nature structural & molecular biology: …...nature structural & molecular biology:...

Supplementary Figure 1

MAL forms filaments in solution

a, Negative-stain EM images of MALTIR

at 60 M, 52.5 M, 45 M, and 30 M. b, SDS-PAGE precipitation analysis of MALTIR

and MAL

FL. SF, soluble fraction; IF, insoluble fraction. c-d, Turbidity assays of MAL

TIR (c) and MAL

FL (d) at 30°C. MAL polymerization could

be quantified by following the increase in absorbance at 350 nm as a function of t ime. Typically, with 60 μM MALTIR

at 30°C, an initial lag time of 10 min was observed, followed by a rapid increase in turbidity that approached saturation after approximately 20 min. This suggests that MAL-filament growth proceeds through small early assembly intermediates, for which disassembly is energetically favored over assembly. After a sufficiently large oligomer assembles, MAL-filament growth becomes energetically favorable and proceeds rapidly. The increase in turbidity correlated well with the observation of filaments by negative-stain EM. The critical concentration for MAL

TIR-filament formation is estimated to be 50-60 μM. e-f, Effect of NaCl (e) and pH (f) on MAL

TIR filament formation.

The original image of the gel in (b) can be found in Supplementary Data Set 1.

Nature Structural & Molecular Biology: doi:10.1038/nsmb.3444


Single molecule fluorescence analyses of MALFL and MALTIR, and analyses of MALTIR interaction with TLR3TIR

a-b, MALFL

and MALTIR

tagged with GFP were expressed in LTE and fluorescence time traces were acquired. Representative traces

are shown. The trace for MALFL

shows the presence of larger peaks (>4000 photons per ms or cpms) compared to MALTIR

(peaks at 2000 cpms). c, Schematic diagram of the seeding assay. d, mCherry MAL

FL was used to create the seeds, which were added to a

solution of monomeric GFP MALFL

. Formation of MALFL

polymers was detected immediately after addition of seeds and increase in the intensities of the peaks upon time indicate polymer elongation. e, The ragged profile of the peak in the insert suggests that the objects

are fibrillar. f, As a control for the TLR4TIR

-MALTIR

interaction, we incubated equal amounts of TLR3TIR

with MALTIR

(60 M, 30 °C, 1

hour). TLR3 only engages the TRIF-dependent pathway, which does not require MAL. SDS-PAGE analysis revealed that both proteins were present in the insoluble fraction but similar amounts of TLR3 was observed in the TLR3

TIR control sample without MAL, suggesting

that co-precipitation of TLR3TIR

and MALTIR

is not due to formation of co-filaments. g, Supporting this conclusion, SDS-PAGE analyses

of TLR3TIR

and MALTIR

incubated at a concentration below the critical concentration for MALTIR

oligomerization (30 μM of each, 30°C for 1 h) only revealed the presence of a faint MAL

TIR band in the insoluble fraction. Results in (f-g) are representative of three experiments.

h, Negative-stain EM images of TLR3TIR

+ MALTIR

, 60 M of each, and TLR3TIR

(60 M). Ordered assemblies were not observed in the

TLR3TIR

samples, while only the MALTIR

filaments were observed in the TLR3TIR

:MALTIR

samples. Original gel images in (f-g) can be found in Supplementary Data Set 1.



MALTIR induced formation of MyD88TIR assemblies, and single molecule fluorescence analyses of MyD88FL and MyD88TIR

a, SDS-PAGE analyses of the soluble fraction (SF) and insoluble fraction (IF) after incubation of MALTIR

(60 M) + MyD88TIR

(60 M) at

30°C for 1 hour. b, Negative-stain EM images of MALTIR

+ MyD88TIR

, 60 M of each. c, Turbidity assay of MyD88TIR

(30-80 M)

assembly formation in the presence MALTIR

(2 M). d-e, Negative-stain EM image of a MAL induced MyD88TIR

assembly (d), and the corresponding fast Fourier transform analysis (e). f-g, MyD88

