Site-specific interactions between PCAT1 and its substrate
Bacteria employ an array of efflux pumps to achieve the secretion of toxins, including the membrane-transporter family known as peptidase-containing ATP-binding cassette transporters (PCATs). PCATs are a subfamily of the ATP-binding cassette (ABC) transporter superfamily and have an additional N-terminal peptidase domain that exhibits cysteine protease activity necessary for substrate processing. Thus far, PCAT1 of Clostridium thermocellum is the only transporter of this subfamily to have its crystal structure resolved. However, the mechanistic details of PCAT1’s binding, cleavage, and transport of its substrate have yet to be established. We hypothesize that the substrate makes well defined contacts with the lumen of PCAT1 and its subsequent transport is carried out by an alternating access mechanism. In order to observe PCAT1’s interaction with its substrate and understand its catalytic transport cycle, we will engineer single cysteine mutants of PCAT1’s substrate via PCR and label them with the fluorophore monobromobimane (bimane-substrate). Fluorescence spectroscopy will then be used to analyze interactions of the bimane-substrate mutants with the PCAT1 variant Cysless Core , which lacks its peptidase domain, in the presence of various nucleotides. Knowing that changes in peak fluorescence result from differing site-specific interactions of bimane-substrate mutants with PCAT1, we can conclude which residues of the wild-type substrate fit entirely in the binding cavity, allowing for alternating access transport. These analyses will provide a more complete transport mechanism for PCAT1, and the knowledge gained from this study can be extended to other ABC transporters and their role in protein translocation in bacteria.