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- EMDB-42897: LapB cytoplasmic domain in complex with LpxC -

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Basic information

Entry
Database: EMDB / ID: EMD-42897
TitleLapB cytoplasmic domain in complex with LpxC
Map data
Sample
  • Complex: LapB/LpxC
    • Protein or peptide: Lipopolysaccharide assembly protein B
    • Protein or peptide: UDP-3-O-acyl-N-acetylglucosamine deacetylase
  • Ligand: ZINC ION
  • Ligand: uridine-5'-diphosphate-3-O-(R-3-hydroxymyristoyl)-glucosamine
  • Ligand: ACETATE ION
Keywordsadaptor / complex / deacetylase / LPS / LpxC / LapB(YciM) / PROTEIN BINDING
Function / homology
Function and homology information


lipopolysaccharide metabolic process / UDP-3-O-acyl-N-acetylglucosamine deacetylase / UDP-3-O-acyl-N-acetylglucosamine deacetylase activity / regulation of lipid biosynthetic process / lipid A biosynthetic process / cytoplasmic side of plasma membrane / iron ion binding / metal ion binding / membrane
Similarity search - Function
Lipopolysaccharide assembly protein B / LapB, rubredoxin metal binding domain / Rubredoxin metal binding domain / : / Tetratricopeptide repeat / UDP-3-O-acyl N-acetylglucosamine deacetylase / UDP-3-O-acyl N-acetylglucosamine deacetylase, C-terminal / UDP-3-O-acyl N-acetylglucosamine deacetylase, N-terminal / UDP-3-O-acyl N-acetylglycosamine deacetylase / Tetratricopeptide repeat ...Lipopolysaccharide assembly protein B / LapB, rubredoxin metal binding domain / Rubredoxin metal binding domain / : / Tetratricopeptide repeat / UDP-3-O-acyl N-acetylglucosamine deacetylase / UDP-3-O-acyl N-acetylglucosamine deacetylase, C-terminal / UDP-3-O-acyl N-acetylglucosamine deacetylase, N-terminal / UDP-3-O-acyl N-acetylglycosamine deacetylase / Tetratricopeptide repeat / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / Tetratricopeptide-like helical domain superfamily / Ribosomal protein S5 domain 2-type fold
Similarity search - Domain/homology
UDP-3-O-acyl-N-acetylglucosamine deacetylase / Lipopolysaccharide assembly protein B
Similarity search - Component
Biological speciesEscherichia coli CFT073 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsMi W / Shu S
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM137068 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RM1GM149406 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Dual function of LapB (YciM) in regulating lipopolysaccharide synthesis.
Authors: Sheng Shu / Yuko Tsutsui / Rajkanwar Nathawat / Wei Mi /
Abstract: Levels of lipopolysaccharide (LPS), an essential glycolipid on the surface of most gram-negative bacteria, are tightly controlled-making LPS synthesis a promising target for developing new ...Levels of lipopolysaccharide (LPS), an essential glycolipid on the surface of most gram-negative bacteria, are tightly controlled-making LPS synthesis a promising target for developing new antibiotics. adaptor protein LapB (YciM) plays an important role in regulating LPS synthesis by promoting degradation of LpxC, a deacetylase that catalyzes the first committed step in LPS synthesis. Under conditions where LPS is abundant, LapB recruits LpxC to the AAA+ protease FtsH for degradation. LapB achieves this by simultaneously interacting with FtsH through its transmembrane helix and LpxC through its cytoplasmic domain. Here, we describe a cryo-EM structure of the complex formed between LpxC and the cytoplasmic domain of LapB (LapB). The structure reveals how LapB exploits both its tetratricopeptide repeat (TPR) motifs and rubredoxin domain to interact with LpxC. Through both in vitro and in vivo analysis, we show that mutations at the LapB/LpxC interface prevent LpxC degradation. Unexpectedly, binding to LapB also inhibits the enzymatic activity of LpxC through allosteric effects reminiscent of LpxC activation by MurA in Our findings argue that LapB regulates LPS synthesis in two steps: In the first step, LapB inhibits the activity of LpxC, and in the second step, it commits LpxC to degradation by FtsH.
History
DepositionNov 21, 2023-
Header (metadata) releaseApr 24, 2024-
Map releaseApr 24, 2024-
UpdateMay 8, 2024-
Current statusMay 8, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42897.png

Downloads & links

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Map

FileDownload / File: emd_42897.map.gz / Format: CCP4 / Size: 35.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 210 pix.
= 224.28 Å		1.07 Å/pix.
x 210 pix.
= 224.28 Å		1.07 Å/pix.
x 210 pix.
= 224.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.068 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.19
Minimum - Maximum-1.6003623 - 2.5185008
Average (Standard dev.)0.0015152371 (±0.06434699)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions210210210
Spacing210210210
CellA=B=C: 224.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_42897_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_42897_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : LapB/LpxC