FL and MyD88

TIR tagged with a N-terminal GFP were expressed in LTE

and 180 seconds fluorescence time traces were obtained. Representative traces for MyD88FL

(f) and MyD88TIR

(g) are presented. MyD88

FL (f) forms numerous large objects (>10,000 cpms), whereas MyD88

TIR (g) is mainly oligomeric (peaks at 4,000 cpms). h,

MyD88FL

N-terminally tagged with mCherry was used to create the seeds, which were added to a solution of monomeric GFP MyD88FL

. i, The insert shows, in more detail, the coincidence between the mCherry and the GFP channels.

Note that the difference of intensities

between the two channels indicates that the GFP monomers are elongating the mCherry seeds rather than just coating them. The original gel image in (a) can be found in Supplementary Data Set 1.



Development of a flow cytometry-based assay to quantify MAL-induced clustering of MyD88 in HEK293 cells.

a, HEK293 cells were transfected with a plasmid expressing V5-tagged MyD88FL

alone or with a plasmid expressing Myc-tagged

MALFL

. Cells were immuno-stained with antibodies to V5 and Myc tags (green and red, respectively) and the nuclei stained with DAPI (blue). Representative images are from an experiment performed twice with 1-3 images captured. b-d, Analysis of MyD88 clustering by flow cytometry. b, Plots of MyD88 (V5) stain vs. MAL (Myc) stain for cells transfected with empty vector or with plasmids expressing V5-


tagged MyD88FL

or Myc-tagged MALFL

either together or alone. Cells were immuno-stained and analyzed by flow cytometry. Quadrants representing the MyD88-positive, MAL-positive or double-positive populations are labeled in the panel with empty vector alone. Gates to select broad-expression-window or low-expressing MyD88 cells are indicated in the panels with MyD88. c, Plots of fluorescence

pulse height vs. area for the MyD88 (V5) signal for cells expressing MyD88 alone or with MAL that were gated to obtain cells with low or broad level expression of MyD88 as indicated in (b). In each plot, cells with an elevated height-to-area ratio are boxed and represent the percentage of cells with a MyD88 cluster. d, Gating of cells for Fig. 1i. Transfected cells were immuno-stained and analysed by flow

cytometry. Cells expressing a low level of MyD88 were first selected (green gate) and within this gate, cells expressing MALFL

or MALTIR

at a similar expression level were selected (red gate). Clustering of MyD88 was quantified, within the red gate for samples expressing MAL and MyD88 or within the green gate for samples expressing MyD88 alone, as described in (c).



Power spectra analyses, model-map comparison, and structural analyses of the MALTIR

filament

a, Comparison of the observed versus the calculated power spectra, annotated with Bessel order assignments: on the left is the power spectrum from the projection of the reconstruction, while on the right is the averaged power spectrum from the image segments. b, FSC between the fitted model and the cryoEM map. c, Superposition of the inner MAL

TIR subunit (cyan) and the TIR domain of TLR1 (PDB

ID 1FYV), TLR2 (PDB ID 1FYW), TLR6 (PDB ID 4OM7), TLR10 (PDB ID 2J67), IL-1RAPL (PDB ID 1T3G), MyD88 (PDB ID 2Z5V), MyD88 (PDB ID 4EO7), TRAM (PDB ID 2M1W), TRIF (PDB ID 2M1X) and TRR2 (PDB ID 4W8G).



Structural comparisons of the MALTIR filament with the crystal structures of TRR-2

a, Comparison of the intrastrand and interstrand interactions in the MALTIR

proto-filament (teal) with TIR:TIR interfaces in the TRR-2TIR

(PDB ID 4W8G) crystal structure (violet). b, TIR:TIR interactions in crystal form II of the TRR-2 TIR domain (PDB ID 4W8H). c,

Structure-based sequence alignment of MALTIR

and TRR-2 (PDB ID 4W8G). The alignment was formatted using ESPript. Green and purple circles indicated residues involved in intrastrand, or interstrand TIR:TIR interactions, respectively. Strictly conserved residues are indicated in white letters with a red box and similar residues are indicated in red letters with a blue frame.



