As the first step of MOLECULAR MUCUS, we determined using cryo-electron microscopy the high-resolution structures of key segments of the respiratory mucins, MUC5B and MUC5AC. These structures supported the conclusion that secreted mucins share a fundamental mechanism for orchestrating intermolecular disulfide bonding to generate glycoprotein polymers. Specifically, key segments of all three mucins formed beaded filaments in vitro, at moderately low pH as found in the Golgi apparatus and secretory granules, consistent with the mechanism we discovered in the context of the intestinal mucin, MUC2, for aligning reactive cysteines at the center of the beads (Javitt et al., Cell 2020). However, we observed striking differences in the higher-order supramolecular assembly modes of the beaded filaments for the three mucins. We had previously observed that MUC2 forms isolated, elongated filaments (Javitt et al., Cell 2020). In the context of MOLECULAR MUCUS, we found that one of the lung mucins, MUC5AC, forms single or paired loose coils (Haberman et al., PNAS 2025), whereas the other lung mucin, MUC5B, forms bundles of elongated filaments (unpublished results). Analysis of the structural differences between the three mucins showed how slight changes in amino acid sequence during evolution dramatically altered supramolecular assembly, likely leading to differences in the properties of the respective mucus gels after secretion.
These structural studies revealed general principles of mucin organization. In particular, we identified three important regions of the mucins where diversification can be accommodated while preserving the fundamental polymerization mechanism: one is the packing angles between the domains that determine the relative orientation of adjacent beads in the filaments, the second is in segments known as CysD domains, which in some cases make intermolecular interactions stabilizing the beaded filament, and the third is the length of the first glycosylated, natively-disordered region of the mucins, which determines the reach of the adjacent CysD domain and directs its docking onto the beaded filament.
The two next steps of MOLECULAR MUCUS, which are already underway, are to determine the structural contribution of additional CysD domains, which are scattered along the lengths of the secreted mucin glycoproteins, and to quantify the extensibility of mucin glycosylated segments, which we hypothesize serve as “entropic spacers” between specific CysD adhesion domains.
