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Structural details of CcdA, a membrane transporter of thiol-reducing potential


We propose to provide structural details of how thiol-reducing electrons are transported across biological membranes. Bisulfide bonds formed between pairs of cysteines are important for the native function of many proteins in the extracytoplasmic compartments. Living organisms have evolved to acquire elaborate thiol- redox pathways to promote the correct pairing of cysteines in proteins. In bacteria, a number of key enzymes, such as DsbA, DsbB, DsbC, and DsbD, catalyze a network of thiol-redox reactions. Among them, DsbD probably has the most intriguing structural features because it contains a membrane-embedded domain (DsbD-b) that uses a pair of cysteines to translocate electrons, or reducing power, from the cytoplasm to the periplasm. DsbD-b moves electrons across the membrane in two steps, 1) reduction of its disulfide-bonded cysteines by cytoplasmic thioredoxin (Trx), and 2) reformation of the disulfide bond by reducing the Trx-like domain of DsbD (DsbD-g) on the periplasmic side. Clearly, for the pair of cysteines to be accessible to substrates from both sides of the membrane, DsbD-b must either adopt an unusual architecture in membrane or undergo a large conformational change between the oxidized and reduced states. To provide the first structural insights into this process, we will carry out structural studies on a 27 kD sequence and functional homolog of DsbD-b that is suitable for NMR studies, named CcdA. We will use solution NMR methods to determine the structure of CcdA in both oxidized and reduced states. Based on our initial success in obtaining a high quality NMR spectrum of CcdA, we are in a good position to provide the first structural details of this fascinating class of transmembrane electron transporters. Upon the completion of the structures of oxidized and reduced CcdA, we will examine how CcdA recognizes its cytoplasmic Trx and periplasmic substrate CcmG, also a Trx-like protein. We will attach paramagnetic centers to different parts of the proteins for identifying interaction surfaces between CcdA and its substrates. Additionally, C-X- X-C to C-X-X-A mutations in Trx-like substrates will be used to trap the CcdA-Trx mixed disulfide intermediate to slow down the turnover rates for detailed structural studies. The proposed study will likely produce the first novel membrane protein fold determined by solution NMR, thereby demonstrating the general application of this technology in studying membrane protein structures.


Funded by the NIH National Center for Advancing Translational Sciences through its Clinical and Translational Science Awards Program, grant number UL1TR002541.