MALTIR

proto-filament interactions and structure-based mutagenesis

a, Detailed interactions in the MALTIR

proto-filament:proto-filament interface. b, Precipitation analysis of MALTIR

W156A Y159A double

mutant, and W156A Y159A L165A triple mutant (100 μM incubated at 30°C for 12 hours). The soluble and insoluble fraction of each mutant was analyzed by 15% SDS PAGE. c, Negative-stain EM analysis of MAL

TIR alanine mutants. d, Cartoon representation of a

MALTIR

subunit (outer strand). Residues mutated to alanine (stick representation) are highlighted in green (intrastrand interface), orange (interstrand interface), blue (interface between proto-filaments) and magenta (surface exposed residues outside the interaction interfaces of the filament). The original image of gel in (b) can be found in Supplementary Data Set 1.



Modeling, cryoEM analysis, and structure-based mutagenesis of MyD88TIR

assemblies, and modeling of TRAMTIR

:TRAMTIR

interactions.

a, Cryo-EM image of a MyD88TIR

assembly. b, Power spectrum calculated from the raw image in (a). Spots extending beyond 10 Å resolution can be observed in the power spectrum. c, Overall size of a intrastrand MAL

TIR dimer in the proto-filament. The MyD88

TIR

assemblies were found to have unit cell dimensions of approximately a = 60 Å b = 30 Å, γ = 105, which are similar to the dimensions of a single interstrand MAL

TIR dimer as arranged in the MAL

TIR proto-filament. d, Sequence alignment of MAL

TIR and MyD88

TIR. Strictly


conserved residues are indicated in white letters with a red box and similar residues are indicated in red letters with a blue frame. e, Negative-stain EM images of MyD88

TIR mutants. The scale bar is indicated on the images. f, Turbidity assays of MyD88

TIR mutants.

The assays were performed in triplicate (blue, green, and red lines). g-h, Superposition of the inner MALTIR

subunit (cyan) with TRAMTIR

(light-orange, PDB ID 2M1W). Both the monomer (g) and the proto-filament of MAL

TIR are displayed (h). i, Model of TRAM

TIR intrastrand

and interstrand interactions using the MALTIR

proto-filament as a template.


Supplementary note 1: Sequence alignments of TIR domains. a, Multiple-sequence alignment of human TIR domains involved in TLR or IL-1R signaling. b, Structure-based sequence alignment of human TIR domains of known structure involved in TLR signaling. The positions of the secondary structure elements in the MALTIR cryoEM structure are shown at the top. The alignment was formatted using ESPript. Strictly conserved residues are indicated in white letters with a red box and similar residues are indicated in red letters with a blue frame.

......... ...

