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- PDB-9j03: Cyro-EM Structure of Human TLR4/MD-2/DLAM1 Complex -

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Basic information

Entry
Database: PDB / ID: 9j03
TitleCyro-EM Structure of Human TLR4/MD-2/DLAM1 Complex
Components
  • Lymphocyte antigen 96
  • Toll-like receptor 4
KeywordsIMMUNE SYSTEM / Innate immune system / Toll-like receptors / TLR4 agonist / Vaccine adjuvants / Disaccharide-based Lipid A Mimetics
Function / homology
Function and homology information


detection of fungus / nitric oxide production involved in inflammatory response / MHC class II biosynthetic process / positive regulation of cellular response to macrophage colony-stimulating factor stimulus / lipopolysaccharide immune receptor activity / positive regulation of nucleotide-binding oligomerization domain containing 1 signaling pathway / positive regulation of matrix metallopeptidase secretion / Toll-like receptor 4 binding / detection of lipopolysaccharide / regulation of dendritic cell cytokine production ...detection of fungus / nitric oxide production involved in inflammatory response / MHC class II biosynthetic process / positive regulation of cellular response to macrophage colony-stimulating factor stimulus / lipopolysaccharide immune receptor activity / positive regulation of nucleotide-binding oligomerization domain containing 1 signaling pathway / positive regulation of matrix metallopeptidase secretion / Toll-like receptor 4 binding / detection of lipopolysaccharide / regulation of dendritic cell cytokine production / lipopolysaccharide receptor complex / MyD88-independent TLR4 cascade / negative regulation of interleukin-23 production / cellular response to oxidised low-density lipoprotein particle stimulus / TRIF-mediated programmed cell death / wound healing involved in inflammatory response / B cell proliferation involved in immune response / nucleotide-binding oligomerization domain containing 1 signaling pathway / positive regulation of nucleotide-binding oligomerization domain containing 2 signaling pathway / positive regulation of stress-activated MAPK cascade / Toll Like Receptor 4 (TLR4) Cascade / intestinal epithelial structure maintenance / Caspase activation via Death Receptors in the presence of ligand / positive regulation of interleukin-1 production / macrophage activation / Regulation of TLR by endogenous ligand / astrocyte development / TRIF-dependent toll-like receptor signaling pathway / microglia differentiation / nucleotide-binding oligomerization domain containing 2 signaling pathway / NAD+ nucleosidase activity, cyclic ADP-ribose generating / positive regulation of MHC class II biosynthetic process / positive regulation of macrophage activation / positive regulation of platelet activation / positive regulation of lipopolysaccharide-mediated signaling pathway / negative regulation of interleukin-17 production / MyD88 deficiency (TLR2/4) / positive regulation of cytokine production involved in inflammatory response / positive regulation of chemokine (C-X-C motif) ligand 2 production / positive regulation of extrinsic apoptotic signaling pathway / IRAK4 deficiency (TLR2/4) / negative regulation of cold-induced thermogenesis / positive regulation of macrophage cytokine production / positive regulation of smooth muscle cell migration / MyD88-dependent toll-like receptor signaling pathway / MyD88:MAL(TIRAP) cascade initiated on plasma membrane / T-helper 1 type immune response / toll-like receptor 4 signaling pathway / toll-like receptor signaling pathway / RSV-host interactions / positive regulation of NLRP3 inflammasome complex assembly / negative regulation of osteoclast differentiation / positive regulation of reactive oxygen species biosynthetic process / cellular response to lipoteichoic acid / negative regulation of interleukin-6 production / negative regulation of type II interferon production / Respiratory syncytial virus (RSV) attachment and entry / positive regulation of interferon-alpha production / positive regulation of interleukin-10 production / negative regulation of tumor necrosis factor production / phagocytosis / phagocytic cup / stress-activated MAPK cascade / positive regulation of chemokine production / JNK cascade / cellular response to platelet-derived growth factor stimulus / ruffle / positive regulation of B cell proliferation / ERK1 and ERK2 cascade / nitric oxide biosynthetic process / positive regulation of interleukin-12 production / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / positive regulation of smooth muscle cell proliferation / TRAF6-mediated induction of TAK1 complex within TLR4 complex / positive regulation of interferon-beta production / lipopolysaccharide-mediated signaling pathway / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / IKK complex recruitment mediated by RIP1 / positive regulation of interleukin-1 beta production / positive regulation of interleukin-8 production / positive regulation of JNK cascade / lipopolysaccharide binding / Heme signaling / cellular response to mechanical stimulus / positive regulation of NF-kappaB transcription factor activity / negative regulation of ERK1 and ERK2 cascade / cellular response to type II interferon / positive regulation of interleukin-6 production / positive regulation of type II interferon production / cellular response to amyloid-beta / positive regulation of inflammatory response / positive regulation of nitric oxide biosynthetic process / positive regulation of tumor necrosis factor production / transmembrane signaling receptor activity / signaling receptor activity / amyloid-beta binding / ER-Phagosome pathway / cellular response to lipopolysaccharide / response to lipopolysaccharide
Similarity search - Function
Lymphocyte antigen 96 / ML domain / MD-2-related lipid-recognition domain / Domain involved in innate immunity and lipid metabolism. / Toll-like receptor / Leucine rich repeat 4 / Leucine Rich repeats (2 copies) / TIR domain / Leucine Rich repeat / Cysteine-rich flanking region, C-terminal ...Lymphocyte antigen 96 / ML domain / MD-2-related lipid-recognition domain / Domain involved in innate immunity and lipid metabolism. / Toll-like receptor / Leucine rich repeat 4 / Leucine Rich repeats (2 copies) / TIR domain / Leucine Rich repeat / Cysteine-rich flanking region, C-terminal / Leucine rich repeat C-terminal domain / Toll - interleukin 1 - resistance / TIR domain profile. / Leucine-rich repeat, SDS22-like subfamily / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily / Leucine rich repeat / Leucine-rich repeat, typical subtype / Leucine-rich repeats, typical (most populated) subfamily / Leucine-rich repeat profile. / Leucine-rich repeat / Leucine-rich repeat domain superfamily / Immunoglobulin E-set
Similarity search - Domain/homology
(3R)-3-(dodecanoyloxy)tetradecanoic acid / Chem-GP4 / : / Lymphocyte antigen 96 / Toll-like receptor 4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsFu, Y. / Kim, H. / Zamyatina, A. / Kim, H.M.
Funding support1items
OrganizationGrant numberCountry
Other government
CitationJournal: Nat Commun / Year: 2025
Title: Structural insight into TLR4/MD-2 activation by synthetic LPS mimetics with distinct binding modes.
Authors: Yaoyao Fu / Hyojin Kim / Dong Sun Lee / Ah-Reum Han / Holger Heine / Alla Zamyatina / Ho Min Kim /
Abstract: The mammalian pattern-recognition receptor TLR4/MD-2 (Toll-like receptor 4/myeloid differentiation factor-2) can be activated by a wide variety of pathogen-associated and endogenous molecules, with ...The mammalian pattern-recognition receptor TLR4/MD-2 (Toll-like receptor 4/myeloid differentiation factor-2) can be activated by a wide variety of pathogen-associated and endogenous molecules, with Gram-negative bacterial lipopolysaccharide (LPS) being the primary natural TLR4 agonist. Activation of TLR4 triggers cellular signaling that enables the beneficial innate immune responses and enhances adaptive immunity, thereby emphasizing the potential of TLR4 agonists for the management of diseases with an immunopathological background and for use as vaccine adjuvants. Given the challenges associated with LPS-derived products, including structural complexity, heterogeneity, toxicity, and species specificity, synthetic molecules targeting TLR4/MD-2 offer a promising alternative. Here, we elucidate the structural basis for the recognition of synthetic LPS-mimicking glycolipids, Disaccharide Lipid A Mimetics (DLAMs), by human and mouse TLR4/MD-2 through cryo-EM structures of six dimeric [TLR4/MD-2/ligand] complexes resolved at 2.2-3.1 Å. We reveal that the specific binding modes of DLAMs, distinct from those of LPS, are essential for the species-independent TLR4 agonistic activity. DLAMs function as a molecular bridge, effectively induce the dimerization of TLR4/MD-2 complexes through specific carbohydrate structure-relevant ligand-protein interactions. Our findings reveal the distinct molecular modes of TLR4 activation, and provide a structural basis for the rationale design and development of innovative, highly potent TLR4-targeting immunotherapeutics and adjuvants.
History
DepositionAug 2, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 14, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Lymphocyte antigen 96
D: Lymphocyte antigen 96
B: Toll-like receptor 4
A: Toll-like receptor 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)178,68026
Polymers170,4494
Non-polymers8,23122
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 4 molecules CDBA

