Areas of Interest
ABC transporters share a common architecture of two nucleotide-binding domains (NBD’s) and two transmembrane domains (TMD’s). CFTR is unique in also having a central PKA-sensitive regulatory domain. There is significant sequence similarity in the ~200-residue core of the NBD cassettes between even the most remotely-related members of the ABC transporter superfamily. However, homology between the transmembrane domains is weak, consistent with their proposed role in determining the diverse transport-substrate specificities of the various family members. A high number of CF-causing mutations reside in the first NBD of CFTR, including the common DF508 mutation, which is involved in >90% of CF cases.
Since CFTR NBD1 has been refractory to high-resolution structural studies, several labs have instead focused on solving the structure of bacterial NBD subunits. We have taken such an approach in collaboration with Dr. John Hunt at Columbia University and solved the crystal structure of two NBD’s of the hyperthermophilic archaeon Methanococcus jannaschii, MJ1267 and MJ0796. MJ1267 and MJ0796 are the NBD subunits of branched-chain amino acid and Lol transporters, respectively. The homology between these two proteins and CFTR NBD1 (Figure 1) allows the position of disease-associated residues critical for folding and function to be mapped (Figure 2). Comparison of the effects of mutations at these positions in MJ1267 and CFTR also allows us to investigate the folding pathway and functional mechanism of the ABC-transporters. Current efforts are also underway to improve the biochemical behavior of CFTR NBD1 to allow solution of the crystal structure of the CFTR domain.
Figure 1 Figure 2
In addition to its function as an ATP-dependent chloride channel, CFTR also regulates other transport systems including the epithelial sodium channel (EnaC) and ROMK potassium channel as well as exchangers critical for epithelial bicarbonate secretion and maintenance of extracellular pH (Figure 3). Recently, in collaboration with Dr. Shmuel Muallem here at UT Southwestern, we have begun addressing the molecular mechanism of CFTR-regulated bicarbonate secretion in the pathology of CF. These studies have established that CFTR activates the SLC26 anion exchangers and thus, bicarbonate secretion. Disease-causing mutations in CFTR lead to loss of one or more of these activities due to a variety of molecular mechanisms, the most common of which is defective folding of CFTR (see also Folding section). In this regard, we found that CF mutations which most strongly impair bicarbonate secretion correlate with a more severe disease, especially in the pancreas. The studies are providing important new information directly relevant to the development of effective CF treatments.
In addition to these physiological studies, we are investigating the mechanochemistry of the ABC transporters using microbial systems such as the MJ0769 and MJ1267 NBD of Methanococcus jannaschii and the prt metalloprotease secretion system of Erwinia chrysantemi. For example, we have been able to find mutations in the NBD subunit that impair transport without measurable effect on ATP hydrolysis and mutations that trap a NBD dimer. These studies are providing valuable insight into the (likely common) molecular mechanism whereby ABC transporters use the energy released by ATP hydrolysis is used to drive solute.
Folding & Misfolding
Defective protein folding is becoming increasingly recognized as a significant cause of human disease, and cystic fibrosis (CF) provides a prime example. A number of CF-causing mutations in CFTR result in a protein that does not reach its proper destination in the plasma membrane, but is instead retained by the cellular quality control system and degraded by the ubiquitin-proteasome system. Misfolded CFTR that cannot be degraded accumulates in the cell as centrosome-associated inclusions of aggregated protein that are replete with proteasome components (Figure 4), reminiscent of perinuclear inclusions associated with numerous neurodegenerative disorders such as Parkinson's disease. In fact, the centrosomal region is a significant site of proteasome concentration even in resting cells, suggesting a novel role for this subcellular location in the quality control of protein expression. Currently, in collaboration with Dr. George DeMartino, our efforts are directed at revealing the mechanisms by which the proteasome recognizes misfolded mutant protein substrates and prepares them for proteolysis or refolding.
Several CF-causing mutations, including the common DF508 mutation in NBD1, result in misfolded CFTR proteins that never reach their proper location in the apical membrane. Proper folding and maturation of CFTR in vivo is an inefficient process that requires the participation of several molecular chaperones, including Hsp70, Hsp90 and calnexin. Since the DF508 mutation is involved in >90% of CF cases, we have used an in vitro system to analyze the folding pathway of NBD1 and the effect of DF508 on this process. We found that the F508 residue makes crucial contacts during the folding process, but plays little role in stabilization of the NBD native state. The DF508 mutation also imparts a temperature sensitive defect in NBD1 folding in vitro, which recapitulates the temperature-sensitive defect seen in the maturation of full-length DF508 CFTR in vivo. Current efforts are focused on further characterizing the NBD1 folding pathway, with the ultimate goal being the development of agents that can manipulate the folding of DF508 NBD1 as potential therapies for CF. We are currently extending these approaches to elucidate the molecular pathology of Parkinson's Disease due to misfolding of a-synuclein.