MAL 79 Y V V D A LV LE .....SSRWSKD D C CHSEE ....LV AQ.........D SY ...GSTASLRCF

MyD88 155 F A I D V MI LE ......GHMPER D F CYCPS ...IQF Q..........E RQ ...QTNYRLKLC

TRAM 75 I D V L ........EEVFLKFV LHAED TDEALR QN.........L QDDFG....IKPGIIFA

TRIF 338 Y I D V L L .....SSSEQKF NFV LHARA EHIALR R..........EK EA G....VPDGATFC

tlr3 751 Y A I D V ME .......TEQFE A Y IHAYK ...KDW W..........EHFSS ...KEDQSLKFC

tlr5 691 Y A L D V LL LD ..........YK D Y CFSSK ...FTW QN.........A KH TQYSDQNRFNLC

TRR2 95 Y V I D V LV VE ..........KT D N CYAPE ...REW IN.........T FE .....RAGIKTF

tlr9 861 Y A V V L LE ...SGRDEDALP D F VFDKTQSAVADW YN.........E RGQ .ECRGRWALRLC

tlr7 881 Y A I D V LV LE ..YQRLISPDCC D F VYDTK PAVTEW LA.........E AK ..DPREKHFNLC

tlr8 891 Y A I D V L LE .....LSTSQTF D Y SYDTK ASVTDW IN.........E RYH ..ESRDKNVLLC

tlr4 671 Y A V D V LV LE .........ENI D F IYSSQ ...EDW RN.........E KN ...EGVPPFQLC

tlr2 646 A E L E .........SRNICYD FVSYS RD...AYWVEN......... MVQ LE..NFNPPFKL

tlr10 630 L .........RNVRFHAFISYSEHD...SLWVKN.........E IPNLE...KEDGSILI

tlr1 635 F A I D V LL LE NIPLEELQRNLQ H F SYSGH ...SFW KN.........E PN .....KEGMQIC

tlr6 640 F A I D V LV LE ..........LQ H F SYSEH ...SAW KS.........E PY .....KEDIQIC

IL1RL1 391 V IL V ......TDGASRVEHF H.......................Q PD L...ENKCGYTLC

IL1-RAPL 403 Y A L D IL M ..........KD D Y SYTKV P..DQWNQETGEEERFALE PD L...EKHYGYKLF

. .

MAL 118 L RD P G V L L S V LIT FL C A A QL AT. . GAI SE C.QA SS. HCR L PG QDPW .KYQMLQ LTE P..G

MyD88 193 V RD P G A I R V VVS YL C A SD VL. . TCVWSI S.EL EKRCR M V DD QSKE .DFQTKF LSLSPGAH

TRAM 114 P G L L V S I LLT FL C EM...... C RQH QN D.DA NG. AWT L EN RDTW .NFQFYTSLMNSVNRQ

TRIF 379 D P G L L I S I LLT F C V M ...E FQV . RGE SC Q.DA DH. AFI L SN D.... .RLSLHQ NQA MSNL

tlr3 788 L RD G L I I S K I VIT L C A A EE FE.A. VFE EA V.NS KR. R I F HHL KDPL KRFKVHH VQQ IEQN

tlr5 729 RD P G I I I S K V LVS FL C A FEE FV. . ENR AN Q.DA WN. R I C RH RDGW .LEAFSY QGRCLSDL

TRR2 128 V RD P G A I I S R I VMS F C NI DT. . NFF EN M.DA EN. N T V PD FKNNI .DKTLQIGLSHQ....

tlr9 908 L RD P G L V S K L VL A L EE WL. . KTLFEN W.AS YG. R T F AHTDRVSGLL.RASFLL QQR LEDR

tlr7 928 L RD P G L L I S K V VMT YA L EE WL. . QPV EN S.QS QL. K T F DK KTENF.KIAFYLSHQR MDEK

tlr8 935 L RD P G I L I S K V VLT YA A L EE WD. . LAI DN M.QS NQ. K T F KK KSWNF.KTAFYL LQR MDEN

tlr4 707 L RD P G A I S K I VVS FI C A HY FI. . VAI AN IHEGFHK. R V V QH QSRW .IFEYEI QTWQFLSS

tlr2 683 K P G I I I S K V VLS FV C L CLH RDFI . KWI DN I.DS EK. H T F EN KSEW .KYELDFSHFR FDEN

tlr10 666 P G I I S K I VLS FV C A L CLYESYFD . KSISEN V.SF EK. Y S F PN QNEW .HYEFYF HHN FHEN

tlr1 678 L R P G V I I S K I VLS FV C A L HE NFV. . KSI EN I.TC EK. Y S F PN QSEW .HYELYF HHN FHEG

tlr6 673 L R P G V I I S K I VLS FV C A L HE NFV. . KSI EN I.NC EK. Y S F PN QSEW .HYELYF HHN FHEG

IL1RL1 419 I RD P G V V I S R I ILT A A YG ML. . EDV TA E.TN RK. R H F PQITHNKEF.AYEQEV LHC LIQN

IL1-RAPL 448 I RD P I V V S R I VMT YV M PD LI. .TGTY ED A.RC DQ. K L I PN VRRGWSIFELETRLRN LVTG

....... ...

