Structure and dynamics of NBD1 from CFTR characterized using crystallography and hydrogen/deuterium exchange mass spectrometry.

The DeltaF508 mutation in nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the predominant cause of cystic fibrosis. Previous biophysical studies on human F508 and DeltaF508 domains showed only local structural changes restricted to residues 509-511 and only minor differences in folding rate and stability. These results were remarkable because DeltaF508 was widely assumed to perturb domain folding based on the fact that it prevents trafficking of CFTR out of the endoplasmic reticulum. However, the previously reported crystal structures did not come from matched F508 and DeltaF508 constructs, and the DeltaF508 structure contained additional mutations that were required to obtain sufficient protein solubility. In this article, we present additional biophysical studies of NBD1 designed to address these ambiguities. Mass spectral measurements of backbone amide (1)H/(2)H exchange rates in matched F508 and DeltaF508 constructs reveal that DeltaF508 increases backbone dynamics at residues 509-511 and the adjacent protein segments but not elsewhere in NBD1. These measurements also confirm a high level of flexibility in the protein segments exhibiting variable conformations in the crystal structures. We additionally present crystal structures of a broader set of human NBD1 constructs, including one harboring the native F508 residue and others harboring the DeltaF508 mutation in the presence of fewer and different solubilizing mutations. The only consistent conformational difference is observed at residues 509-511. The side chain of residue V510 in this loop is mostly buried in all non-DeltaF508 structures but completely solvent exposed in all DeltaF508 structures. These results reinforce the importance of the perturbation DeltaF508 causes in the surface topography of NBD1 in a region likely to mediate contact with the transmembrane domains of CFTR. However, they also suggest that increased exposure of the 509-511 loop and increased dynamics in its vicinity could promote aggregation in vitro and aberrant intermolecular interactions that impede trafficking in vivo.

CYP2E1 expression in human lymphocytes from various ethnic populations.

BACKGROUND: Monitoring CYP2E1 levels in alcoholic individuals holds inherent appeal because such determinations might indicate individuals at increased risk for alcoholic liver disease. We previously demonstrated that lymphocyte CYP2E1 expression reflects in vivo activity of the hepatic enzyme. METHODS: To further validate this approach, the current investigation compared lymphocyte CYP2E1 content and chlorzoxazone pharmacokinetics in 51 alcoholic and nonalcoholic White, Navajo, and Mexican American subjects. After an oral dose of chlorzoxazone, blood samples were collected and lymphocytes isolated. RESULTS: Alcoholics exhibited a 2-fold elevation in lymphocyte CYP2E1 messenger ribonucleic acid (mRNA) and protein compared to nonalcoholics. Chlorzoxazone clearance rates were 1.9-fold higher and area under the concentration curve (AUC) values 1.8-fold lower in alcoholic individuals compared to nonalcoholics. Furthermore, chlorzoxazone clearance rates correlated (r = 0.55, p < 0.01, n = 38) with lymphocyte CYP2E1 mRNA content, and transcript levels further correlated (r = 0.52, p < 0.001, n = 38) with CYP2E1 protein content. To compare phenotype with genotype, restriction fragment length polymorphism analyses on deoxyribonucleic acid samples were performed to identify polymorphisms in the CYP2E1 gene. No subjects were homozygous for rare alleles c2 or C. Nonetheless, 27% of the Navajos and 15% of the Mexican Americans were heterozygous for the c2 allele. Two White subjects appeared heterozygous (c1/c2) when RsaI was used to characterize CYP2E1 genotype but homozygous (c1/c1) at the PstI locus. Fifteen percent of Mexican American subjects, 20% of Navajo subjects, and 6% of White subjects were heterozygous for the C allele. Neither CD nor cl/c2 genotypes were associated with alcoholism. CONCLUSIONS: Human lymphocyte CYP2E1 mRNA levels may be useful predictors of alcohol-mediated alterations in hepatic CYP2E1 activity. Moreover, ethnicity does not appear to play a major role in the levels of expression of lymphocyte CYP2E1.