPDSVPQKFSKLRKLLKRKTY
CHYELYFAHHKLFSE-NSNSLILILLEPIPPYVIPARYHKLKALMAKRTY
CHYELYFAHHKLYTE-NNDNLILILLEPIPHYLIPSKYYKLK-------CKYELEFSHFRLFDE-NNDSAILILLEPIENETVPKRFCKLRKLMNTKTY
RRFKVQHAFQQAIEQ-NRDSIILIFLEDIPDYKLYHTIHLRRGMFKSRCI
VEALN-YAQSRYFTD-LKDVLIMVVVGSLSQYQLMKYQPIR-AYVKRCQY
FKTAFYMAHQRLIEE-KVDVIILILLEKTLQRSRYLRLRKR---LCANSV
FKTAFYMAHQRLIEE-KVDVIILILLEKTLQRSRYLRLRKR---LCANSV
FKTAFYLALQKLMDE-NMDVIVIVLLEPVLQNSQYLRLRRK---ICKSSI
LRHAFFMSNQRLLDE-KKDVVALVVLDHKMKMSQYFLTRKR---LCPKSF
CHYELYFANHQKLTR-GMDSILLILLEPLPLYLIPSKYYQLKTMMSRRTY
CRYELDFSHFRIMDE-HNDSAVLVLLEPIKKETIPKRFCKLRKIMNSRTY
RQFKAHHALHHVMED-NRDSLILIFLEDVTDYNLNRSLHLRRGMLKPKCV
CRFEFELAQSRFLME-RNANIIIIILEDVAERKTKKILGLH-KHLKKNTY
CRFEFELAQSRFLME-RNANIIIIILEDVAERKTKKVFGLH-KHLKKNTY
LETFT-LAQKRMQAE-LEDILVVLVVGNIPQYRLLKYKQVR-SFIENRSY
VRQAFLLVQQRLLDE-KVDVAVLVLLDFLFPKFKYLQMRKR---LCKKSV
CNYELYFAQQRAIGK-TFSDVILVVKEPIDPTSLPSKFCKLKRMLNTKTY
CSLEMQLATYRLQVE-HRDILILVFLENIPSRLLSSHHRLA-RLVKTRTY
CSLEIQLASYRLFQE-MQDVLLLVFLEPIPERQLSAYHRMR-KVMLKKTY
CSSEVQVASFRLFDE-QKDVLILVFLEDIPTHQLSPYHRLR-KLVKKRTY
GRLEFRLALHATSRD-RCKRLIVVLYPNVKNFDSLDSELRTYMAFN--TY
GRLEFRLALHATSRD-RCKRLIVVLYPDVEHFDDLDSELRAYMVLN--TY
ARMEFRIAYQATLQD-KRKRIIIILYRELEHMNGIDSELRA--YLKLNTY
SRFEYKSALHELVK--RRKRVVFILYGDLP-QRDIDMDMRHYLRTS--TC
ARCELRRSVHDALRG-RPQKLVIIEEPEVAFEAESDIELLPYLKTSAVHR
CRFEFKSAHQSVLRD-RRRRLIVIVLGEVP-QKELDPDLRLYLKTN--TY
CQFEMYLAQHRIFEV-SKEHLILVFLEDIPRRKRPKTLQYL---MDVKTY
ARLEFRAAHRSALNE-GRSRIIVIIYSDIGDVEKLDEELKA--YLKMNTY
KTLQIKTSHQLFAKN-RAKRVIAVLGDGVDANLLDDELGQI---LRKHTR
QDACMAKVVRFITGELNRTTPTTKEMPSISRKTTQQRWQTTNTVSRTGPS
QDACMAKVVRFITGELNRTTPTTKEMPSISRKTTQQRWQTTNTVSRTGPS
QDACMAKVVRFITGELNRTTPTTKEMPSISRKTTQQRWQTTNTVSRTGPS
755
761
876
794
815
760
1012
1019
992
754
758
761
877
792
816
760
1013
992
992
897
884
954
768
770
808
818
1022
825
768
85
754
872
832
1017
1017
1003
992
761
762
875
724
795
828
1019
773
208
914
913
763
1092
761
1228
1217
1193
867
977
1175
544
939
886
6
sp|Q15399|TLR1_HUMAN
sp|O60603|TLR2_HUMAN
sp|O15455|TLR3_HUMAN
sp|O00206|TLR4_HUMAN
sp|O60602|TLR5_HUMAN
sp|Q9Y2C9|TLR6_HUMAN
sp|Q9NYK1|TLR7_HUMAN
sp|Q9NR97|TLR8_HUMAN
sp|Q9NR96|TLR9_HUMAN
sp|Q9BXR5|TLR10_HUMAN
sp|Q9EPQ1|TLR1_MOUSE
sp|Q9QUN7|TLR2_MOUSE
sp|Q99MB1|TLR3_MOUSE
sp|Q9QUK6|TLR4_MOUSE
sp|Q9JLF7|TLR5_MOUSE
sp|Q9EPW9|TLR6_MOUSE
sp|P58681|TLR7_MOUSE
sp|P58682|TLR8_MOUSE
sp|Q9EQU3|TLR9_MOUSE
sp|Q6R5P0|TLR11_MOUSE