MAL 171 F I L L LR M Y AE......GCT PL SG SRAAYP...P...E .......F Y ..VDGRGPDGG...

MyD88 249 F LI I M I I QK......R P KYKA KK.....EFP...S LRF TVCDY......TNPCTKSW...

TRAM 165 F VI M L R HK......YNS P RP NN.....PLP...RE TPFALQTINAL....EEESRG....

TRIF 428 F I L L L V TRQGSPDCV PF P ESSPAQLSSDTAS...L SGL RLDEHSQI..............

tlr3 844 F IILV L I R L W LD......S F EE PDYKLNHALC...LR GMFKSHCI N ..PVQKERIGA...

tlr5 784 F LIMV V L IR V L W NS......A V GS SQYQLM.KHQ...S GF QKQQY R ..PEDLQDVGW...

TRR2 179 F II I V L L W ......... P LYRP..........C...E PYF NHMTY D ...CDKDVRPV...

tlr9 963 F VVLV L LR L L W KD......V I SPDGRRS...RYV...R QR CRQSV L ..PHQPSGQRS...

tlr7 983 F IILI L LR L L W VD......V F EKPFQKS...KFL...Q KR CGSSV E ..PTNPQAHPY...

tlr8 990 F II I L V LR I L W MD......V F L EP LQHS...QYL...R QR CKSSI Q ..PDNPKAEGL...

tlr4 763 F II I L V L L L W RA......G F V QK EKTLLRQQ.V...E YRL SRNTY E ..EDSVLGRHI...

tlr2 739 F AILI L I LR M L W ND......A L EP EKKAIPQRFC...K KI NTKTY E ..PMDEAQREG...

tlr10 722 F IILI L I LK L L W SD......H L EP PFYCIPTRYH...K AL EKKAY E ..PKDRRKCGL...

tlr1 733 F LILI L I LK M L W SN......S L EP PQYSIPSSYH...K SL ARRTY E ..PKEKSKRGL...

tlr6 728 F LILI L I LK M L W SN......N L EP PQNSIPNKYH...K AL TQRTY Q ..PKEKSKRGL...

IL1RL1 474 F VILI M L L M I W DA......K E EA SELDMLQAEALQDS QHL KVQGT K REDHIANKRSLNSK

IL1-RAPL 504 F VILI L LK I I W EI......K ECSE RGIMNYQEVE...A HT KLLTV K HGPKCNKLNSK...

. . . . .....

MAL 79 Y V V A L LE L L .....SSRWSKD D C. CXSEE.DLV AQD VSY .G.STAS .R.AF. ..Q.....

TLR2 646 Y A V V M LE L .......SRNIC D F. SYSERDAYW ENL VQE NF.NP.PF.K.LC. ..HK....

TLR1 635 F A I V L LE M L NIPLEELQRNLQ H F. SYSGHDSFW KNE LPN .K.EG.. .Q.IC. H.E.....

TLR6 640 F A I V L LE I L ..........LQ H F. SYSEHDSAW KSE VPY .K.ED.. .Q.IC. ..H.....

TLR10 630 F A I V L LE I L ........RNVR H F. SYSEHDSLW KNE IPN .K.EDGS .L.IC. ..Y.....

MyD88 157 F A I V I L V ........MPER D F. CYCPSDIQF QEM RQLEQ.TNYR.. .K.LC. ..S.....

TRIF 388 Y I I V LE V A .....SSSEQKF N.FV LHARA.DEH ALR REK .ALG... .PDG.. T.FCEDFQ

TRAM 75 I V L I ........EEVFLKFV. LHAED.DTDEALR QNL Q.DDFG.. KP.GIIFAEM.....

