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- PDB-9dki: Crystal structure of the TIR domain C117H mutant from human TRAM -

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Basic information

Entry
Database: PDB / ID: 9dki
TitleCrystal structure of the TIR domain C117H mutant from human TRAM
ComponentsTIR domain-containing adapter molecule 2
KeywordsIMMUNE SYSTEM / Toll-like receptor / TIR domain / TRAM
Function / homology
Function and homology information


TRAM-dependent toll-like receptor 4 signaling pathway / positive regulation of interleukin-18-mediated signaling pathway / negative regulation of chemokine (C-C motif) ligand 5 production / MyD88-independent TLR4 cascade / negative regulation of toll-like receptor 4 signaling pathway / positive regulation of toll-like receptor 4 signaling pathway / TRIF-mediated programmed cell death / positive regulation of chemokine (C-C motif) ligand 5 production / Caspase activation via Death Receptors in the presence of ligand / Toll Like Receptor 4 (TLR4) Cascade ...TRAM-dependent toll-like receptor 4 signaling pathway / positive regulation of interleukin-18-mediated signaling pathway / negative regulation of chemokine (C-C motif) ligand 5 production / MyD88-independent TLR4 cascade / negative regulation of toll-like receptor 4 signaling pathway / positive regulation of toll-like receptor 4 signaling pathway / TRIF-mediated programmed cell death / positive regulation of chemokine (C-C motif) ligand 5 production / Caspase activation via Death Receptors in the presence of ligand / Toll Like Receptor 4 (TLR4) Cascade / TRIF-dependent toll-like receptor signaling pathway / toll-like receptor 4 signaling pathway / phagocytic cup / positive regulation of type I interferon production / phagocytosis / signaling adaptor activity / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / TRAF6-mediated induction of TAK1 complex within TLR4 complex / secretory granule membrane / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / IKK complex recruitment mediated by RIP1 / cell projection / late endosome membrane / late endosome / cellular response to lipopolysaccharide / early endosome membrane / molecular adaptor activity / positive regulation of canonical NF-kappaB signal transduction / early endosome / endosome / endosome membrane / inflammatory response / innate immune response / Neutrophil degranulation / endoplasmic reticulum / Golgi apparatus / plasma membrane
Similarity search - Function
: / TIR domain / TIR domain profile. / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily
Similarity search - Domain/homology
TIR domain-containing adapter molecule 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3 Å
AuthorsPan, M. / Gu, W. / Nanson, J.D. / Kobe, B.
Funding support Australia, 2items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)2025931 Australia
Australian Research Council (ARC)FL180100109 Australia
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Structural basis for TIR domain-mediated innate immune signaling by Toll-like receptor adaptors TRIF and TRAM.
Authors: Mohammad K Manik / Mengqi Pan / Le Xiao / Weixi Gu / Hyoyoung Kim / Sabrina Pospich / Andrew Hedger / Parimala R Vajjhala / Morris Y L Lee / Xiaoqi Qian / Michael J Landsberg / Thomas Ve / ...Authors: Mohammad K Manik / Mengqi Pan / Le Xiao / Weixi Gu / Hyoyoung Kim / Sabrina Pospich / Andrew Hedger / Parimala R Vajjhala / Morris Y L Lee / Xiaoqi Qian / Michael J Landsberg / Thomas Ve / Jeffrey D Nanson / Stefan Raunser / Katryn J Stacey / Hao Wu / Bostjan Kobe /
Abstract: Innate immunity relies on Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns. The TIR (Toll/interleukin-1 receptor) domain-containing TLR adaptors TRIF (TIR domain-containing ...Innate immunity relies on Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns. The TIR (Toll/interleukin-1 receptor) domain-containing TLR adaptors TRIF (TIR domain-containing adaptor-inducing interferon-β) and TRAM (TRIF-related adaptor molecule) are essential for MyD88-independent TLR signaling. However, the structural basis of TRIF and TRAM TIR domain-based signaling remains unclear. Here, we present cryo-EM structures of filaments formed by TRIF and TRAM TIR domains at resolutions of 3.3 Å and 5.6 Å, respectively. Both structures reveal two-stranded parallel helical arrangements. Functional studies underscore the importance of intrastrand interactions, mediated by the BB-loop, and interstrand interactions in TLR4-mediated signaling. We also report the crystal structure of the monomeric TRAM TIR domain bearing the BB loop mutation C117H, which reveals conformational differences consistent with its inactivity. Our findings suggest a unified signaling mechanism by the TIR domains of the four signaling TLR adaptors MyD88, MAL, TRIF, and TRAM and reveal potential therapeutic targets for immunity-related disorders.
History
DepositionSep 9, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: TIR domain-containing adapter molecule 2


Theoretical massNumber of molelcules
Total (without water)16,3541
Polymers16,3541
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)87.021, 87.021, 87.021
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number198
Space group name H-MP213
Space group name HallP2ac2ab3

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Components

#1: Protein TIR domain-containing adapter molecule 2 / TICAM-2 / Putative NF-kappa-B-activating protein 502 / TRIF-related adapter molecule / TRAM / Toll- ...TICAM-2 / Putative NF-kappa-B-activating protein 502 / TRIF-related adapter molecule / TRAM / Toll-like receptor adaptor protein 3 / Toll/interleukin-1 receptor domain-containing protein / MyD88-4


Mass: 16354.437 Da / Num. of mol.: 1 / Fragment: TIR domain / Mutation: C117H
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TICAM2, TIRAP3, TIRP, TRAM / Production host: Escherichia coli (E. coli) / References: UniProt: Q86XR7
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.36 Å3/Da / Density % sol: 63.37 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, hanging drop
Details: 0.2 M Li2SO4, 0.1 M Bis-Tris (pH 6.5) and 16% w/v PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Australian Synchrotron / Beamline: MX2 / Wavelength: 0.954 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jul 3, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.954 Å / Relative weight: 1
ReflectionResolution: 3→35.53 Å / Num. obs: 4567 / % possible obs: 99.65 % / Redundancy: 38.7 % / CC1/2: 1 / Rmerge(I) obs: 0.06086 / Rpim(I) all: 0.0099 / Rrim(I) all: 0.0617 / Net I/σ(I): 42.35
Reflection shellResolution: 3→3.108 Å / Rmerge(I) obs: 1.725 / Mean I/σ(I) obs: 2.44 / Num. unique obs: 433 / CC1/2: 0.798 / Rpim(I) all: 0.29 / Rrim(I) all: 1.75 / % possible all: 98.18

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Processing

Software
NameVersionClassification
PHENIX1.21.1_5286refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3→35.53 Å / SU ML: 0.1075 / Cross valid method: FREE R-VALUE / σ(F): 1.35 / Phase error: 26.5545
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.266 247 5.41 %
Rwork0.232 4320 -
obs0.2335 4567 99.54 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 116 Å2
Refinement stepCycle: LAST / Resolution: 3→35.53 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1153 0 0 0 1153
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00251181
X-RAY DIFFRACTIONf_angle_d0.65241604
X-RAY DIFFRACTIONf_chiral_restr0.0446175
X-RAY DIFFRACTIONf_plane_restr0.0074213
X-RAY DIFFRACTIONf_dihedral_angle_d12.7706440
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3-3.780.35091400.26082090X-RAY DIFFRACTION99.2
3.78-35.530.24071070.22572230X-RAY DIFFRACTION99.87

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