|Entry||Database: EMDB / ID: EMD-10336|
|Title||Respiratory mucin MUC5B: C-terminal dimerization domain structure|
|Sample||Dimeric structure of C-terminal MUC5B with C2 symmetry:|
|Biological species||Homo sapiens (human)|
|Method||single particle reconstruction / cryo EM / Resolution: 8.9 Å|
|Authors||Lockhart-Lockhart MP / Ridley C / Collins RF / Baldock C / Thornton DJ|
|Funding support|| United Kingdom, 1 items |
|Citation||Journal: J. Biol. Chem. / Year: 2019|
Title: The C-terminal dimerization domain of the respiratory mucin MUC5B functions in mucin stability and intracellular packaging before secretion.
Authors: Caroline Ridley / Michael P Lockhart-Cairns / Richard F Collins / Thomas A Jowitt / Durai B Subramani / Mehmet Kesimer / Clair Baldock / David J Thornton /
Abstract: Mucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways. Accordingly, knowledge of MUC5B structure and its interactions with itself and other proteins is ...Mucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways. Accordingly, knowledge of MUC5B structure and its interactions with itself and other proteins is critical to better understand airway mucus biology and improve the management of lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). The role of an N-terminal multimerization domain in the supramolecular organization of MUC5B has been previously described, but less is known about its C-terminal dimerization domain. Here, using cryogenic electron microscopy (cryo-EM) and small-angle X-ray scattering (SAXS) analyses of recombinant disulfide-linked dimeric MUC5B dimerization domain we identified an asymmetric, elongated twisted structure, with a double globular base. We found that the dimerization domain is more resistant to disruption than the multimerization domain suggesting the twisted structure of the dimerization domain confers additional stability to MUC5B polymers. Size-exclusion chromatography-multi-angle light scattering (SEC-MALS), SPR-based biophysical analyses and microscale thermophoresis of the dimerization domain disclosed no further assembly, but did reveal reversible, calcium-dependent interactions between the dimerization and multimerization domains that were most active at acidic pH, suggesting that these domains have a role in MUC5B intragranular organization. In summary, our results suggest a role for the C-terminal dimerization domain of MUC5B in compaction of mucin chains during granular packaging via interactions with the N-terminal multimerization domain. Our findings further suggest that the less stable multimerization domain provides a potential target for mucin depolymerization to remove mucus plugs in COPD and other lung pathologies.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_10336.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 2.0371 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire Dimeric structure of C-terminal MUC5B with C2 symmetry
|Entire||Name: Dimeric structure of C-terminal MUC5B with C2 symmetry|
Number of components: 2
-Component #1: protein, Dimeric structure of C-terminal MUC5B with C2 symmetry
|Protein||Name: Dimeric structure of C-terminal MUC5B with C2 symmetry|
Recombinant expression: No
|Mass||Experimental: 260 MDa|
|Source||Species: Homo sapiens (human)|
|Source (engineered)||Expression System: Homo sapiens (human) / Vector: pCEP-His / Cell of expression system: HEK293-EBNA|
-Component #2: protein, MUC5B
|Protein||Name: MUC5B / Recombinant expression: No|
|Source||Species: Homo sapiens (human)|
|Source (engineered)||Expression System: Homo sapiens (human)|
|Specimen||Specimen state: Particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.25 mg/mL|
Buffer solution: Buffers were filtered and degassed before use.
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: OTHER|
|Lens||Imaging mode: BRIGHT FIELD|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Processing||Method: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 16590|
|3D reconstruction||Software: cryoSPARC / Resolution: 8.9 Å / Resolution method: FSC 0.143 CUT-OFF|
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