TRR2 96 Y V I V L LE I V ...........T D N. CYAPEDREW INT VFK .R.AG.. .K.TF. ..N.....

..... .......... .... .

MAL 120 L S V LIT FL .....L..........R....DATPGGAIVSELCQA SS XCR L PG QDPWCK.Y

TLR2 687 I S V VLS FV .....R..........D....FIPG.KWIIDNIIDS EK HKT F EN KS..EW.C

TLR1 681 I S I VLS FV .....R..........N....FVPG.KSIVENIITC EK YKS F PN QS..EW.C

TLR6 675 I S I VLS FV .....E..........R....NFVPGKSIVENIINC EK YKS F PN QS..EW.C

TLR10 669 I S I VLS FV .....E..........S....YFDPGKSISENIVSF EK YKS F PN QN..EW.C

MyD88 195 V VVS YL .....DRDVLPGTCVWS....IA.....SE....LIEKRCRRM V DD QS.KEC.D

TRIF 433 I S I LLT F VHGRGE..........L....SC.....LQ....DA DH AFI L SN DCR..LSLH

TRAM 116 V S I LLT FL .....P..........HGRQHLQ.....NLD...DA NG AWT L EN R...DTWC

TRR2 130 I S I VMS F .....I..........R....DDTPGNFFAENIMDA EN NRT V PD FKNNICD.K

.... ... .

MAL 160 I L LL QMLQ....A.. .....T.......EAPG..AEGCT P S...G.LSRAAYPPE.....

TLR2 724 I A IL K.YEL...D..FSHFRLF.......DEN....NDA L L...E.P...IEKKAIPQRF

TLR1 718 I A L IL H.YEL..YF.. H...HN.......LFHE..GSNS L L...E.P...IPQYSIPSSY

TLR6 713 I A L IL H.YEL..YF.. .....H.......HNLFHEGSNN L L...E.P...IPQNSIPNKY

TLR10 707 I A I IL H.YEF...YF. .....H.......HNLFHENSDH L L...E.P...IPFY......

MyD88 235 I L L I FQTK....FA. .....S.......LSPGA.HQKR P KY...KAMK..KEFPS.....

TRIF 468 I M V L Q.VN....QAM .....SNLTRQGSPD.......C PF PL.ES.SPAQLSSD......

TRAM 150 I M V M N.FQFYTSL.. .....N.......SVNRQHKYNS P RPLNNP.LPRERTP.......

TRR2 170 I L I IL T.LQ....IG. .....S.......H........Q P Y...R.P...CEVPY.....

... ... . .

MAL 191 VD V ......LRFM....Y.Y GRG...PD..GGFRQ...VK.EA .MRYLQTLS........

TLR2 760 LE L A CKLRKIMNTK....T.Y WPMD..EAQ.R..EGFWVN .RA IKS..............

TLR1 754 LE L A HKLKSLMARR....T.Y WPKE..KSK.R..GLFWAN .RA INIKLTEQAK.......

TLR6 749 L I A HKLKALMTQR....T.Y QWPKE..KSK.R..GLFWAN .RA .FN..............

TLR10 737 LE L A .......EKK....A.Y WPKD..RRK.C..GLFWAN .RA .IN..............

MyD88 267 D L L .....ILRF.....ITVC YTNPCTKSW.F..WT...R AKA .SLP.............

TRIF 503 LD V ......TASLLSGLV.R EHS....QI.F..AR...K .ANT.FKPHRLQARKAMWRKE

TRAM 187 LE V I .......FALQ..TINA EES....RG.F..PT...Q .ER .FQESVYKTQQTIWKET

TRR2 192 LD L .....FLNHM....T.Y WCD....KDVR..PVFWRN .FRD.IRN.............

MAL ..........

TLR2 ..........

TLR1 ..........

TLR6 ..........

TLR10 ..........

MyD88 ..........

TRIF 544 QD........

