ATP-dependent interactions of a cargo protein with the transmembrane domain of a polypeptide processing and secretion ABC transporter.

Powered by the energy of ATP binding and hydrolysis, protease-containing ABC transporters (PCATs) export amphipathic and hydrophilic bacteriocin and quorum-sensing proteins across the membrane hydrophobic barrier. The cargo proteins have N-terminal leader peptides that are cleaved off by the cysteine protease domain, referred to as the C39 domain, or referred to as the peptidase (PEP) domain. The sequence and structural determinants of the interaction between PCATs and cargo proteins are poorly understood, yet this interaction is a central aspect of the transport mechanism. Here, we demonstrate the ATP-dependent, equilibrium binding of the cargo protein to the transmembrane domain (TMD) of a PCAT subsequent to the removal of the leader peptide by the PEP domain. Binding of the cargo protein to PCAT1 variants devoid of the PEP domain is detected through changes in the spectroscopic properties of fluorescent or spin label. Moreover, we find similar energetics of binding regardless of the presence of the leader peptide, suggesting that although the PEP domain serves for recognition and orientation, interaction with the TMD is the main contributor to the affinity. These findings are in direct contradiction with a recent study claiming that the TMD does not interact with the cargo protein; rather acting as a "Teflon-like" conduit across the bilayer (Kieuvongngam, V., Olinares, P. D. B., Palillo, A., Oldham, M. L., Chait, B. T., and Chen, J. (2020) Structural basis of substrate recognition by a polypeptide processing and secretion transporter. eLife 9, e51492). A distinctive feature of the transport model emerging from our data invokes a stable complex between PCATs and their cargo proteins following processing of the leader peptide and prior to ATP-dependent alternating access that translocates the cargo protein to the extracellular side.