sp|Q6QNU9|TLR12_MOUSE
sp|Q6R5N8|TLR13_MOUSE
tr|Q5WA51|Q5WA51_CHICK
sp|Q9DD78|TLR21_CHICK
tr|Q7ZTG5|Q7ZTG5_CHICK
tr|Q5GR02|Q5GR02_CHICK
tr|Q5ZJD0|Q5ZJD0_CHICK
tr|E0AEW6|E0AEW6_CHICK
tr|A5YBP4|A5YBP4_CHICK
tr|A4UXC9|A4UXC9_XENLA
tr|F7C313|F7C313_XENTR
tr|F6YQH2|F6YQH2_XENTR
tr|Q5U5B1|Q5U5B1_XENLA
tr|F6ZAC8|F6ZAC8_XENTR
tr|F6QHI4|F6QHI4_XENTR
tr|B2GUH5|B2GUH5_XENTR
tr|F6U8S1|F6U8S1_XENTR
tr|F7EDI8|F7EDI8_XENTR
tr|B3DIW3|B3DIW3_DANRE
tr|Q6TS42|Q6TS42_DANRE
tr|Q6IWL5|Q6IWL5_DANRE
tr|B3U3W0|B3U3W0_DANRE
tr|Q6TS41|Q6TS41_DANRE
tr|B3DIN1|B3DIN1_DANRE
tr|B3DJW3|B3DJW3_DANRE
tr|B3DKG5|B3DKG5_DANRE
tr|B3DKB1|B3DKB1_DANRE
tr|B0S591|B0S591_DANRE
tr|B3DJL6|B3DJL6_DANRE
tr|Q9NBK6|Q9NBK6_DROME
tr|Q9VLE6|Q9VLE6_DROME
tr|Q9NBK8|Q9NBK8_DROME
tr|Q9NBK9|Q9NBK9_DROME
tr|Q7KIN0|Q7KIN0_DROME
tr|Q9V477|Q9V477_DROME
tr|Q9VPH1|Q9VPH1_DROME
sp|P08953|TOLL_DROME
tr|Q9N5Z3|Q9N5Z3_CAEEL
tr|Q5I9V9|Q5I9V9_CAEEL
tr|Q5I9W0|Q5I9W0_CAEEL
tr|Q5I9W1|Q5I9W1_CAEEL
LEWP-----KEKSKRGLFWANLRAAINIKLTEQAKK-------------LEWP-----MDEAQREGFWVNLRAAIKS---------------------LNWP-----VQKERIGAFRHKLQVALGSK---NSVH-------------LEWE-----DSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI
LRWP-----EDFQDVGWFLHKLSQQILKK---EKEKKKDNNIPLQTVATI
LQWP-----KEKSKRGLFWANIRAAFNMKLTLVTENNDVKS--------LEWP-----TNPQAHPYFWQCLKNALATDNHVAYSQVFKETV-------LQWP-----DNPKAEGLFWQTLRNVVLTENDSRYNNMYVDSIKQY----LLWP-----HQPSGQRSFWAQLGMALTRDNHHFYNRNFCQGPTAE----LEWP-----KDRRKCGLFWANLRAAINVNVLATREMYELQTFTELNEESR
LEWP-----TEKNKHGLFWANLRASINVKLVNQAEGTCYTQQ-------LEWP-----LDEGQQEVFWVNLRTAIKS---------------------LNWP-----VQKERINAFHHKLQVALGSR---NSAH-------------LEWE-----DNPLGRHIFWRRLKNALLDGKASNPEQTAEE--EQETATWT
LRWP-----EDLQDVGWFLDKLSGCILKE---EKGKKRSSSIQLRTIATI
LEWP-----TEKGKRGLFWANLRASFIMKLALVNED-DVKT--------LEWP-----ANPQAHPYFWQCLKNALTTDNHVAYSQMFKETV-------LQWP-----NNPKAENLFWQSLKNVVLTENDSRYDDLYIDSIRQY----LFWP-----QQPNGQGGFWAQLSTALTRDNRHFYNQNFCRGPTAE----FDLS-----QGKVEWNSFCEQLKRRLSKAGQERD---------------CLWP-----EEEERKSGFWTWLRSRLG----------------------ITWP-----DSVHQQPLFWARIRNALGKETVEKENTHLIVVE-------LEWP-----KERSKHALFWANLRAAVNIKLPTSFETDEEQSDVTSTSSIT
LEWP-----PDEEQQQMFWENLKAALKS---------------------LEWK-----NKEISRHIFWRQLTSVLLEGKKWNHEEIKLM---------LRWP-----EDYQDIGWFLDNLSSQILKE---KKVQRNVSGIELQTIATV