#1: Protein Lymphocyte antigen 96


Mass: 16385.941 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: LY96 / Cell (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: B3Y6A6
#2: Protein Toll-like receptor 4


Mass: 68838.328 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TLR4 / Cell (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: O00206

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Sugars , 6 types, 16 molecules

#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#4: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#5: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2- ...2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 627.594 Da / Num. of mol.: 2 / Source method: obtained synthetically
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,3,2/[a2122h-1b_1-5_2*NCC/3=O]/1-1-1/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#7: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#8: Sugar ChemComp-GP4 / 2-amino-2-deoxy-4-O-phosphono-alpha-D-glucopyranose / GLUCOSAMINE 4-PHOSPHATE / N-acetyl-4-O-phosphono-alpha-D-glucosamine / 2-amino-2-deoxy-4-O-phosphono-alpha-D-glucose / 2-amino-2-deoxy-4-O-phosphono-D-glucose / 2-amino-2-deoxy-4-O-phosphono-glucose


Type: D-saccharide, alpha linking / Mass: 259.151 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C6H14NO8P / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
a-D-GlcpN4PO3IUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
#9: Sugar ChemComp-X6Z / [(2~{R},3~{S},4~{S},5~{S})-5-methoxy-3,4,6-tris(oxidanyl)oxan-2-yl]methyl dihydrogen phosphate


Type: D-saccharide, alpha linking / Mass: 274.162 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C7H15O9P / Feature type: SUBJECT OF INVESTIGATION

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Non-polymers , 1 types, 6 molecules

#6: Chemical
ChemComp-2IL / (3R)-3-(dodecanoyloxy)tetradecanoic acid


Mass: 426.673 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C26H50O4 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: human TLR4/MD-2/DLAM1 complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTrisTris1
2200 mMsodium chlorideNaCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 10mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 58962 X / Nominal defocus max: 1700 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 67.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 13308

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Processing

EM softwareName: PHENIX
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 181542 / Symmetry type: POINT
RefinementHighest resolution: 2.7 Å

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