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- PDB-8sah: Huntingtin C-HEAT domain in complex with HAP40 -

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Basic information

Entry
Database: PDB / ID: 8sah
TitleHuntingtin C-HEAT domain in complex with HAP40
Components
  • 40-kDa huntingtin-associated protein
  • Huntingtin
KeywordsPROTEIN BINDING / Huntingtin / scaffold / HEAT repeats / Structural Genomics / Structural Genomics Consortium / SGC
Function / homology
Function and homology information


vesicle cytoskeletal trafficking / positive regulation of CAMKK-AMPK signaling cascade / microtubule-based transport / positive regulation of inositol 1,4,5-trisphosphate-sensitive calcium-release channel activity / vocal learning / negative regulation of proteasomal protein catabolic process / regulation of CAMKK-AMPK signaling cascade / positive regulation of mitophagy / profilin binding / vesicle transport along microtubule ...vesicle cytoskeletal trafficking / positive regulation of CAMKK-AMPK signaling cascade / microtubule-based transport / positive regulation of inositol 1,4,5-trisphosphate-sensitive calcium-release channel activity / vocal learning / negative regulation of proteasomal protein catabolic process / regulation of CAMKK-AMPK signaling cascade / positive regulation of mitophagy / profilin binding / vesicle transport along microtubule / positive regulation of cilium assembly / retrograde vesicle-mediated transport, Golgi to endoplasmic reticulum / positive regulation of aggrephagy / positive regulation of lipophagy / dynein intermediate chain binding / Golgi organization / beta-tubulin binding / establishment of mitotic spindle orientation / dynactin binding / phosphoprotein phosphatase activity / Regulation of MECP2 expression and activity / postsynaptic cytosol / presynaptic cytosol / inclusion body / heat shock protein binding / centriole / cytoplasmic vesicle membrane / autophagosome / negative regulation of extrinsic apoptotic signaling pathway / protein destabilization / kinase binding / p53 binding / late endosome / transmembrane transporter binding / early endosome / nuclear body / positive regulation of apoptotic process / axon / apoptotic process / dendrite / perinuclear region of cytoplasm / Golgi apparatus / endoplasmic reticulum / protein-containing complex / nucleoplasm / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Factor VIII intron 22 protein / Huntingtin / Huntingtin, middle-repeat / Huntingtin family / : / : / : / Huntingtin, N-terminal HEAT / Huntingtin, bridge / Huntingtin, N-terminal HEAT 1 ...Factor VIII intron 22 protein / Huntingtin / Huntingtin, middle-repeat / Huntingtin family / : / : / : / Huntingtin, N-terminal HEAT / Huntingtin, bridge / Huntingtin, N-terminal HEAT 1 / Huntingtin, C-terminal HEAT / Armadillo-like helical / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold
Similarity search - Domain/homology
40-kDa huntingtin-associated protein / Huntingtin
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsHarding, R.J. / Deme, J.C. / Alteen, M.G. / Arrowsmith, C.H. / Lea, S.M. / Structural Genomics Consortium (SGC)
Funding support1items
OrganizationGrant numberCountry
Other government
Citation
Journal: Structure / Year: 2023
Title: Delineation of functional subdomains of Huntingtin protein and their interaction with HAP40.
Authors: Matthew G Alteen / Justin C Deme / Claudia P Alvarez / Peter Loppnau / Ashley Hutchinson / Alma Seitova / Renu Chandrasekaran / Eduardo Silva Ramos / Christopher Secker / Mona Alqazzaz / ...Authors: Matthew G Alteen / Justin C Deme / Claudia P Alvarez / Peter Loppnau / Ashley Hutchinson / Alma Seitova / Renu Chandrasekaran / Eduardo Silva Ramos / Christopher Secker / Mona Alqazzaz / Erich E Wanker / Susan M Lea / Cheryl H Arrowsmith / Rachel J Harding /
Abstract: The huntingtin (HTT) protein plays critical roles in numerous cellular pathways by functioning as a scaffold for its many interaction partners and HTT knock out is embryonic lethal. Interrogation of ...The huntingtin (HTT) protein plays critical roles in numerous cellular pathways by functioning as a scaffold for its many interaction partners and HTT knock out is embryonic lethal. Interrogation of HTT function is complicated by the large size of this protein so we studied a suite of structure-rationalized subdomains to investigate the structure-function relationships within the HTT-HAP40 complex. Protein samples derived from the subdomain constructs were validated using biophysical methods and cryo-electron microscopy, revealing they are natively folded and can complex with validated binding partner, HAP40. Derivatized versions of these constructs enable protein-protein interaction assays in vitro, with biotin tags, and in cells, with luciferase two-hybrid assay-based tags, which we use in proof-of-principle analyses to further interrogate the HTT-HAP40 interaction. These open-source biochemical tools enable studies of fundamental HTT biochemistry and biology, will aid the discovery of macromolecular or small-molecule binding partners and help map interaction sites across this large protein.
#1: Journal: bioRxiv / Year: 2022
Title: Expanding the Huntingtons disease research toolbox; validated huntingtin subdomain constructs for biochemical and structural investigation of the huntingtin protein
Authors: Alteen, M.G. / Deme, J.C. / Alvarez, C.P. / Loppnau, P. / Hutchinson, A. / Seitova, A. / Chandrasekaran, R. / Silva Ramos, E. / Secker, E. / Alqazzaz, M. / Wanker, E.E. / Lea, S.M. / ...Authors: Alteen, M.G. / Deme, J.C. / Alvarez, C.P. / Loppnau, P. / Hutchinson, A. / Seitova, A. / Chandrasekaran, R. / Silva Ramos, E. / Secker, E. / Alqazzaz, M. / Wanker, E.E. / Lea, S.M. / Arrowsmith, C.H. / Harding, R.J.
History
DepositionMar 31, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 26, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 26, 2023Group: Database references / Category: citation / citation_author
Revision 1.2Sep 20, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Huntingtin
B: 40-kDa huntingtin-associated protein


Theoretical massNumber of molelcules
Total (without water)158,8222
Polymers158,8222
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: light scattering
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Huntingtin / Huntington disease protein / HD protein


Mass: 117479.758 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HTT, HD, IT15 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P42858
#2: Protein 40-kDa huntingtin-associated protein / HAP40 / CpG island protein / Factor VIII intron 22 protein


Mass: 41342.254 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: F8A1, F8A2, F8A3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P23610

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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: Huntingtin C-HEAT domain in complex with HAP40 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 400 nm
Image recordingElectron dose: 51.3 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 134849 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0079370
ELECTRON MICROSCOPYf_angle_d0.69112750
ELECTRON MICROSCOPYf_dihedral_angle_d19.7631255
ELECTRON MICROSCOPYf_chiral_restr0.0421505
ELECTRON MICROSCOPYf_plane_restr0.0051621

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