LEWP-----TNPRSQPYFWQRLKNAIAMNNTLSYNKLLQETV-------LKWS-----PEEHKQKIFWHQLAAVLKTTNEPLVRAENGPNEDVIEME-LEWP-----KERSKHALFWANLRAAISINLSVADEQN--RTEV-------------------------------------------------------LEWP-----TDEEQQEVFWDNLKTALQS---------------------LDWP-----DQMERIKTFYQRLKIALGST---NLVN-------------LKWP-----EDIQDVEWFLGRLSYQILKENKVEKKLKKSSNHELQTIETI
LEWP-----SEKSKHGLFWANLRAAISIDLTHAESE-------------LYWP-----SNPNSQSYFWHCLKSAIATENQMGYDKLFKDHT-------LYWP-----SNPNSQSYFWHCLKSAIATENQMAYDKLFKDHT-------MEWP-----KNPNTKSFFWQRMKNVLLTDNCNRYNNFYTDTIAN-----LNWP-----CNPKAHSHFWHMLRIYIRQDSRRCCGSQLKKYV-------LEWP-----QEGAKQKLFWANLRAALQAELPNTPDREEE----------LEWP-----EDEDKRDEFWSNLRAALQRDEC------------------LYWP-----LHKERIPAFHQKLRSALAST---NKVN-------------LKWS-----RDPLSNMRFWIRLRKAIVATKQ------------------LKWS-----RDPLSNMRFWIRLRKAIVAT--------------------LVWP-----DDGQDLEWFYDQLLHKIRKDIKINQTTKETKREEANFNTNT
LSWP-----RNPRVQPLFWNDLRVALVSDNVRAYNKNVTESFF------LEWP-----QQPTEQNFFWIQLRSVLGKPNSIRPRTISRHSRLSSARSVS
LDWP----QEPE-MHDAFWDRLWCKLSSNKAN-----------------LQWPGSNCSDPNSAKELFWNQLKRALRSSNSGSQDEQKMDDNELRRKEKV
IRWP-----KPGEDNKIFWQKLKMALETKDSHKSENCIL----------LERS----------HPNFWNKLIYSMPHTKLR-----------------LDRN----------NPNFWNKLMYSMPHASHLKRSRSDAETKV------LKWG----------DPLFWSKLYYAMPHNRRVLKGQKKHAGPLI-----IEWD----------DKKFWQKLRLALPLPNGRGNNNKRVVSGCLSGRTPS
IRRS----------DRHFWEKLRYALPVDYPTFRGNNYTLELDHHNHERV
LQWG----------DKLFWQKLRFALPDVSSSQRSN-------VAGQSCH
IKWP-----TAKEDRKLFWKRLKRSLEVIGINSREISV-----------LKWG----------DPWFWDKLRFALPHRRPVGNIGNGALIKTALKGSTD
IEMR----------SHLFWTLLHSSLPSRLPLPSNSGDDSSQLYSDIYGI
RSIGG---PRMEPPTPTFFSVTPTGSQERATSTRRKIQPSASTTSDRN-RSIGG---PRMEPPTPTFFSVTPTGSQERATSTRRKIQPSASTTSDRN-RSIGG---PRMEPPTPTFFSVTPTGSQERATSTRRKIQPSASTTSDRN—-
786
784
904
839
857
796
1049
1059
1032
799
795
784
905
835
858
795
1050
1032
1032
926
906
991
813
793
843
860
1059
868
804
777
900
877
783
1054
1054
1042
1029
795
788
903
750
819
873
1057
818
235
964
947
785
1125
795
1268
1257
1226
900
1017
1215
589
984
931
7
Supplementary Figure 5: Two-dimensional maps representing the cross-peaks (shown in black
symbols for backbone and red for sidechain interactions) arising as a result of the long range
interactions computed for the TIR domain of the ten hTLRs. The BB and DD loop segments have
been marked in green lines. The frequency of involvement of a particular residue in interactions
through its backbone and side chain is shown as histograms.
