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- EMDB-37831: Cryo-EM Structure of Human TLR4/MD-2/DLAM3 Complex -

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Entry
Database: EMDB / ID: EMD-37831
TitleCryo-EM Structure of Human TLR4/MD-2/DLAM3 Complex
Map data
Sample
  • Complex: human TLR4/MD2/DLAM3 complex
    • Protein or peptide: Toll-like receptor 4
    • Protein or peptide: Lymphocyte antigen 96
  • Ligand: 2-amino-2-deoxy-4-O-phosphono-alpha-D-glucopyranose
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (3R)-3-(dodecanoyloxy)tetradecanoic acid
  • Ligand: (3R)-3-(tetradecanoyloxy)tetradecanoic acid
  • Ligand: 2-(hydroxymethyl)-5-methoxy-3,6-bis(oxidanyl)pyran-4-one
KeywordsInnate immune system / Toll-like receptors / TLR4 agonist / Vaccine adjuvants / Disaccharide-based lipid A mimetices / IMMUNE SYSTEM
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 / lipopolysaccharide receptor complex / detection of lipopolysaccharide ...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 / lipopolysaccharide receptor complex / detection of lipopolysaccharide / regulation of dendritic cell cytokine production / MyD88-independent TLR4 cascade / I-kappaB phosphorylation / negative regulation of interleukin-23 production / TRIF-mediated programmed cell death / cellular response to oxidised low-density lipoprotein particle stimulus / wound healing involved in inflammatory response / B cell proliferation involved in immune response / positive regulation of nucleotide-binding oligomerization domain containing 2 signaling pathway / nucleotide-binding oligomerization domain containing 1 signaling pathway / Caspase activation via Death Receptors in the presence of ligand / Toll Like Receptor 4 (TLR4) Cascade / positive regulation of stress-activated MAPK cascade / intestinal epithelial structure maintenance / positive regulation of interleukin-1 production / macrophage activation / Regulation of TLR by endogenous ligand / TRIF-dependent toll-like receptor signaling pathway / astrocyte development / microglia differentiation / NAD+ nucleosidase activity, cyclic ADP-ribose generating / nucleotide-binding oligomerization domain containing 2 signaling pathway / 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 / positive regulation of cytokine production involved in inflammatory response / MyD88 deficiency (TLR2/4) / positive regulation of chemokine (C-X-C motif) ligand 2 production / positive regulation of extrinsic apoptotic signaling pathway / negative regulation of cold-induced thermogenesis / IRAK4 deficiency (TLR2/4) / MyD88-dependent toll-like receptor signaling pathway / positive regulation of macrophage cytokine production / MyD88:MAL(TIRAP) cascade initiated on plasma membrane / toll-like receptor 4 signaling pathway / T-helper 1 type immune response / toll-like receptor signaling pathway / positive regulation of smooth muscle cell migration / positive regulation of reactive oxygen species biosynthetic process / positive regulation of NLRP3 inflammasome complex assembly / RSV-host interactions / negative regulation of osteoclast differentiation / cellular response to lipoteichoic acid / negative regulation of interleukin-6 production / negative regulation of type II interferon production / positive regulation of interferon-alpha production / Respiratory syncytial virus (RSV) attachment and entry / positive regulation of interleukin-10 production / negative regulation of tumor necrosis factor production / phagocytic cup / phagocytosis / stress-activated MAPK cascade / positive regulation of chemokine production / ruffle / JNK cascade / cellular response to platelet-derived growth factor stimulus / positive regulation of B cell proliferation / nitric oxide biosynthetic process / positive regulation of interleukin-12 production / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / ERK1 and ERK2 cascade / positive regulation of MAP kinase activity / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / 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) / positive regulation of interleukin-1 beta production / IKK complex recruitment mediated by RIP1 / positive regulation of interleukin-8 production / positive regulation of smooth muscle cell proliferation / positive regulation of JNK cascade / lipopolysaccharide binding / Heme signaling / positive regulation of non-canonical NF-kappaB signal transduction / 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 mechanical stimulus / positive regulation of inflammatory response / cellular response to amyloid-beta / positive regulation of NF-kappaB transcription factor activity / positive regulation of tumor necrosis factor production / positive regulation of nitric oxide biosynthetic process / transmembrane signaling receptor activity / signaling receptor activity / amyloid-beta binding
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
Lymphocyte antigen 96 / Toll-like receptor 4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsFu Y / Kim H / Kim HM
Funding support Korea, Republic Of, 1 items
OrganizationGrant numberCountry
Other governmentInstitute for Basic Science, IBS Korea, Republic Of
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
DepositionOct 18, 2023-
Header (metadata) releaseOct 23, 2024-
Map releaseOct 23, 2024-
UpdateMay 14, 2025-
Current statusMay 14, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37831.png

Downloads & links

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Map

FileDownload / File: emd_37831.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.12 Å/pix.
x 300 pix.
= 336. Å		1.12 Å/pix.
x 300 pix.
= 336. Å		1.12 Å/pix.
x 300 pix.
= 336. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.12 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07
Minimum - Maximum-1.0432326 - 3.0361142
Average (Standard dev.)-0.00089893624 (±0.06452457)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 336.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_37831_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_37831_half_map_2.map
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Sample components