EntireName: LapB/LpxC
Components
  • Complex: LapB/LpxC
    • Protein or peptide: Lipopolysaccharide assembly protein B
    • Protein or peptide: UDP-3-O-acyl-N-acetylglucosamine deacetylase
  • Ligand: ZINC ION
  • Ligand: uridine-5'-diphosphate-3-O-(R-3-hydroxymyristoyl)-glucosamine
  • Ligand: ACETATE ION

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Supramolecule #1: LapB/LpxC

SupramoleculeName: LapB/LpxC / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Escherichia coli CFT073 (bacteria)

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Macromolecule #1: Lipopolysaccharide assembly protein B

MacromoleculeName: Lipopolysaccharide assembly protein B / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli CFT073 (bacteria) / Strain: CFT073 / ATCC 700928 / UPEC
Molecular weightTheoretical: 44.588969 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MLELLFLLLP VAAAYGWYMG RRSAQQNKQD EANRLSRDYV AGVNFLLSNQ QDKAVDLFLD MLKEDTGTVE AHLTLGNLFR SRGEVDRAI RIHQTLMESA SLTYEQRLLA IQQLGRDYMA AGLYDRAEDM FNQLTDETDF RIGALQQLLQ IYQATSEWQK A IDVAERLV ...String:
MLELLFLLLP VAAAYGWYMG RRSAQQNKQD EANRLSRDYV AGVNFLLSNQ QDKAVDLFLD MLKEDTGTVE AHLTLGNLFR SRGEVDRAI RIHQTLMESA SLTYEQRLLA IQQLGRDYMA AGLYDRAEDM FNQLTDETDF RIGALQQLLQ IYQATSEWQK A IDVAERLV KLGKDKQRVE IAHFYCELAL QHMASDDLDR AMTLLKKGAA ADKNSARVSI MMGRVFMAKG EYAKAVESLQ RV ISQDREL VSETLEMLQT CYQQLGKTAE WAEFLQRAVE ENTGADAELM LADIIEARDG SEAAQVYITR QLQRHPTMRV FHK LMDYHL NEAEEGRAKE SLMVLRDMVG EKVRSKPRYR CQKCGFTAYT LYWHCPSCRA WSTIKPIRGL DGL

UniProtKB: Lipopolysaccharide assembly protein B

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Macromolecule #2: UDP-3-O-acyl-N-acetylglucosamine deacetylase

MacromoleculeName: UDP-3-O-acyl-N-acetylglucosamine deacetylase / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli CFT073 (bacteria) / Strain: CFT073 / ATCC 700928 / UPEC
Molecular weightTheoretical: 33.995871 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MIKQRTLKRI VQATGVGLHT GKKVTLTLRP APANTGVIYR RTDLNPPVDF PADAKSVRDT MLCTCLVNEH DVRISTVEHL NAALAGLGI DNIVIEVNAP EIPIMDGSAA PFVYLLLDAG IDELNCAKKF VRIKETVRVE DGDKWAEFKP YNGFSLDFTI D FNHPAIDS ...String:
MIKQRTLKRI VQATGVGLHT GKKVTLTLRP APANTGVIYR RTDLNPPVDF PADAKSVRDT MLCTCLVNEH DVRISTVEHL NAALAGLGI DNIVIEVNAP EIPIMDGSAA PFVYLLLDAG IDELNCAKKF VRIKETVRVE DGDKWAEFKP YNGFSLDFTI D FNHPAIDS SNQRYAMNFS ADAFMRQISR ARTFGFMRDI EYLQSRGLCL GGSFDCAIVV DDYRVLNEDG LRFEDEFVRH KM LDAIGDL FMCGHNIIGA FTAYKSGHAL NNKLLQAVLA KQEAWEYVTF QDDAELPLAF KAPSAVLA

UniProtKB: UDP-3-O-acyl-N-acetylglucosamine deacetylase

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Macromolecule #3: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 3 / Number of copies: 4 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #4: uridine-5'-diphosphate-3-O-(R-3-hydroxymyristoyl)-glucosamine

MacromoleculeName: uridine-5'-diphosphate-3-O-(R-3-hydroxymyristoyl)-glucosamine
type: ligand / ID: 4 / Number of copies: 2 / Formula: 24G
Molecular weightTheoretical: 791.672 Da
Chemical component information

ChemComp-24G:
uridine-5'-diphosphate-3-O-(R-3-hydroxymyristoyl)-glucosamine

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Macromolecule #5: ACETATE ION

MacromoleculeName: ACETATE ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: ACT
Molecular weightTheoretical: 59.044 Da
Chemical component information

ChemComp-ACT:
ACETATE ION

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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

Concentration10 mg/mL
BufferpH: 7.8
GridModel: C-flat-1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: GATAN CRYOPLUNGE 3

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average exposure time: 2.6 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.01) / Number images used: 1171191
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.01)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.01)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 4.01)
FSC plot (resolution estimation)

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