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 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-multiangle 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.
Highlights
Mucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways
We have previously described a role for the N-terminal multimerization domain of MUC5B in forming noncovalent, reversible calcium-dependent tetramers that are involved in the organization of the mucin chains within the secretory granule [19, 21]
During packaging of MUC5B within secretory granules, the mucin chains are organized around protein nodes formed from pH and calcium-dependent, noncovalent, reversible, homotypic interactions between N-terminal multimerization domains [19, 21]
Summary
To investigate the structure and further define the role of the C-terminal dimerization domain of MUC5B, we stably expressed a C-terminal construct of MUC5B (CT5B), consisting of D4-B-C-CK domains (Fig. 1A). 2D classification of CT5B showed an elongated shape consisting of a globular base connected to an extended stalk region (Fig. 3C and Fig. S1B). The B-C-CK domains form the stalk structure It was apparent from the 2D class averages (Fig. 3C and Fig. S1B) and 3D reconstruction (Fig. 2, A and C) that the two molecules within the stalk region twist around each other. This may increase the interface and the number of domain-domain interactions between the monomers in the stalk region, potentially increasing the stability of this C-terminal region. CT5B dimer by size exclusion chromatography at different our data suggest that the twist in the stalk of the molecule,. Which represents the B-C domains, could be a feature that provides enhanced stability to the CT5B dimer
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