Philip J. Thomas

ATP binding to the motor domain from an ABC transporter drives formation of a nucleotide sandwich dimer.

It has been proposed that the reaction cycle of ATP binding cassette (ABC) transporters is driven by dimerization of their ABC motor domains upon binding ATP at their mutual interface. However, no such ATP sandwich complex has been observed for an ABC from an ABC transporter. In this paper, we report the crystal structure of a stable dimer formed by the E171Q mutant of the MJ0796 ABC, which is hydrolytically inactive due to mutation of the catalytic base. The structure shows a symmetrical dimer in which two ATP molecules are each sandwiched between the Walker A motif in one subunit and the LSGGQ signature motif in the other subunit. These results establish the stereochemical basis of the power stroke of ABC transporter pumps.

Defective protein folding as a basis of human disease

The ability of a polypeptide to fold into a unique, functional, three-dimensional structure in vivo is dependent upon its amino acid sequence and the function of molecular chaperone proteins and enzymes that catalyse folding. Intense study of the physical chemistry and cell biology of folding have greatly aided our understanding of the mechanisms normally employed. Evidence is accumulating that many disease-causing mutations and modifications exert their effects by altering protein folding. Here we discuss the pathobiology of these processes.

Causes of venous ulceration: a new hypothesis

Previous hypotheses about the causes of venous ulceration are inconsistent with recently published data. In patients with chronic venous insufficiency the number of functioning capillary loops visible in the skin on microscopy fell after the legs had been dependent for 30 minutes. Another study had shown that leucocytes became trapped in the circulation in dependent legs. A new hypothesis linking these two findings proposes that the trapped while cells occlude the capillaries and result in ischaemia of the skin of the leg.

Gating of CFTR by the STAS domain of SLC26 transporters.

Chloride absorption and bicarbonate secretion are vital functions of epithelia, as highlighted by cystic fibrosis and diseases associated with mutations in members of the SLC26 chloride-bicarbonate exchangers. Many SLC26 transporters (SLC26T) are expressed in the luminal membrane together with CFTR, which activates electrogenic chloride-bicarbonate exchange by SLC26T. However, the ability of SLC26T to regulate CFTR and the molecular mechanism of their interaction are not known. We report here a reciprocal regulatory interaction between the SLC26T DRA, SLC26A6 and CFTR. DRA markedly activates CFTR by increasing its overall open probablity (NPo) sixfold. Activation of CFTR by DRA was facilitated by their PDZ ligands and binding of the SLC26T STAS domain to the CFTR R domain. Binding of the STAS and R domains is regulated by PKA-mediated phosphorylation of the R domain. Notably, CFTR and SLC26T co-localize in the luminal membrane and recombinant STAS domain activates CFTR in native duct cells. These findings provide a new understanding of epithelial chloride and bicarbonate transport and may have important implications for both cystic fibrosis and diseases associated with SLC26T.

Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP‐binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide‐binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF‐causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP‐ and ATP‐bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1‐transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1–NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.

Aberrant CFTR-dependent HCO3- transport in mutations associated with cystic fibrosis

Cystic fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Initially, Cl- conductance in the sweat duct was discovered to be impaired in CF, a finding that has been extended to all CFTR-expressing cells. Subsequent cloning of the gene showed that CFTR functions as a cyclic-AMP-regulated Cl- channel; and some CF-causing mutations inhibit CFTR Cl- channel activity. The identification of additional CF-causing mutants with normal Cl- channel activity indicates, however, that other CFTR-dependent processes contribute to the disease. Indeed, CFTR regulates other transporters, including Cl--coupled HCO-3 transport. Alkaline fluids are secreted by normal tissues, whereas acidic fluids are secreted by mutant CFTR-expressing tissues, indicating the importance of this activity. HCO-3 and pH affect mucin viscosity and bacterial binding. We have examined Cl--coupled HCO-3 transport by CFTR mutants that retain substantial or normal Cl- channel activity. Here we show that mutants reported to be associated with CF with pancreatic insufficiency do not support HCO-3 transport, and those associated with pancreatic sufficiency show reduced HCO-3 transport. Our findings demonstrate the importance of HCO-3 transport in the function of secretory epithelia and in CF.