TRAM 226 RNMVQRQFIA

TRR2 ..........

F

Intrastrand interface

Interstrand interface

a b


Table S1. Summary of structural comparisons between TIR-domain structures.

Name PDB ID Chain Zscore RMSD lali nres id MAL (crystal) 2Y92 A 15 2.2 119 127 84 TLR2 1O77A A 12.6 2.4 119 141 20 TLR1 1FYV A 12.5 2.4 127 161 17 TLR6 4OM7 A 12.1 2.4 117 143 20 TLR10 2J67 B 12.4 2.6 117 134 16 TLR2 1FYW A 12 2.7 121 149 19 MyD88 (NMR) 2Z5V A 10.5 2.7 120 141 20 MyD88 (crystal) 4EO7 A 11 3.4 120 143 19 TRAM 2M1W A 9.9 2.8 126 166 15 TRIF 2M1X A 8.1 3.4 116 160 22 ILRACP 1T3G A 10.6 2.9 121 152 16 MAL (NMR) 2NDH A 8.3 3.7 118 144 79 TTR2 4W8G A 17.2 1.9 120 125 26 MAL outer subunit 18.8 1.9 135 141 93

The inner subunit of the MAL filament is compared with the indicated structures using DALI 61. Zscore = structural similarity score, RMSD = root-mean-square deviation, lali = length of alignment, nres = number of residues, id = sequence identity.


TableS2:FunctionaleffectsofmutationsinMAL.

Interface/ protein region

Structure-based mutants (effect on assembly formation)

Previously characterized mutants (effect on NF-kB activation - relative

to wild-type MAL)

Residue * In vitro † In cells ‡ Lin et al., 2012 20, §

Bovijn et al., 2013 21, ¶

Intrastrand interface E94 E94A (+) E94A (~65%)

Q119 Q119A (~110%) Q119R (15 ± 5%)

L120 L120A (~95%)

R121 R121A (-) R121D122LC (-) R121A (-) R121A (~10%)

D122 D122A (~10%) D122G (16 ± 6%)

A123 A123V (16 ± 8%)

T124 T124A (~80%)

P125 P125A (-) P125A (±) P125H (-)

P125A (~35%) P125H (~10%) P125H (4 ± 2%)

G126

G127

I129 I129A (~30%)

E132 E132A (+) E132A (~35%)

A168

T175

I176

R184

Y195 Y195A (±) Y195N (16 ± 5%)

Y196 Y196A (-) Y196A (~80%) Y196H (16 ± 2%)

V197

D198 D198A (+) D198E (21 ± 5%)

D208

A212

R215 R215A (±) R215C (21 ± 8%)

Y216 Interstrand interface A128 A128P (18 ± 11%)

V130

C134

P155 P155A (±) P155T (7 ± 5%)

W156 W156A (±) W156Y159AA (-) W156A (~25%) W156A (14 ± 10%)

Y159 Y159 (+) Y159A (~10%)

L162 L162A (-)

Q160

Q163 Q163A (~60%) Q163H (18 ± 2%)

L165 L165A (-) L165A (~20%) L165Q (16 ± 6%)

T166 T166A (+) T166I (24 ±7%)


E167

P169

P189 P189H (13 ± 5%)

E190 E190A (+) E190A (~50%) E190K (2 ± 1%)

R192 R192A (-) R192A (~55%) R192L (2 ± 1%) R192A (29 ±3%)

F193 F193A (-) F193A (-) F193A (60 ± 2%) F193L (31 ± 37%)

M194 M194T (84 ± 16%) M194I (76 ± 22%)

BB loop E108 E108A (±) E108A (~25%)

G109

S110

T111

A112 A112G (12 ± 3%)

S113

L114

R115 R115A (+) R115A (~25%)

C116 C116F (14 ± 6%)

F117 F117A (-) F117A (~10%) F117I (9 ± 4%)

