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- EMDB-25799: cryo-EM structure of MBP-KIX-apoferritin complex with peptide 7 -

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Basic information

Entry
Database: EMDB / ID: EMD-25799
Titlecryo-EM structure of MBP-KIX-apoferritin complex with peptide 7
Map datacryo-EM structure of MBP-KIX-apoferritin complex with peptide 7
Sample
  • Complex: MBP-KIX-apoferritin complex with peptide 7
    • Protein or peptide: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
    • Protein or peptide: LEU-SER-ARG-ARG-PRO-SEP-TYR-ARG-LYS-ILE-LEU-ASN-ASP-LEU-SER-SER-ASP-ALA-PRO
Function / homology
Function and homology information


Iron uptake and transport / Golgi Associated Vesicle Biogenesis / NFE2L2 regulating ER-stress associated genes / peptide N-acetyltransferase activity / Activation of the TFAP2 (AP-2) family of transcription factors / NFE2L2 regulating inflammation associated genes / regulation of smoothened signaling pathway / iron ion sequestering activity / histone H3K18 acetyltransferase activity / LRR FLII-interacting protein 1 (LRRFIP1) activates type I IFN production ...Iron uptake and transport / Golgi Associated Vesicle Biogenesis / NFE2L2 regulating ER-stress associated genes / peptide N-acetyltransferase activity / Activation of the TFAP2 (AP-2) family of transcription factors / NFE2L2 regulating inflammation associated genes / regulation of smoothened signaling pathway / iron ion sequestering activity / histone H3K18 acetyltransferase activity / LRR FLII-interacting protein 1 (LRRFIP1) activates type I IFN production / N-terminal peptidyl-lysine acetylation / histone H3K27 acetyltransferase activity / NFE2L2 regulates pentose phosphate pathway genes / MRF binding / NFE2L2 regulating MDR associated enzymes / RUNX1 regulates transcription of genes involved in differentiation of myeloid cells / Regulation of gene expression in late stage (branching morphogenesis) pancreatic bud precursor cells / RUNX3 regulates NOTCH signaling / NOTCH4 Intracellular Domain Regulates Transcription / Regulation of FOXO transcriptional activity by acetylation / Regulation of gene expression by Hypoxia-inducible Factor / Nuclear events mediated by NFE2L2 / Regulation of NFE2L2 gene expression / autolysosome / negative regulation of transcription by RNA polymerase I / NOTCH3 Intracellular Domain Regulates Transcription / NFE2L2 regulating anti-oxidant/detoxification enzymes / TRAF6 mediated IRF7 activation / peptide-lysine-N-acetyltransferase activity / FOXO-mediated transcription of cell death genes / NFE2L2 regulating tumorigenic genes / embryonic digit morphogenesis / homeostatic process / Notch-HLH transcription pathway / ferroxidase / protein acetylation / Formation of paraxial mesoderm / positive regulation of transforming growth factor beta receptor signaling pathway / intracellular sequestering of iron ion / non-canonical NF-kappaB signal transduction / Zygotic genome activation (ZGA) / ferroxidase activity / stimulatory C-type lectin receptor signaling pathway / acetyltransferase activity / cellular response to nutrient levels / TP53 Regulates Transcription of Genes Involved in Cytochrome C Release / histone acetyltransferase complex / Attenuation phase / regulation of cellular response to heat / negative regulation of fibroblast proliferation / histone acetyltransferase activity / Transferases; Acyltransferases; Transferring groups other than aminoacyl groups / histone acetyltransferase / endocytic vesicle lumen / RORA activates gene expression / NPAS4 regulates expression of target genes / Neutrophil degranulation / Regulation of lipid metabolism by PPARalpha / CD209 (DC-SIGN) signaling / ferric iron binding / BMAL1:CLOCK,NPAS2 activates circadian gene expression / Activation of gene expression by SREBF (SREBP) / SUMOylation of transcription cofactors / ferrous iron binding / Formation of the beta-catenin:TCF transactivating complex / protein destabilization / Heme signaling / Transcriptional activation of mitochondrial biogenesis / transcription coactivator binding / PPARA activates gene expression / Cytoprotection by HMOX1 / NOTCH1 Intracellular Domain Regulates Transcription / chromatin DNA binding / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / Pre-NOTCH Transcription and Translation / Transcriptional regulation of white adipocyte differentiation / Activation of anterior HOX genes in hindbrain development during early embryogenesis / positive regulation of protein localization to nucleus / transcription corepressor activity / cellular response to UV / rhythmic process / Circadian Clock / p53 binding / TRAF3-dependent IRF activation pathway / HATs acetylate histones / iron ion transport / protein-containing complex assembly / DNA-binding transcription factor binding / Estrogen-dependent gene expression / transcription regulator complex / RNA polymerase II-specific DNA-binding transcription factor binding / damaged DNA binding / transcription coactivator activity / response to hypoxia / nuclear body / immune response / iron ion binding / negative regulation of cell population proliferation / chromatin binding
