LAUSR

Significance Recent advances in cryo-electron microscopy (cryo-EM) enabled us to determine multiple structures from the same sample. At equilibrium, the Boltzmann distribution law can be applied to identify the lowest energy state of the protein. We found that the conformational distributions of PCAT1, the peptidase-containing ATP-binding cassette (ABC) transporter 1, are completely different in the presence and absence of the Mg2+ ion. This difference reflects energy inflow from ATP hydrolysis, shifting the system out of equilibrium. The conformational distribution under ATP turnover condition is determined by the transition rates along the transport pathway rather than the energy of each state. This study demonstrates how cryo-EM data can be used to understand thermodynamic and kinetic properties of an active transporter. ATP-binding cassette (ABC) transporters are ubiquitous molecular pumps that transport a broad range of substrates across biological membranes. Although the structure and function of ABC transporters has been studied extensively, our understanding of their energetics and dynamics remains limited. Here, we present studies of the peptidase-containing ABC transporter 1 (PCAT1), a polypeptide processing and secretion ABC transporter that functions via the classic alternating access mechanism. PCAT1 is a homodimer containing two peptidase (PEP) domains, two transmembrane domains, and two nucleotide-binding domains (NBDs). Using cryo-electron microscopy, we analyzed the structures of wild-type PCAT1 under conditions that either prevent or permit ATP hydrolysis and observed two completely different conformational distributions. In the presence of ATP but absence of Mg2+, PCAT1 adopts an NBD-dimerized, outward-facing conformation. The two PEP domains are dissociated from the transporter core, preventing uncoupled substrate cleavage. The addition of Mg2+ to promote ATP hydrolysis shifts the majority of the particles into NBD-separated, inward-facing conformations. Under this ATP turnover condition, only a small fraction of PCAT1 adopts the NBD-dimerized conformation. These data give rise to two mechanistic conclusions: 1) the ATP-bound, NBD-dimerized conformation is the lowest energy state, and 2) the rate-limiting step in the PCAT1 transport cycle is the formation of the NBD dimer. The thermodynamic conclusion is likely a general property shared by many ABC transporters. The kinetic bottleneck, however, varies from transporter to transporter.