Folding of pig gastric mucin non-glycosylated domains: a discrete molecular dynamics study

Abstract

Mucin glycoproteins consist of tandem-repeating glycosylated regions flanked by non-repetitive protein domains with little glycosylation. These non-repetitive domains are involved in polymerization of mucin and play an important role in the pH-dependent gelation of gastric mucin, which is essential for protecting the stomach from autodigestion. We examine folding of the non-repetitive sequence of PGM-2X (242 amino acids) and the von Willebrand factor vWF-C1 domain (67 amino acids) at neutral and low pH using discrete molecular dynamics (DMD) in an implicit solvent combined with a four-bead peptide model. Using the same implicit solvent parameters, folding of both domains is simulated at neutral and low pH. In contrast to vWF-C1, PGM-2X folding is strongly affected by pH as indicated by changes in the contact order, radius of gyration, free-energy landscape, and the secondary structure. Whereas the free-energy landscape of vWF-C1 shows a single minimum at both neutral and low pH, the free-energy landscape of PGM-2X is characterized by multiple minima that are more numerous and shallower at low pH. Detailed structural analysis shows that PGM-2X partially unfolds at low pH. This partial unfolding is facilitated by the C-terminal region GLU236-PRO242, which loses contact with the rest of the domain due to effective "mean-field" repulsion among highly positively charged N- and C-terminal regions. Consequently, at low pH, hydrophobic amino acids are more exposed to the solvent. In vWF-C1, low pH induces some structural changes, including an increased exposure of CYS at position 67, but these changes are small compared to those found in PGM-2X. For PGM-2X, the DMD-derived average β-strand propensity increases from 0.26 ± 0.01 at neutral pH to 0.38 ± 0.01 at low pH. For vWF-C1, the DMD-derived average β-strand propensity is 0.32 ± 0.02 at neutral pH and 0.35 ± 0.02 at low pH. The DMD-derived structural information provides insight into pH-induced changes in the folding of two distinct mucin domains and suggests plausible mechanisms of the aggregation/gelation of mucin.

Fig. 1

Sketch of porcine gastric mucin PGM showing different domains in the apoprotein (top), a S-S linked dimer (middle), and a multimer (bottom) illustrating the multi-block structure. Adapted from Dekker et al. [35]

Fig. 2

The average β-strand propensity as a function of the implicit solvent parameter E_CH at neutral and low pH for DMD-derived folded structures of PGM-2X and vWF-C1. The error bars correspond to SEM values

Fig. 3

Sampling efficiency of DMD simulations of PGM-2X folding at a neutral and b low pH and the corresponding PMF surfaces at c neutral and d low pH using the reaction coordinates CO and R_g. In a and b, each point corresponds to a different conformation along a trajectory and different colors are used for different trajectories. In c and d, we use bin sizes of 0.16% and 0.026 nm along the CO and R_g axes, respectively. The 2D plots were generated with the GNUPLOT software package

Fig. 4

Representative conformations of a folded PGM-2X domain obtained by DMD simulations at a neutral and b low pH. First, the DMD-derived folded structures with the lowest PMF value were selected (129 at neutral and 71 at low pH). Second, the resulting structures were clustered based on their pairwise RMSD values (with cut-off 7 Å), as implemented using the GROMOS algorithm within the GROMACS software package. Third, the centroid of the largest resulting cluster was identified as a representative conformation. The N-terminal amino acid ASP1 is colored red and the C-terminal amino acid PRO242 is colored blue. The images were generated by the VMD software package [25]

Fig. 5

Intramolecular contact maps of PGM-2X folded structures at a neutral and b low pH. The color scale quantifying the average number of contacts between two residues is displayed on the right. The SEM values are small and not visible within this color scheme

Fig. 6

The average a turn and b β-strand propensities per residue within PGM-2X folded structures at neutral and low pH as calculated by using the STRIDE algorithm within the VMD software package. The error bars correspond to SEM values

Fig. 7

a The average coarse-grained SASA per residue of PGM-2X folded structures at neutral and low pH. The error bars correspond to SEM values. The inset shows normalized histograms of the combined coarse-grained SASA, which is summed over all hydrophobic residues, for neutral (black) and low (red) pH. b The difference in coarse-grained SASA values between low and neutral pH

Fig. 8

Sampling efficiency of DMD simulations of vWF-C1 folding at a neutral and b low pH and the corresponding PMF surfaces at c neutral and d low pH using the reaction coordinates CO and R_g. In a and b, each point corresponds to a different conformation along a trajectory and different colors are used for different trajectories. In c and d, we use bin sizes of 0.20% and 0.007 nm along the CO and R_g axes, respectively. The 2D plots were generated with the GNUPLOT software package

Fig. 9

Representative conformations of a folded vWF-C1 domain obtained by DMD simulations at a neutral and b low pH. First, the DMD-derived folded structures with the lowest PMF value were selected (19 at neutral and 19 at low pH). Second, the resulting structures were clustered based on their pairwise RMSD values (with cut-off 7 Å), as implemented using the GROMOS algorithm within the GROMACS software package [36, 37]. Third, the centroid of the largest resulting cluster was identified as a representative conformation. The N-terminal amino acid CYS1 is colored red and the C-terminal amino acid CYS67 is colored blue. The images were generated by the VMD software package [25]

Fig. 10

Intramolecular contact maps of vWF-C1 folded structures at a neutral and b low pH. The color scale quantifying the average number of contacts between two residues is displayed on the right. The SEM values are small and not visible within this color scheme

Fig. 11

The average a turn and b β-strand propensities per residue within vWF-C1 folded structures at neutral and low pH as calculated by using the STRIDE algorithm within the VMD software package. The error bars correspond to SEM values

Fig. 12

a The average coarse-grained SASA per residue of vWF-C1 folded structures at neutral and low pH. The error bars correspond to SEM values. The inset shows normalized histograms of the combined coarse-grained SASA, which is summed over all hydrophobic residues, for neutral (black) and low (red) pH. b The difference in coarse-grained SASA values between low and neutral pH