Foldase and inhibitor functionalities of the pepsinogen prosegment are encoded within discrete segments of the 44 residue domain

Abstract

Pepsin is initially produced as the zymogen pepsinogen, containing a 44 residue prosegment (PS) domain. When folded without the PS, pepsin forms a thermodynamically stable denatured state (refolded pepsin, Rp). To guide native folding, the PS binds to Rp, stabilizes the folding transition state, and binds tightly to native pepsin (Np), thereby driving the folding equilibrium to favor the native state. It is unknown whether these functionalities of the PS are encoded within the entire sequence or within discrete segments. PS residues 1p–29p correspond to a highly conserved region in pepsin-like aspartic proteases and we hypothesized that this segment is critical to PS-catalyzed folding. This notion was tested in the present study by characterizing the ability of various truncated PS peptides to bind Rp, catalyze folding from Rp to Np, and to inhibit Np. Four PS truncations were examined, corresponding to PS residues 1p–16p (PS1–16), 1p–29p (PS1–29), 17p–44p (PS17–44) and 30p–44p (PS30–44). The three PS functionalities could be ascribed primarily to discrete regions within the highly conserved motif: 1p–16p dictated Rp binding, 17p–29p dictated Np binding/inhibition, while the entire 1p–29p dictated transition state binding/catalyzing folding. Conversely, PS30–44 played no obvious role in PS-catalyzed folding; it is hypothesized that this more variable region may serve as a linker between PS1–29 and the mature domain. The high sequence conservation of PS1–29 and its role in catalyzing pepsin folding strongly suggest that there is a conserved PS-catalyzed folding mechanism shared by pepsin-like aspartic proteases with this motif.