hTLR1
hTLR2
450
400
350
Frequency
Frequency
hTLR2-TIR
400
hTLR1-TIR
350
300
250
200
150
100
50
300
250
200
150
100
50
0
780
0
780
760
Backbone
Backbone
760
740
720
700
680
740
720
700
680
660
660
640
640
640
660
680
700
720
740
760
7800
5
10
15
20
25
30
640
hTLR3
680
700
720
740
760
7800
5
10
15
20
5
10
15
25
30
35
40
Frequency
Sidechain
hTLR4
400
400
hTLR3-TIR
350
350
300
300
Frequency
Frequency
660
Frequency
Sidechain
250
200
150
100
50
hTLR4-TIR
250
200
150
100
50
0
0
900
880
Backbone
Backbone
800
860
840
820
800
780
760
740
720
700
780
680
760
760
780
800
820
840
Sidechain
860
880
9000
5
10
15
20
25
30
Frequency
35
680
700
720
740
760
Sidechain
780
800
0
20
25
30
35
Frequency
8
Supplementary Figure 5 (Continued.)
hTLR5
hTLR6
350
400
300
Frequency
350
250
200
150
100
50
200
150
100
50
0
0
840
780
820
760
800
Backbone
hTLR6-TIR
250
Backbone
Frequency
hTLR5-TIR
300
780
760
740
720
740
720
700
680
660
700
640
700
720
740
760
780
800
8400
820
Sidechain
5
10
15
20
25
640
30
660
680
700
720
740
760
780 0
5
hTLR7
10
15
20
25
30
35
Frequency
Sidechain
Frequency
hTLR8
400
300
hTLR8-TIR
350
Frequency
Frequency
hTLR7-TIR
250
200
150
100
50
300
250
200
150
100
50
0
0
1040
1020
Backbone
Backbone
1020
1000
980
960
940
1000
980
960
940
920
920
900
900
900
920
940
960
980
1000
1020
0
5
10
15
20
25
30
900
960
980
1000
1020
0
1040
5
10
15
20
25
30
Frequency
hTLR10
400
350
hTLR9-TIR
hTLR10-TIR
350
Frequency
300
250
200
150
100
50
300
250
200
150
100
50
0
0
1000
760
980
740
Backbone
Frequency
940
Sidechain
hTLR9
Backbone
920
Frequency
Sidechain
960
940
920
900
720
700
680
660
880
640
880
900
920
940
960
Sidechain
980
1000
0
5
10
15
20
25
Frequency
30
35
640
660
680
700
720
Sidechain
740
760
0
5
10
15
20
25
30
35
Frequency
9
Supplementary Figure 6: Contact maps representing the cross-peaks of the mutants of hTLR1TIR (G676A) and hTLR2-TIR compared to their native forms. Panel A shows difference in crosspeaks seen for hTLR1-TIR native and its G676A mutant. Panels B-H show the cross peaks of
hTLR2-TIR mutants compared to their native form. Every contact map has been labelled
according to the residue substitution (shown at top right corner of the map), the position of which
is shown with a red line running through the contact-map. The color coding of the symbols used
are: gray for cross-peaks arising from native-like interactions, blue for those present in native only
and red for those unique to the mutant form.
A.
760
740
720
700
660
640
660
680
700
720
740
760
780
680
620
620
800
hTLR2-TIR:F701V-K743E
800
D.
