Cystic fibrosis, a disease of altered water and salt secretion across epithelial tissues, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is a member of the ABC-transporter family of proteins and functions as a chloride channel;loss of the functional chloride channel activity is causative of CF. The ATP-Binding Cassette (ABC-) transporter superfamily of proteins is highly conserved across prokaryotes and eukaryotes, facilitating solute transport across biological membranes. ABC-transporter proteins are minimally composed of a dimer of highly conserved, cytosolic nucleotide-binding domains (NBDs), which provides the energy for solute transport through a dimer of transmembrane domains (TMDs). ATP binding and hydrolysis within the NBDs is regulated by the canonical Walker A and B nucleotide-binding sequences, as well as a sequence unique to the ABC-transporter family of proteins ? the signature sequence, or LSGGQRxR. While the Walker A and B sequences are well characterized, the structural and functional properties of the LSGGQRxR sequence are not known. Preliminary data suggest that the LSGGQRxR sequence critically contributes to the biosynthesis and function of CFTR and other ABC-transporter proteins. Functional regulation of NBD-NBD association events is critically altered by substitution of the glycine residues, resulting in either hyperactive or inactive channels. Alterations to the RxR sequence alter channel biosynthesis with only minor effects on channel activity. The major goal of this project is to elucidate the structural and functional roles of the LSGGQRxR sequence in regulating CFTR-channel and ABC-transporter biosynthesis and function. To accomplish this, we have developed methods for the expression, purification and biophysical characterization of the isolated NBD proteins from CFTR and two homologues (human ABCC6 and the bacterial Mj0796). Using a combination of X-ray crystallographic, nucleotide binding and hydrolysis, and biochemical approaches, we will evaluate the specific structural and functional roles of the LSGGQRxR signature sequence. These in vitro data will complement experiments evaluating the biosynthesis and function of full-length protein to provide a detailed model for the regulation of ABC-transporter biogenesis and mechanochemistry by the signature sequence.
The specific aims of this application are: (1) Characterize the nucleotide-binding and hydrolysis properties of CFTR NBD1 and NBD2, (2) Elucidate the role of the signature sequence di-glycine residues in ATP-mediated NBD association and function, and (3) Characterize the role of the RxR sequence on local NBD structure and CFTR biosynthesis. The research proposed in this application will provide novel insight into the previously undefined structural and functional roles of this highly conserved LSGGQRxR sequence. The innovative use of in vitro biochemical, structural and enzymatic assays to complement studies of full-length protein biosynthesis and function will refine models of the biogenesis and mechanochemistry of these medically important proteins.
ABC transporters play a key role in human disease by contributing to disease pathophysiology when mutated and by contributing to virulence of opportunistic pathogens. Understanding the biosynthetic and functional regulation of these proteins will contribute to our understanding of how these proteins directly and indirectly impact human disease.
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