Exploring the structural constraints at cleavage site of mucin 1 isoform through molecular dynamics simulation.

In silico alanine scanning mutagenesis on the cleavable isoform of mucin 1 revealed isoleucine 67 as one of the key factors contributing to the strain at the autoproteolytic cleavage site. In this study, we demonstrate the structural basis of isoleucine-induced rigidity towards the strain-driven autoproteolysis at G(-1)S(+1) cleavage site of mucin 1. We further evaluated the gain in flexibility upon isoleucine 67 mutation through molecular dynamics and essential dynamics studies. The results show that the mutant exhibits stability in its secondary structural elements while the native displays a less-bonded network, however the cleavage site of native remains constrained. Essential dynamics revealed that large motions of the mutant were confined to the loop although the internal domain of the structure remains unaffected. Also, the mutation exerted a larger effect on the intraprotein interactions and consequently resulted in a stabilized motif at the cleavage. Analyses on MD trajectory conformations illustrate a completely disrupted motif in native as an effect of the peptide strain. The study also revealed that in mutant, the cleavage competent catalytic groups C=O and OG were in geometrical aspects unfavorable for a nucleophilic attack. The results support the earlier speculation that the presence of bulky isoleucine proximal G(-1)S(+1) cleavage site limits the conformational sampling of residues and therefore maintains the residues in a torsionally restrained conformation.