Similarity search - Function
Nuclear receptor coactivator, CREB-bp-like, interlocking / Nuclear receptor coactivator, CREB-bp-like, interlocking domain superfamily / Creb binding / Zinc finger, TAZ-type / TAZ domain superfamily / TAZ zinc finger / Zinc finger TAZ-type profile. / TAZ zinc finger, present in p300 and CBP / Coactivator CBP, KIX domain / CREB-binding protein/p300, atypical RING domain ...Nuclear receptor coactivator, CREB-bp-like, interlocking / Nuclear receptor coactivator, CREB-bp-like, interlocking domain superfamily / Creb binding / Zinc finger, TAZ-type / TAZ domain superfamily / TAZ zinc finger / Zinc finger TAZ-type profile. / TAZ zinc finger, present in p300 and CBP / Coactivator CBP, KIX domain / CREB-binding protein/p300, atypical RING domain / CBP/p300-type histone acetyltransferase domain / CBP/p300, atypical RING domain superfamily / KIX domain / CREB-binding protein/p300, atypical RING domain / KIX domain profile. / CBP/p300-type histone acetyltransferase (HAT) domain profile. / Coactivator CBP, KIX domain superfamily / Histone acetyltransferase Rtt109/CBP / Histone acetylation protein / Histone acetylation protein / Zinc finger ZZ-type signature. / Zinc-binding domain, present in Dystrophin, CREB-binding protein. / Zinc finger, ZZ type / Zinc finger, ZZ-type / Zinc finger, ZZ-type superfamily / Zinc finger ZZ-type profile. / Ferritin iron-binding regions signature 1. / Ferritin iron-binding regions signature 2. / Ferritin, conserved site / Ferritin / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Ferritin/DPS protein domain / Ferritin-like domain / Nuclear receptor coactivator, interlocking / Ferritin-like / Ferritin-like superfamily / Bromodomain, conserved site / Bromodomain signature. / Bromodomain / Bromodomain profile. / bromo domain / Bromodomain / Bromodomain-like superfamily / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
Ferritin heavy chain / CREB-binding protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsZhang K / Horikoshi N / Li S / Powers A / Hameedi M / Pintilie G / Chae H / Khan Y / Suomivuori C / Dror R ...Zhang K / Horikoshi N / Li S / Powers A / Hameedi M / Pintilie G / Chae H / Khan Y / Suomivuori C / Dror R / Sakamoto K / Chiu W / Wakatsuki S
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM079429 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021600 United States
CitationJournal: ACS Cent Sci / Year: 2022
Title: Cryo-EM, Protein Engineering, and Simulation Enable the Development of Peptide Therapeutics against Acute Myeloid Leukemia.
Authors: Kaiming Zhang / Naoki Horikoshi / Shanshan Li / Alexander S Powers / Mikhail A Hameedi / Grigore D Pintilie / Hee-Don Chae / Yousuf A Khan / Carl-Mikael Suomivuori / Ron O Dror / Kathleen M ...Authors: Kaiming Zhang / Naoki Horikoshi / Shanshan Li / Alexander S Powers / Mikhail A Hameedi / Grigore D Pintilie / Hee-Don Chae / Yousuf A Khan / Carl-Mikael Suomivuori / Ron O Dror / Kathleen M Sakamoto / Wah Chiu / Soichi Wakatsuki /
Abstract: Cryogenic electron microscopy (cryo-EM) has emerged as a viable structural tool for molecular therapeutics development against human diseases. However, it remains a challenge to determine structures ...Cryogenic electron microscopy (cryo-EM) has emerged as a viable structural tool for molecular therapeutics development against human diseases. However, it remains a challenge to determine structures of proteins that are flexible and smaller than 30 kDa. The 11 kDa KIX domain of CREB-binding protein (CBP), a potential therapeutic target for acute myeloid leukemia and other cancers, is a protein which has defied structure-based inhibitor design. Here, we develop an experimental approach to overcome the size limitation by engineering a protein double-shell to sandwich the KIX domain between apoferritin as the inner shell and maltose-binding protein as the outer shell. To assist homogeneous orientations of the target, disulfide bonds are introduced at the target-apoferritin interface, resulting in a cryo-EM structure at 2.6 Å resolution. We used molecular dynamics simulations to design peptides that block the interaction of the KIX domain of CBP with the intrinsically disordered pKID domain of CREB. The double-shell design allows for fluorescence polarization assays confirming the binding between the KIX domain in the double-shell and these interacting peptides. Further cryo-EM analysis reveals a helix-helix interaction between a single KIX helix and the best peptide, providing a possible strategy for developments of next-generation inhibitors.
History
DepositionDec 22, 2021-
Header (metadata) releaseMar 16, 2022-
Map releaseMar 16, 2022-
UpdateMar 16, 2022-
Current statusMar 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7tbh
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7tbh
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25799.png

