The biocatalytic reactions coupled in this work: substrate segregationunfolding and substrate proteolysis. In the first reaction, p97 binds the substrate protein I3 (25 kDa), trapped in a complex with PP1 (37 kDa) and SDS22 (50 kDa), via the adapter protein p37 (ca. 40 kDa). Binding occurs at the N-terminal domains of p97. Addition of ATP drives the unidirectional threading of I3 through the central p97 channel and its ejection at the C-terminal tails, resulting in the unfolding of I3 and segregation from PP1 and SDS22. In the second reaction, the proteolytic degradation of the substrate is catalysed by aCt (25 kDa). All proteins involved in the modular chimera and their complexes are reported in Supplementary Table 1.