L118 L118A (~35%) * Interface residues in MALTIR filament with a buried surface area of more then >30% as calculated by PISA (http://www.ebi.ac.uk/pdbe/pisa/). † (-): disruption of filament formation; (±): weakening of filament formation; (+) no effect on filament formation. Fig. 4a. ‡ (-): disruption of punctate distribution; (±): weakening of punctate distribution; (+) no effect on punctate distribution. See Fig. 4b. § NF-kB activity in HEK293T cells with overexpressed wild-type MAL and mutants as described in Lin et al., 2012 20. Percentage activity estimated from bar-graph in Fig. 3 of Lin et al., 2012 20. ¶ NF-kB activity in HEK293T cells with overexpressed wild-type MAL and mutants as described in Bovijn et al., 2013 21. Percentage activity and associated error is taken from Table S2 in Bovijn et al., 2013 21.


TableS3:FunctionaleffectsofmutationsinMyD88

Interface

Structure-based mutants (effect on assembly

formation)

Previously characterized mutants (effect on NF-kB activation - relative to wild-type

MyD88)

Residue* In vitro† In cells‡ Loiarro et al., 2013 22,

§ Vyncke et al., 2016 23, ¶ Interstrand interface D195 D195A (85-100%)

R196 R196A (-) R196A (-) R196A (56 ± 2.9%) R196S (47 ± 2.1%)

D197 D197A (-) D197A (65 ± 0.4%)

V198 V198A (57 ± 5.0%)

L199

P200 P200A (-) P200A (-) P200H (65 ± 0.4%)

G201

T202 C203R (31 ± 6.1%)

C203

V204

W205 W205 (-)

L252 L252P (200-220%) L252P (208 ± 18.6%)

I253 I253D (-)

I271

T272

T272G (132± 0.0%) T272H (107 ± 1.6%) T272S (130 ± 25.9%)

V273 V273A (120 ± 12.0%)

C274 C274A (116 ± 7.6%)

K282 K282A (+) K282A (+) K282I (95-100%) K282E (90%)

W284 W284A (-) W284A (-) W284R (43 ±1.2%)

F285

R288 R288A (-) R288A (-) R288G (50-60%) R288H (50 ± 5.3%)

L289

A292 A292V (72 ± 0.9%) Intrastrand interface

S206

S209 S209R (140 ± 11.5%)

K231

D234 D234A (+) D234S (74 ± 2.8%)

F235

K238 K238A (-) K238A (+) K238A (31 ± 5.6%)

F239 F239A (+)

L241 L241A (-) L241A (+) L241H (25 ± 2.8%)

S242 S242A (45-55%) S242A (71 ± 6.3%)

S244 S244A (54 ± 2.3%) S244D (214 ± 14.5%)

P245 P245S (81 ± 4.3%)


G246

H248 H248A (85-100%)

Q249 Q249E (141 ± 8.7%)

S266 S266A (-) S266C (88 ± 2.9%)

R269 R269A (-) R269A (+) R269G (49 ± 1.7%)

F270 F270A (+) F270I (85 ± 6.1%)

* Interface residues in the modelled MyD88TIR assembly with a buried surface area of more then >30% as calculated by PISA (http://www.ebi.ac.uk/pdbe/pisa/). † (-): disruption of MyD88 TIR assembly formation; (+) no effect on MyD88 TIR assembly formation. See Fig. 5b. ‡ (-): disruption of MyD88 clustering; (+) no effect on MyD88 clustering. See Fig. 5c. § NF-kB activity in HEK293T cells with overexpressed wild-type MyD88 and mutants as described in Loiarro et al., 2013 22. Percentage activity range estimated from bar-graph in Fig. 2b in Loiarro et al., 2013 22. ¶ NF-kB activity in HEK293T cells with overexpressed wild-type MyD88 and mutants as described in Vyncke et al., 2016 23. Percentage activity and associated error is taken from Table S1 in Vyncke et al., 2016 23.


nature structural & molecular biology: …...nature structural & molecular biology:...

Documents