760
740
720
680
660
640
660
680
700
E.
720
740
760
780
740
760
780
800
hTLR2-TIR:F701V
740
720
700
680
620
620
800
760
720
700
680
760
780
620
620
800
640
660
H.
720
700
680
Backbone residues
740
800
680
640
740
780
700
660
720
760
720
640
700
740
740
660
Sidechain residues
720
760
780
800
L
760
R
780
760
680
700
hTLR2-TIR:L762Q
800
780
660
680
Sidechain residues
hTLR2-TIR:R753Q
640
800
TLR2-TIR:F749A
Sidechain residues
800
780
680
640
G.
760
700
660
740
740
720
640
720
720
740
660
700
700
F
K
740
Backbone residues
780
680
680
800
760
660
660
F.
780
640
640
Sidechain residues
hTLR2-TIR:K743E
800
Backbone residues
720
640
Sidechain residues
Backbone residues
700
660
640
620
620
680
F
F
700
Backbone residues
780
760
620
620
660
800
780
620
620
640
Sidechain residues
K
Backbone residues
700
640
Sidechain residues
C.
720
660
640
620
620
740
P
Backbone residues
780
760
680
hTLR2-TIR:P681H
800
780
G
Backbone residues
B.
hTLR1-TIR:G676A
800
620
620
640
660
680
700
720
740
760
780
800
Sidechain residues
10
hTLR1-TIR
G676A
200
210
220
230
Mean Residue Ellipticity
Mean Residue Ellipticity
Supplementary Figure 7: Circular dichroism spectra acquired from (left) mCherry-TLR1 (TIR
domain) and its Ala substitution mutant, and (right) mCherry-TLR2 (TIR domain) and some of its
mutants. The ellipticity axis has been translated for clarity.
Native hTLR2-TIR
F701V-K743E
P681H
L762Q
R753Q
F749A
200
240
210
220
230
240
Wavelength(nm)
Wavelength(nm)
Estimated secondary structural content in the different recombinant mCherry-TLR1 (TIR) domain
and its point mutant based on deconvulation of the measured CD data using K2D2 server.
Protein
Native TLR1
G676A
α-helical Content
20
19
β-sheet Content
38
33
Native TLR2
F701V-K743E
P681H
L762Q
R753Q
F749A
20
19
21
26
23
17
38
33
40
31
35
37
11
Log10 Intensity
Log10 Intensity
Supplementary Figure 8A: SAXS scattering intensity profile from a solution of (left) mCherryTLR1-TIR and, (right) mCherry-TLR2-TIR are shown below.
0.01
0.1
-1
Q (A )
1
0.01
0.1
1
-1
Q (A )
Supplementary Figure 8B: Three rotated views show the SAXS data based chain-ensemble
model of the mCherry-TLR1 (TIR domain) (gray cpk). In the volume of the SAXS data based
model, crystal structure of the mCherry (PDB ID: 2H5Q; red ribbon) and that of TLR1-TIR (PDB
ID: 1FYV; green ribbon) have been placed to show their relative positioning in space.
Supplementary Figure 8C: Three rotated views show the SAXS data based chain-ensemble
model of the mCherry-TLR2 (TIR domain) (green cpk). In the volume of the SAXS data based
model, crystal structures of the mCherry (PDB ID: 2H5Q; red ribbon) and hTLR2-TIR domain
(PDB ID: 1FYW; green ribbon) have been placed to show their relative positioning in space.
12
Supplementary Table 1: Shape parameters of the mCherry-tagged TIR domains of hTLR1 and
TLR2, and their mutants as deduced from indirect Fourier transformation of the measured SAXS
data from protein solutions are tabulated below.
Protein
Native TLR1
G676A
Native TLR2
F701V-K743E
P681H
L762Q
R753Q
F749A
Conc. (mg/ml)
1.2
1.5
2.7
3.0
2.5
2.2
3.5
3.1
Dmax
80
82
RG (Å)
26. 8 ± 0.3
23.9 ± 0.7
83
79
80
78
77
85
28.2 ± 0.2
25.7 ± 0.2
23.9 ± 0.5
24.8 ± 0.18
26.5 ± 0.15
24.6 ± 0.9
13
Supplementary Figure 9: Contact maps for native hTLR4-TIR and its comparison with mutants.
