Chaperone-like activity of pepsin. Aggregation assays with thermally denatured citrate synthase (A) and chemically denatured rhodanese (B) in the absence of pepsin (▴); an equal molar amount (•), 2.5-fold (▪), or fivefold (♦) molar excess of pepsin; a 2.5-fold molar excess of pepsin inhibited with pepstatin (B, □); a fivefold molar excess of pepsin inhibited with pepstatin (⋄); a fivefold molar excess of BSA (▵). (Bottom) SDS-PAGE analysis of the assay solutions to show protein composition and to exclude remnant proteolytic activity of pepsin.

Stability of complexes between pepsin and denatured polypeptide. Aggregation of rhodanese in the absence (▴) or with a 2.5-fold molar excess (▪) of pepsin. Arrows indicate addition of pepstatin during the measurement.

Chaperone-like activity of HIV-1 protease. (A) Aggregation assay with chemically denatured rhodanese: Rhodanese in the absence of HIV-1 protease (▴); a 2.5-fold (▪) or fivefold (♦) molar excess of HPR-M, a 2.5-fold () or fivefold () molar excess of HPR-DS; a fivefold molar excess of BSA (▵). (B) Effect of HIV-1 protease inhibitor: Rhodanese in the absence (▴) or presence of a fivefold molar (•) excess of HPR-WT; a fivefold molar excess of HPR-WT inhibited with HIV-1 protease inhibitor (○). (B) (Bottom) SDS-PAGE analysis of the assay solutions to exclude remnant proteolytic activity of HPR-WT.

Structural comparison of double-ψ barrel proteins and aspartic proteases. Topology and structure of HIV-1 protease, pepsin, and Cdc48 N-terminal domain-like double-ψ barrels. Topology diagrams of (A) HIV-1 protease and a (E) double-ψ barrel. β-strands are represented as arrows, α-helices as bars, and loops as lines. Pseudo-symmetric units are depicted as filled and empty forms. The “ψ”-forming loops are in bold. The secondary structure elements are totally interleaved—elements of one symmetric unit make only contacts to elements of the other unit. Structures shown as ribbon diagrams. Order of secondary structure elements is color-coded from blue to red: (B) monomer of HIV-1 protease (5hvp) and (F) N-terminal double-ψ barrel domain of VAT (1cz4) from Thermoplasma acidophilum. (Bottom) Structural context of the domains: (C) One monomer of the HIV-1 protease dimer is colored and the substrate binding site is indicated by an arrow between both domains. (D) One domain of pepsin is colored and the substrate binding site is indicated by an arrow between both domains. (G) Structure of p97 (1r7r) representing the structural context of Cdc48 N-terminal domain-like double-ψ barrels. One monomer of the hexamer is colored with the N-terminal double-ψ barrel domain in red.

Chaperone-like activity of the pepsin C-domain. Aggregation assay with thermally denatured citrate synthase: Citrate synthase in the absence of the pepsin C-domain (▴); an equal molar amount (•), 2.5-fold (▪), or fivefold (♦) molar excess of pepsin C-domain; a fivefold molar excess of BSA (▵).

Complex formation of pepsin with denatured citrate synthase. UV-profiles of gel-size exclusion chromatography runs. The asterisk (*) (bottom, right) indicates the additional peak occurring when pepsin and citrate synthase are incubated together at 50°C. The inset shows SDS-PAGE analysis of the run with the mixture of citrate synthase and pepsin. Loaded samples are: fraction at 10 mL elution (1), fraction at 15 mL elution volume (2), references citrate synthase (3), and pepsin (4).

Scenario of Cdc48 N-terminal domain-like double-ψ barrel and aspartic protease evolution. The scenario originates with an ancestral chaperone showing a symmetrical double-ψ barrel fold. The left branch leads to AAA-ATPases by fusion with AAA-domains, e.g., in VAT. The right branch leads to circular permutation, loss of internal symmetry, and gain of hydrolytic activity. Further branching leads to the retroviral proteases like HIV-1 protease, and via gene duplication and fusion, to the eukaryotic aspartic proteases like pepsin.