Linda Millen

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.

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.

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.

The Cystic Fibrosis-causing Mutation ΔF508 Affects Multiple Steps in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis

The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the ΔF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the ΔF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that ΔF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.

The molecular chaperone Hsc70 assists the in vitro folding of the N-terminal nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator.

The most common disease-causing mutation in the cystic fibrosis transmembrane conductance regulator is a single amino acid deletion (ΔF508) in the N-terminal cytosolic nucleotide-binding domain (NBD1). This mutation has previously been shown to be a temperature-sensitive folding mutation that alters the folding pathway but not the native state stability of the isolated domain (Qu, B.-H., and Thomas, P. J. (1996) J. Biol. Chem. 271, 7261–7264). Here we provide evidence that the molecular chaperone Hsc70 productively interacts with NBD1 to increase the folding yield of the domain and inhibit off-pathway associations leading to the formation of high molecular weight aggregates. Furthermore, we have sublocalized a region within NBD1 where Hsc70 binds. Notably, inhibition of NBD1 aggregation is not dependent upon the presence of Hsc70 in the early stages of folding, indicating that the chaperone may act on a folding intermediate. In the presence of K+ and Mg2+-ATP, conditions where Hsp70 binds substrate rapidly and can release it, Hsc70 is less effective at inhibiting NBD1 aggregation. Thus, the rate of release of unfolded substrate is an important factor in preventing aggregation and promoting folding of the domain. These results demonstrate that Hsc70 promotes the otherwise inefficient folding of ΔF-NBD1 and provide insight into the mechanisms by which molecular chaperones assist proteins in folding.

A Unique Redox-sensing Sensor II Motif in TorsinA Plays a Critical Role in Nucleotide and Partner Binding

Early onset dystonia is commonly associated with the deletion of one of a pair of glutamate residues (ΔE302/303) near the C terminus of torsinA, a member of the AAA+ protein family (ATPases associated with a variety of cellular activities) located in the endoplasmic reticulum lumen. The functional consequences of the disease-causing mutation, ΔE, are not currently understood. By contrast to other AAA+ proteins, torsin proteins contain two conserved cysteine residues in the C-terminal domain, one of which is located in the nucleotide sensor II motif. Depending on redox status, an ATP hydrolysis mutant of torsinA interacts with lamina-associated polypeptide 1 (LAP1) and lumenal domain like LAP1 (LULL1). Substitution of the cysteine in sensor II diminishes the redox-regulated interaction of torsinA with these substrates. Significantly, the dystonia-causing mutation, ΔE, alters the ability of torsinA to mediate the redox-regulated interactions with LAP1 and LULL1. Limited proteolysis experiments reveal redox- and mutation-dependent changes in the local conformation of torsinA as a function of nucleotide binding. These results indicate that the cysteine-containing sensor II plays a critical role in redox sensing and the nucleotide and partner binding functions of torsinA and suggest that loss of this function of torsinA contributes to the development of DYT1 dystonia.

Direct Binding of the Corrector VX-809 to Human CFTR NBD1: Evidence of an Allosteric Coupling between the Binding Site and the NBD1:CL4 Interface

Understanding the mechanism of action of modulator compounds for the cystic fibrosis transmembrane conductance regulator (CFTR) is key for the optimization of therapeutics as well as obtaining insights into the molecular mechanisms of CFTR function. We demonstrate the direct binding of VX-809 to the first nucleotide-binding domain (NBD1) of human CFTR. Disruption of the interaction between C-terminal helices and the NBD1 core upon VX-809 binding is observed from chemical shift changes in the NMR spectra of residues in the helices and on the surface of β-strands S3, S9, and S10. Binding to VX-809 leads to a significant negative shift in NBD1 thermal melting temperature (Tm), pointing to direct VX-809 interaction shifting the NBD1 conformational equilibrium. An inter-residue correlation analysis of the chemical shift changes provides evidence of allosteric coupling between the direct binding site and the NBD1:CL4 interface, thus enabling effects on the interface in the absence of direct binding in that location. These NMR binding data and the negative Tm shifts are very similar to those previously reported by us for binding of the dual corrector-potentiator CFFT-001 to NBD1 (Hudson et al., 2012), suggesting that the two compounds may share some aspects of their mechanisms of action. Although previous studies have shown an important role for VX-809 in modulating the conformation of the first membrane spanning domain (Aleksandrov et al., 2012; Ren et al., 2013), this additional mode of VX-809 binding provides insight into conformational dynamics and allostery within CFTR.

The BiP Molecular Chaperone Plays Multiple Roles during the Biogenesis of TorsinA, an AAA+ ATPase Associated with the Neurological Disease Early-onset Torsion Dystonia

Background: The ΔE mutation in the AAA+ ATPase torsinA is associated with the neurological disease torsion dystonia. Results: BiP and its co-factors maintain torsinA and torsinAΔE stability, glycosylation, and solubility. Conclusion: torsinA/ΔE, a chaperone-like protein, requires the assistance of other chaperones to fold. Significance: Therapeutics that modulate BiP may counteract torsinAΔE-associated physiological defects. Early-onset torsion dystonia (EOTD) is a neurological disorder characterized by involuntary and sustained muscle contractions that can lead to paralysis and abnormal posture. EOTD is associated with the deletion of a glutamate (ΔE) in torsinA, an endoplasmic reticulum (ER) resident AAA+ ATPase. To date, the effect of ΔE on torsinA and the reason that this mutation results in EOTD are unclear. Moreover, there are no specific therapeutic options to treat EOTD. To define the underlying biochemical defects associated with torsinAΔE and to uncover factors that might be targeted to offset defects associated with torsinAΔE, we developed a yeast torsinA expression system and tested the roles of ER chaperones in mediating the folding and stability of torsinA and torsinAΔE. We discovered that the ER lumenal Hsp70, BiP, an associated Hsp40, Scj1, and a nucleotide exchange factor, Lhs1, stabilize torsinA and torsinAΔE. BiP also maintained torsinA and torsinAΔE solubility. Mutations predicted to compromise specific torsinA functional motifs showed a synthetic interaction with the ΔE mutation and destabilized torsinAΔE, suggesting that the ΔE mutation predisposes torsinA to defects in the presence of secondary insults. In this case, BiP was required for torsinAΔE degradation, consistent with data that specific chaperones exhibit either pro-degradative or pro-folding activities. Finally, using two independent approaches, we established that BiP stabilizes torsinA and torsinAΔE in mammalian cells. Together, these data define BiP as the first identified torsinA chaperone, and treatments that modulate BiP might improve symptoms associated with EOTD.

Alteration of CFTR transmembrane span integration by disease-causing mutations

There are distinct molecular pathologies for two cystic fibrosis–causing mutations in the first transmembrane span of the CF transmembrane conductance regulator protein. These results have implications for understanding the mechanisms of membrane protein integration and folding and the effects of disease-causing mutants.