Abstract
Nerita Versicolor carboxypeptidase inhibitor (NvCI) is the strongest inhibitor reported so far for the M14A subfamily of carboxypeptidases. It comprises 53 residues and a protein fold composed of a two-stranded antiparallel β sheet connected by three loops and stabilized by three disulfide bridges. Here we report the oxidative folding and reductive unfolding pathways of NvCI. Much debate has gone on whether protein conformational folding guides disulfide bond formation or instead they are disulfide bonds that favour the arrangement of local or global structural elements. We show here that for NvCI both possibilities apply. Under physiological conditions, this protein folds trough a funnelled pathway involving a network of kinetically connected native-like intermediates, all sharing the disulfide bond connecting the two β-strands. In contrast, under denaturing conditions, the folding of NvCI is under thermodynamic control and follows a “trial and error” mechanism, in which an initial quasi-stochastic population of intermediates rearrange their disulfide bonds to attain the stable native topology. Despite their striking mechanistic differences, the efficiency of both folding routes is similar. The present study illustrates thus a surprising plasticity in the folding of this extremely stable small disulfide-rich inhibitor and provides the basis for its redesign for biomedical applications.
Highlights
Oxidative folding is a very complex process, where an interplay between conformational folding, disulfide bond formation and disulfide isomerization takes place15, 16
Mass spectrometry analysis of disulfide species along Nerita versicolor carboxypeptidase inhibitor (NvCI) folding reaction showed the formation of 1-disulfide species (1S-S) within the first 4 hours of refolding, to reach a maximum around 2 to 3 hours (Fig. 2b)
Oxidative folding experiments are usually performed at pH 8.4, because this is the pKa of the Cys amino acid side chain
Summary
Oxidative folding is a very complex process, where an interplay between conformational folding, disulfide bond formation and disulfide isomerization takes place . Nerita versicolor carboxypeptidase inhibitor (NvCI) is the strongest, natural proteinaceous inhibitor reported so far for the M14A subfamily of carboxypetidases, with inhibitory constants in the picomolar range29 It comprises 53 residues and a protein fold composed of a two-stranded antiparallel β-sheet connected by three loops and stabilized by three disulfide bridges: The Cys27-Cys disulfide bond interconnects the two major secondary structure elements; Cys9-Cys stabilizes the first beta sheet by fixing this element to the N terminus of the inhibitor and Cys15-Cys links the C terminal tail, which corresponds to the active site of NvCI, with the first loop (Fig. 1). The three-dimensional protein structures of these inhibitors are unrelated and completely different, the only conserved motif being the structural conformation of the P1 and P2 residues in the C-terminal tail of the inhibitor that interacts in a competitive manner with the active site of the carboxypeptidase by occlusion of the active site subsites S1’, S1 and S235
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