The cross-peaks formed by the interaction of native hTLR4-TIR have been shown in gray squares
(panel A). Panels B-H show cross-peaks of the mutants of hTLR4-TIR compared to those
exhibited by the native form. Cross-peaks which remain native-like have been shown in light-gray
squares while those which were present in native but absent in mutant have been shown in blue
squares. Red squares depict cross-peaks which are new to mutant i.e. present only in mutant.
Position of mutation has been marked in red lines in the map. Every contact map has been labelled
according to the residue substitution in the mutant (shown at top right corner of the map), green
font represents mutant showing activity comparable to that of wild-type while red shows mutants
with almost complete loss of wild type activity.
A.
hTLR4-TIR
820
B4
800
Backbone residues
800
S8
780
760
B3
S4
S11
720
700
760
740
720
700
S3
B1
S2
680
680
700
720
740
760
680
S9
S6
S1
660
660
S10
S7
S5
B2
740
780
H
Backbone residues
hTLR4-TIR:H728A
B.
820
780
800
660
660
820
680
700
D.
hTLR4-TIR:R780A
820
800
800
780
760
740
720
Backbone residues
820
R
760
780
800
820
hTLR4:FVI677-679AAA
760
740
720
700
FVI
680
680
660
660
660
660
680
700
720
740
760
780
800
680
700
820
E.
F.
hTLR4:LCL705-707AAA
820
800
800
780
760
720
780
800
820
hTLR4:VVS736-738AAA
780
760
740
LCL
720
680
680
700
720
740
760
780
800
660
660
820
Sidechain residues
G.
800
780
IFI
760
740
720
Backbone residues
800
780
800
820
800
820
hTLR4:L815A
720
680
760
780
740
680
740
760
760
700
720
740
780
700
Sidechain residues
720
L
820
700
700
H.
hTLR4:IFI767-769AAA
680
680
Sidechain residues
820
660
660
760
700
680
660
660
740
VVS
740
Backbone residues
820
700
720
Sidechain residues
Sidechain residues
Backbone residues
740
780
700
Backbone residues
Backbone residues
C.
720
Sidechain residues
Sidechain residues
660
660
680
700
720
740
760
780
Sidechain residues
800
820
14
Supplementary Figure 10: Contact maps showing the comparison of cross-peaks of the point mutants of
TIR domain of hTLR4 with those in the native form. Every contact map has been labeled according to the
residue substitution (shown at top right corner of the map), orange color stands for milder mutant and red
for mutants showing almost complete loss of wild type activity. Red lines show the point of mutation while
the BB- and DD-loop regions have been marked in black lines. Gray cross-peaks are for native like
interactions, blue for those present only in the native and red for those unique to mutant forms.
hTLR4-TIR:V678A
820
800
800
Backbone residues
Backbone residues
hTLR4-TIR:F677A
820
780
760
740
720
780
760
740
720
680
660
660
680
700
720
740
760
780
800
660
660
820
V
700
680
F
700
680
700
720
800
Backbone residues
800
780
760
740
720
740
720
680
I
700
740
760
780
800
660
660
820
680
700
720
800
780
760
740
720
780
800
820
780
760
740
720
C
700
Backbone residues
800
680
700
680
680
700
720
740
760
780
800
660
660
820
Sidechain residues
680
700
720
740
760
780
800
820
Sidechain residues
hTLR4-TIR:H724A
hTLR4-TIR:L707A
820
800
800
780
760
740
700
680
780
760
740
H
720
Backbone residues
820
L
Backbone residues
760
hTLR4-TIR:C706S
820
C
Backbone residues
hTLR4-TIR:C706A
820
660
660
740
Sidechain residues
Sidechain residues
660
660
820
760
680
720
800
780
700
700
780
hTLR4-TIR:L705A
820
680
760
L
Backbone residues
hTLR4-TIR:I679A
820
660
660
740
Sidechain residues
Sidechain residues
720
700
680
680
700
720
740
760
780
Sidechain residues
800
820
660
660
680
700
720
740
760
780
800
820
Sidechain residues
15
Supplementary Figure 10 (Continued).
