SEA domain autoproteolysis accelerated by conformational strain: mechanistic aspects.

摘要: Abstract A subclass of SEA (sea urchin sperm protein, enterokinase, and agrin) domain proteins undergoes autoproteolysis between glycine serine in a conserved G − 1 S + 1 VVV motif to generate stable heterodimers. Autoproteolysis has been suggested involve only the intramolecular catalytic action hydroxyl combination with conformational strain glycine–serine peptide bond. We conducted number experiments simulations on from MUC1 mucin test this mechanism. Alanine-scanning mutagenesis polar residues vicinity cleavage site demonstrates that nucleophile at position is required for efficient proteolysis. Molecular modeling shows an uncleaved trans incompatible native heterodimeric structure, resulting disruption secondary structure elements distortion scissile Insertion (to obtain n motifs) appears relieve strain, 100 times slower 1G (  = 1) mutant not measurable 2G 4G mutants. Removal hampers considerably, but S1098A still proceeds presence alone. The precursor populates interconverting partially folded conformations, coincides adoption proper β-sheet completed folding. dynamics show preceding carbonyl carbon can be van der Waals contact same time as bond becomes strained. These observations are all consistent accelerated by N → O acyl shift imposed upon protein folding reaction which free-energy barrier decreased substrate destabilization rather than transition-state stabilization. energetics coupled mechanism accounted accompanying article.