Downloads & links

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Map

FileDownload / File: emd_25799.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM structure of MBP-KIX-apoferritin complex with peptide 7
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.12
Minimum - Maximum-0.17170592 - 0.6200727
Average (Standard dev.)0.010035068 (±0.030972483)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 314.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z314.880314.880314.880
α/β/γ90.00090.00090.000
start NX/NY/NZ139118109
NX/NY/NZ123164187
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.1720.6200.010

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Supplemental data

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Sample components

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Entire : MBP-KIX-apoferritin complex with peptide 7

EntireName: MBP-KIX-apoferritin complex with peptide 7
Components
  • Complex: MBP-KIX-apoferritin complex with peptide 7
    • Protein or peptide: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
    • Protein or peptide: LEU-SER-ARG-ARG-PRO-SEP-TYR-ARG-LYS-ILE-LEU-ASN-ASP-LEU-SER-SER-ASP-ALA-PRO

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Supramolecule #1: MBP-KIX-apoferritin complex with peptide 7

SupramoleculeName: MBP-KIX-apoferritin complex with peptide 7 / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightExperimental: 1.7 MDa

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Macromolecule #1: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-termina...

MacromoleculeName: Isoform 2 of CREB-binding protein,Ferritin heavy chain, N-terminally processed
type: protein_or_peptide / ID: 1 / Number of copies: 24 / Enantiomer: LEVO / EC number: histone acetyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 30.408438 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: HMGVRKGWHE HVTQDLRSHL VHKLVQAIFP CPDPCALKDR RMENLVAYAK KVEGDMYESA NSRDEYYHLL AEKIYKIQKE LEEKSQVRQ NYHQDAEAAI NRQINLELYA SYVYLSMSCY FDRDDVALKN FAKYFLHQSH EEREHAEKLM KLQNQRGGRI F LQDIKKPD ...String:
HMGVRKGWHE HVTQDLRSHL VHKLVQAIFP CPDPCALKDR RMENLVAYAK KVEGDMYESA NSRDEYYHLL AEKIYKIQKE LEEKSQVRQ NYHQDAEAAI NRQINLELYA SYVYLSMSCY FDRDDVALKN FAKYFLHQSH EEREHAEKLM KLQNQRGGRI F LQDIKKPD RDDWCSGLNA MESALHLEKS VNQSLLELHK LATDKNDPHL CDFIETYYLS EQVKSIKELG DHVTNLRKMG AP CAGMAEY LFDKHTLGHG DES

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Macromolecule #2: LEU-SER-ARG-ARG-PRO-SEP-TYR-ARG-LYS-ILE-LEU-ASN-ASP-LEU-SER-SER-A...

MacromoleculeName: LEU-SER-ARG-ARG-PRO-SEP-TYR-ARG-LYS-ILE-LEU-ASN-ASP-LEU-SER-SER-ASP-ALA-PRO
type: protein_or_peptide / ID: 2 / Number of copies: 24 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.272457 KDa
SequenceString:
LSRRP(SEP)YRKI LNDLSSDAP

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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

Concentration4 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/1 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE
DetailsMBP-KIX-apoferritin complex with peptide 7

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2490 / Average exposure time: 6.0 sec. / Average electron dose: 43.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 124114
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 35613

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Atomic model buiding 1

RefinementProtocol: OTHER / Target criteria: Q-score
Output model

PDB-7tbh:
cryo-EM structure of MBP-KIX-apoferritin complex with peptide 7

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