hTLR4-TIR:V736A
800
800
780
760
720
780
760
740
720
700
700
680
680
660
660
680
700
720
740
760
780
800
V
740
Backbone residues
820
G
Backbone residues
hTLR4-TIR:G726C
820
660
660
820
680
700
720
740
800
780
760
V
740
720
700
Backbone residues
800
780
760
740
720
700
680
700
720
740
760
780
800
660
660
820
680
700
720
740
800
780
740
720
760
740
720
700
680
680
740
760
780
800
660
660
820
680
700
720
hTLR4-TIR:F768A
800
800
780
740
720
740
720
700
680
780
Sidechain residues
820
760
680
760
800
780
700
740
780
I
F
760
Backbone residues
820
720
760
hTLR4-TIR:I769A
820
700
740
Sidechain residues
Sidechain residues
680
820
780
700
720
800
I
760
Backbone residues
800
I
Backbone residues
820
700
780
hTLR4-TIR:I767A
hTLR4-TIR:I753A
820
680
760
Sidechain residues
Sidechain residues
Backbone residues
820
680
680
660
660
800
S
Backbone residues
820
660
660
780
hTLR4-TIR:S738A
hTLR4-TIR:V737A
820
660
660
760
Sidechain residues
Sidechain residues
800
820
660
660
680
700
720
740
760
780
800
820
Sidechain residues
16
Supplementary Figure 11: Circular dichroism data on mCherry-TLR4 (TIR domain) and some of its ala
substitution mutants is presented below. The ellipticity axis has been translated for clarity.
Mean Residue Ellipticity
Native
H728A
R780A
FVI677to679AAA
LCL705to707AAA
VVS736to738AAA
IFI767to769AAA
L815A
200
210
220
230
240
Wavelength (nm)
Protein
Native TLR4
H728A
R780A
FVI677-679AAA
LCL705-707AAA
VVS736-738AAA
IFI767-769AAA
L815A
α-helical Content
23
19
23
25
19
20
15
12
β-sheet Content
31
33
35
36
29
28
35
35
17
Log10 Intensity
Supplementary Figure 12A: SAXS scattering intensity profile from a solution of mCherry-TLR4 is shown
below.
0.01
0.1
1
Q (A-1)
Supplementary Figure 12B: Three rotated views show the SAXS data based chain-ensemble model of the
mCherry-TLR4 (TIR domain) (green cpk). In the volume of the SAXS data based model, crystal structure
of the mCherry (PDB ID: 2H5Q; red ribbon) and modelled structure of TLR4 TIR domain (blue ribbon)
have been placed to show their relative positioning in space.
Supplementary Table 2: Shape parameters of the mCherry-TLR4
from indirect Fourier transformation of the measured SAXS data
below.
Protein
Conc. (mg/ml)
Dmax
Native TLR4
4.1
80
H728A
3.7
79
R780A
2.2
82
FVI677-679AAA
3.4
78
LCL705-707AAA
3.2
80
VVS736-738AAA
3.5
80
IFI767-769AAA
3.7
78
L815A
3.6
81
molecule and its mutants as deduced
from protein solutions are tabulated
RG (Å)
24.2 ± 0.2
23.9 ± 0.2
24.5 ± 0.5
23.8 ± 0.3
24.0 ± 0.1
24.2 ± 0.5
23.7 ± 0.2
24.1 ± 0.1
18
References in Supplementary
Ashish, Grover, A. & Kishore, R. (2000). Characterization of a novel type VII beta-turn conformation for a
bio-active tetrapeptide rigin A synergy between theoretical and experimental results. Eur J
Biochem 267: 1455-1463.
Ashish, A. & Kishore, R. (2002). Folded conformation of an immunostimulating tetrapeptide rigin: high
temperature molecular dynamics simulation study. Bioorg Med Chem 10: 4083-4090.
Konarev, P. V., Volkov, V. V., Sokolova, A. V., Koch, M. H. J. & Svergun, D. I. (2003). PRIMUS: a Windows PCbased system for small-angle scattering data analysis. J. Appl. Cryst. 36: 1277-1282.
Perez-Iratxeta, C. & Andrade-Navarro, M. A. (2008). K2D2: estimation of protein secondary structure from
circular dichroism spectra. BMC Struct Biol 8: 25.
Svergun, D. I. (1992). J Appl Crystallogr 25: 495-503.
Svergun, D. I. (1999). Restoring Low Resolution Structure of Biological Macromolecules from Solution
Scattering Using Simulated Annealing. Biophys J 76: 2879-2886.
19