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Entire : human TLR4/MD2/DLAM3 complex

EntireName: human TLR4/MD2/DLAM3 complex
Components
  • Complex: human TLR4/MD2/DLAM3 complex
    • Protein or peptide: Toll-like receptor 4
    • Protein or peptide: Lymphocyte antigen 96
  • Ligand: 2-amino-2-deoxy-4-O-phosphono-alpha-D-glucopyranose
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (3R)-3-(dodecanoyloxy)tetradecanoic acid
  • Ligand: (3R)-3-(tetradecanoyloxy)tetradecanoic acid
  • Ligand: 2-(hydroxymethyl)-5-methoxy-3,6-bis(oxidanyl)pyran-4-one

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Supramolecule #1: human TLR4/MD2/DLAM3 complex

SupramoleculeName: human TLR4/MD2/DLAM3 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Toll-like receptor 4

MacromoleculeName: Toll-like receptor 4 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 68.838328 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: EPCVEVVPNI TYQCMELNFY KIPDNLPFST KNLDLSFNPL RHLGSYSFFS FPELQVLDLS RCEIQTIEDG AYQSLSHLST LILTGNPIQ SLALGAFSGL SSLQKLVAVE TNLASLENFP IGHLKTLKEL NVAHNLIQSF KLPEYFSNLT NLEHLDLSSN K IQSIYCTD ...String:
EPCVEVVPNI TYQCMELNFY KIPDNLPFST KNLDLSFNPL RHLGSYSFFS FPELQVLDLS RCEIQTIEDG AYQSLSHLST LILTGNPIQ SLALGAFSGL SSLQKLVAVE TNLASLENFP IGHLKTLKEL NVAHNLIQSF KLPEYFSNLT NLEHLDLSSN K IQSIYCTD LRVLHQMPLL NLSLDLSLNP MNFIQPGAFK EIRLHKLTLR NNFDSLNVMK TCIQGLAGLE VHRLVLGEFR NE GNLEKFD KSALEGLCNL TIEEFRLAYL DYYLDDIIDL FNCLTNVSSF SLVSVTIERV KDFSYNFGWQ HLELVNCKFG QFP TLKLKS LKRLTFTSNK GGNAFSEVDL PSLEFLDLSR NGLSFKGCCS QSDFGTTSLK YLDLSFNGVI TMSSNFLGLE QLEH LDFQH SNLKQMSEFS VFLSLRNLIY LDISHTHTRV AFNGIFNGLS SLEVLKMAGN SFQENFLPDI FTELRNLTFL DLSQC QLEQ LSPTAFNSLS SLQVLNMSHN NFFSLDTFPY KCLNSLQVLD YSLNHIMTSK KQELQHFPSS LAFLNLTQND FACTCE HQS FLQWIKDQRQ LLVEVERMEC ATPSDKQGMP VLSLNITCQM NK

UniProtKB: Toll-like receptor 4

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Macromolecule #2: Lymphocyte antigen 96

MacromoleculeName: Lymphocyte antigen 96 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 16.385941 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString:
QKQYWVCNSS DASISYTYCD KMQYPISINV NPCIELKGSK GLLHIFYIPR RDLKQLYFNL YITVNTMNLP KRKEVICRGS DDDYSFCRA LKGETVNTTI SFSFKGIKFS KGKYKCVVEA ISGSPEEMLF CLEFVILHQP NSN

UniProtKB: Lymphocyte antigen 96

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Macromolecule #5: 2-amino-2-deoxy-4-O-phosphono-alpha-D-glucopyranose

MacromoleculeName: 2-amino-2-deoxy-4-O-phosphono-alpha-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 2 / Formula: GP4
Molecular weightTheoretical: 259.151 Da
Chemical component information

ChemComp-GP4:
2-amino-2-deoxy-4-O-phosphono-alpha-D-glucopyranose

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 6 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #7: (3R)-3-(dodecanoyloxy)tetradecanoic acid

MacromoleculeName: (3R)-3-(dodecanoyloxy)tetradecanoic acid / type: ligand / ID: 7 / Number of copies: 4 / Formula: 2IL
Molecular weightTheoretical: 426.673 Da
Chemical component information

ChemComp-2IL:
(3R)-3-(dodecanoyloxy)tetradecanoic acid

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Macromolecule #8: (3R)-3-(tetradecanoyloxy)tetradecanoic acid

MacromoleculeName: (3R)-3-(tetradecanoyloxy)tetradecanoic acid / type: ligand / ID: 8 / Number of copies: 2 / Formula: 0IL
Molecular weightTheoretical: 454.726 Da

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Macromolecule #9: 2-(hydroxymethyl)-5-methoxy-3,6-bis(oxidanyl)pyran-4-one

MacromoleculeName: 2-(hydroxymethyl)-5-methoxy-3,6-bis(oxidanyl)pyran-4-one
type: ligand / ID: 9 / Number of copies: 2 / Formula: XIQ
Molecular weightTheoretical: 188.135 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMTrisTris
200.0 mMNaClsodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 5 sec. / Details: 10mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Number grids imaged: 1 / Number real images: 2071 / Average electron dose: 40.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.6 µm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3fxi

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 302687
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3fxi


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-8wta:
Cryo-EM Structure of Human TLR4/MD-2/DLAM3 Complex

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