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1.  Rational Coupled Dynamics Network Manipulation Rescues Disease-Relevant Mutant Cystic Fibrosis Transmembrane Conductance Regulator 
Many cellular functions necessary for life are tightly regulated by protein allosteric conformational change, and correlated dynamics between protein regions has been found to contribute to the function of proteins not previously considered allosteric. The ability to map and control such dynamic coupling would thus create opportunities for the extension of current therapeutic design strategy. Here, we present an approach to determine the networks of residues involved in the transfer of correlated motion across a protein, and apply our approach to rescue disease-causative mutant cystic fibrosis transmembrane regulator (CFTR) ion channels, ΔF508 and ΔI507, which together constitute over 90% of cystic fibrosis cases. We show that these mutations perturb dynamic coupling within the first nucleotide-binding domain (NBD1), and uncover a critical residue that mediates trans-domain coupled dynamics. By rationally designing a mutation to this residue, we improve aberrant dynamics of mutant CFTR as well as enhance surface expression and function of both mutants, demonstrating the rescue of a disease mutation by rational correction of aberrant protein dynamics.
PMCID: PMC4324596  PMID: 25685315
2.  Regulatory insertion removal restores maturation, stability and function of ΔF508 CFTR 
Journal of molecular biology  2010;401(2):194-210.
The cystic fibrosis transmembrane conductance regulator (CFTR) epithelial anion channel is a large multi-domain membrane protein which matures inefficiently during biosynthesis. Its assembly is further perturbed by the deletion of F508 from the first nucleotide binding domain (NBD1) responsible for most cystic fibrosis. The mutant polypeptide is recognized by cellular quality control systems and is proteolyzed. CFTR NBD1 contains a 32 residue segment termed the regulatory insertion (RI) not present in other ABC transporters. We report here that RI deletion enabled ΔF508 CFTR to mature and traffic to the cell surface where it mediated regulated anion efflux and exhibited robust single chloride channel activity. Long term pulse-chase experiments showed that the mature ΔRI/ΔF508 had a T1/2 of ~14h in cells, similar to the wild-type. RI deletion restored ATP occlusion by NBD1 of ΔF508 CFTR and had a strong thermo-stabilizing influence on the channel with gating up to at least 40°C. None of these effects of RI removal were achieved by deletion of only portions of RI. Discrete molecular dynamics simulations of NBD1 indicated that RI might indirectly influence the interaction of NBD1 with the rest of the protein by attenuating the coupling of the F508 containing loop with the F1-like ATP-binding core subdomain so that RI removal overcame the perturbations caused by F508 deletion. Restriction of RI to a particular conformational state may ameliorate the impact of the disease-causing mutation.
PMCID: PMC4361937  PMID: 20561529
ABC transporters; CFTR; cystic fibrosis; ion channel; DMD simulations
3.  Allosteric modulation balances thermodynamic stability and restores function of ΔF508 CFTR 
Journal of molecular biology  2012;419(0):41-60.
Most cystic fibrosis is caused by a deletion of a single residue (F508) in CFTR that disrupts the folding and biosynthetic maturation of the ion channel protein. Progress towards understanding the underlying mechanisms and overcoming the defect remain incomplete. Here we show that the thermal instability of human ΔF508 CFTR channel activity evident in both cell-attached membrane patches and planar phospholipid bilayers is not observed in corresponding mutant CFTRs of several non-mammalian species. These more stable orthologs are distinguished from their mammalian counterparts by the substitution of proline residues at several key dynamic locations in the first nucleotide domain (NBD1), including the structurally diverse region (SDR), the gamma phosphate switch loop and the Regulatory Insertion (RI). Molecular Dynamic analyses revealed that addition of the prolines could reduce flexibility at these locations and increase the temperatures of unfolding transitions of ΔF508 NBD1 to that of the wild-type. Introduction of these prolines experimentally into full-length human ΔF508 CFTR together with the already recognized I539T suppressor mutation, also in the SDR, restored channel function and thermodynamic stability as well as its trafficking to and lifetime at the cell surface. Thus, while cellular manipulations that circumvent its culling by quality control systems leave ΔF508 CFTR dysfunctional at physiological temperature, restoration of the delicate balance between the dynamic protein’s inherent stability and channel activity returns a near-normal state.
PMCID: PMC3891843  PMID: 22406676
ABC transporters; CFTR; protein thermal stability; ion channel; DMD simulations
4.  Computational studies reveal phosphorylation dependent changes in the unstructured R domain of CFTR 
Journal of molecular biology  2008;378(5):1052-1063.
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a cAMP dependent chloride channel that is mutated in cystic fibrosis, an inherited disease of high morbidity and mortality. The phosphorylation of its ∼200 amino acid R domain by protein kinase A is obligatory for channel gating under normal conditions. The R domain contains more than ten PKA phosphorylation sites. No individual site is essential but phosphorylation of increasing numbers of sites enables progressively greater channel activity. In spite of numerous studies of the role of the R domain in CFTR regulation, its mechanism of action remains largely unknown. This is because neither its structure nor its interactions with other parts of CFTR have been completely elucidated. Studies have shown that the R domain lacks well-defined secondary structural elements and is an intrinsically disordered region of the channel protein. Here, we have analyzed the disorder pattern and employed computational methods to explore low energy conformations of the R domain. Specific disorder and secondary structure patterns detected suggest the presence of Molecular Recognition Elements (MoREs) that may mediate phosphorylation regulated intra- and inter-domain interactions. Simulations were performed to generate an ensemble of accessible R domain conformations. Although the calculated structures may represent more compact conformers than occur in vivo, their secondary structure propensities are consistent with predictions and published experimental data. Equilibrium simulations of a mimic of a phosphorylated R domain showed that it exhibited an increased radius of gyration. In one possible interpretation of these findings, by changing its size, the globally unstructured R domain may act as an entropic spring to perturb the packing of membrane-spanning sequences that constitute the ion permeability pathway and thereby activate channel gating.
PMCID: PMC2556564  PMID: 18423665
CFTR; R domain; phosphorylation; disordered protein; molecular dynamics
5.  Diminished Self-Chaperoning Activity of the ΔF508 Mutant of CFTR Results in Protein Misfolding 
PLoS Computational Biology  2008;4(2):e1000008.
The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF). Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1). Biochemical and cell biological studies show that the ΔF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the ΔF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-ΔF508 variants exhibited significantly higher folding probabilities than the original NBD1-ΔF508, thereby partially rescuing folding ability of the NBD1-ΔF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of NBD1-ΔF508 are essential information in correcting this pathogenic mutant.
Author Summary
Deletion of a single residue, phenylalanine at position 508, in the first nucleotide binding domain (NBD1) of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is present in approximately 90% of cystic fibrosis (CF) patients. Experiments show that this mutant protein exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant incorrect interactions of other domains. However, little is known about the direct effect of the Phe508 deletion on NBD1 folding. Here, using molecular dynamics simulations of NBD1-WT, NBD1-F508A, and NBD1-ΔF508, we show that the deletion of Phe508 indeed alters the kinetics of NBD1 folding. We also find that the intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. Moreover, we identified critical interactions not necessarily localized near position 508, such as Q493/P574 and F575/F587, to be significant structural elements influencing the kinetic difference between wild type and mutant NBD1. We propose that these observed alterations in folding kinetics of mutant NBD1 result in misassembly of the whole multi-domain protein, thereby causing its premature degradation.
PMCID: PMC2265529  PMID: 18463704

Results 1-5 (5)