Structure and Mechanism of PCAT1, a Polypeptide Processing and Secretion Transporter

ATP-binding cassette (ABC) transporters utilize the energy from ATP to transport substrates across biological membranes. Various ABC transporters perform diverse biological functions across all forms of life ranging from importing essential nutrients to exporting toxic drugs (Ford and Beis 2019; ter Beek, Guskov, and Slotboom 2014). Bacterial cells utilize a class of ABC transporters for exporting proteins or peptides. Unlike the Sec translocon machinery, the ABC peptide exporters are dedicated to specific peptides (Fath and Kolter 1993). These peptides function as quorum sensing peptides, biofilms, or antimicrobial peptides. Among these ABC peptide exporters are Peptidase Containing ABC Transporters (PCATs) that perform dual functions of peptide maturation through proteolytic cleavage and peptide export (Gebhard 2012). As the name connotes, these transporters contain an accessory cysteine protease domain that interacts with the core ABC transporter. This structural feature is essential for the function and is unique among ABC transporters, making it a biologically interesting target for investigation. Although PCATs, which were first described 20 years ago, are essential to prokaryotic life, structural and functional studies of these proteins have been lacking. Until recently, only soluble parts of the proteins have been crystallized. The first full-length structure of PCATs was described in 2015 (Lin, Huang, and Chen 2015). However, structures of PCATs in complex with their substrates are needed to understand how PCATs recognize and transport their peptide substrates. To understand how PCATs work at the atomic level, I mainly took a structural approach. To this end, I decided to use cryo-electron microscopy (cryo-EM) and single-particle reconstruction techniques to obtain high-resolution structures of PCATs in various conformational states in complex with their substrates. These structures, together with the biochemical evidence, give us a clearer mechanistic picture of PCATs. First, to understand how PCATs bind substrate, I determined the structure of a PCAT from Clostridium thermocellum (abbreviated as PCAT1) in the substrate-bound inward-facing conformation. I have identified structural features that enable the substrate to bind, translocate into the transmembrane cavity, and orient properly for cleavage. Next, to understand how substrate binding coordinates with ATP binding, I have determined three structures of PCAT1 in the active-turnover condition, where PCAT1 is allowed to transition freely through the transport cycle. In addition, I have determined the structure of a clear outward-facing structure PCAT1 trapped in the Mg2+ condition that elucidates how the behavior of the core transporter affects the accessory peptidase domain. These structures together enable us to propose a mechanism of how the ATP binding and hydrolysis cycle is synchronized with substrate binding and processing, a unique feature crucial for strict coupling of cleavage and translocation. In addition to the cryo-EM work, I collaborated with Dr. Paul Dominic Olinares in Professor Brian T. Chait’s laboratory to study the stoichiometry of the PCAT1-substrate complex and intermediates of PCAT1 in the transport cycle. This work allows us to delineate the steps along the transport cycles that are short-lived and cannot be captured using structural study