Defining the disease liability of variants in the cystic fibrosis transmembrane conductance regulator gene

Allelic heterogeneity in disease-causing genes presents a substantial challenge to the translation of genomic variation into clinical practice. Few of the almost 2,000 variants in the cystic fibrosis transmembrane conductance regulator gene CFTR have empirical evidence that they cause cystic fibrosis. To address this gap, we collected both genotype and phenotype data for 39,696 individuals with cystic fibrosis in registries and clinics in North America and Europe. In these individuals, 159 CFTR variants had an allele frequency of ł0.01%. These variants were evaluated for both clinical severity and functional consequence, with 127 (80%) meeting both clinical and functional criteria consistent with disease. Assessment of disease penetrance in 2,188 fathers of individuals with cystic fibrosis enabled assignment of 12 of the remaining 32 variants as neutral, whereas the other 20 variants remained of indeterminate effect. This study illustrates that sourcing data directly from well-phenotyped subjects can address the gap in our ability to interpret clinically relevant genomic variation.

A molecular mechanism for aberrant CFTR-dependent HCO(3)(-) transport in cystic fibrosis.

Aberrant HCO3− transport is a hallmark of cystic fibrosis (CF) and is associated with aberrant Cl−‐dependent HCO3− transport by the cystic fibrosis transmembrane conductance regulator (CFTR). We show here that HCO3− current by CFTR cannot account for CFTR‐activated HCO3− transport and that CFTR does not activate AE1–AE4. In contrast, CFTR markedly activates Cl− and OH−/HCO3− transport by members of the SLC26 family DRA, SLC26A6 and pendrin. Most notably, the SLC26s are electrogenic transporters with isoform‐specific stoichiometries. DRA activity occurred at a Cl−/HCO3− ratio ≥2. SLC26A6 activity is voltage regulated and occurred at HCO3−/Cl− ≥2. The physiological significance of these findings is demonstrated by interaction of CFTR and DRA in the mouse pancreas and an altered activation of DRA by the R117H and G551D mutants of CFTR. These findings provide a molecular mechanism for epithelial HCO3− transport (one SLC26 transporter—electrogenic transport; two SLC26 transporters with opposite stoichiometry in the same membrane domain—electroneutral transport), the CF‐associated aberrant HCO3− transport, and reveal a new function of CFTR with clinical implications for CF and congenital chloride diarrhea.

A molecular mechanism for aberrantCFTR-dependent HCO3– transport in cystic fibrosis

Aberrant HCO3− transport is a hallmark of cystic fibrosis (CF) and is associated with aberrant Cl−‐dependent HCO3− transport by the cystic fibrosis transmembrane conductance regulator (CFTR). We show here that HCO3− current by CFTR cannot account for CFTR‐activated HCO3− transport and that CFTR does not activate AE1–AE4. In contrast, CFTR markedly activates Cl− and OH−/HCO3− transport by members of the SLC26 family DRA, SLC26A6 and pendrin. Most notably, the SLC26s are electrogenic transporters with isoform‐specific stoichiometries. DRA activity occurred at a Cl−/HCO3− ratio ≥2. SLC26A6 activity is voltage regulated and occurred at HCO3−/Cl− ≥2. The physiological significance of these findings is demonstrated by interaction of CFTR and DRA in the mouse pancreas and an altered activation of DRA by the R117H and G551D mutants of CFTR. These findings provide a molecular mechanism for epithelial HCO3− transport (one SLC26 transporter—electrogenic transport; two SLC26 transporters with opposite stoichiometry in the same membrane domain—electroneutral transport), the CF‐associated aberrant HCO3− transport, and reveal a new function of CFTR with clinical implications for CF and congenital chloride diarrhea.

CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices

The regulatory (R) region of the cystic fibrosis transmembrane conductance regulator (CFTR) is intrinsically disordered and must be phosphorylated at multiple sites for full CFTR channel activity, with no one specific phosphorylation site required. In addition, nucleotide binding and hydrolysis at the nucleotide-binding domains (NBDs) of CFTR are required for channel gating. We report NMR studies in the absence and presence of NBD1 that provide structural details for the isolated R region and its interaction with NBD1 at residue-level resolution. Several sites in the R region with measured fractional helical propensity mediate interactions with NBD1. Phosphorylation reduces the helicity of many R-region sites and reduces their NBD1 interactions. This evidence for a dynamic complex with NBD1 that transiently engages different sites of the R region suggests a structural explanation for the dependence of CFTR activity on multiple PKA phosphorylation sites.