ABSTRACT
The folding and misfolding mechanism of multidomain proteins remains poorly understood. Although thermodynamic instability of the first nucleotide-binding domain (NBD1) of ΔF508 CFTR (cystic fibrosis transmembrane conductance regulator) partly accounts for the mutant channel degradation in the endoplasmic reticulum and is considered as a drug target in cystic fibrosis, the link between NBD1 and CFTR misfolding remains unclear. Here, we show that ΔF508 destabilizes NBD1 both thermodynamically and kinetically, but correction of either defect alone is insufficient to restore ΔF508 CFTR biogenesis. Instead, both ΔF508-NBD1 energetic and the NBD1-MSD2 (membrane-spanning domain 2) interface stabilization are required for wild-type-like folding, processing, and transport function, suggesting a synergistic role of NBD1 energetics and topology in CFTR-coupled domain assembly. Identification of distinct structural deficiencies may explain the limited success of ΔF508 CFTR corrector molecules and suggests structure-based combination corrector therapies. These results may serve as a framework for understanding the mechanism of interface mutation in multidomain membrane proteins.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Humans , Models, Molecular , Mutation , Protein Folding , Protein Structure, TertiaryABSTRACT
In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Binding Sites , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Humans , Hydrolysis , Kinetics , Molecular Sequence Data , Protein Structure, TertiaryABSTRACT
Understanding the residue-dependent effects of disease-phenotypic mutations in multi-spanning membrane proteins is an essential step toward the development of corrective therapies. As a systematic approach to further elucidate mutant-dependent mis-folding consequences, we prepared two libraries: one consisting of 20 helix-loop-helix ("hairpin") constructs derived from helices 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR) (residues 194-241) in which the CF-phenotypic position Val-232 was substituted individually to each of the 20 commonly-occurring amino acids; and a second library consisting of 20 single-stranded TM4 peptides (CFTR residues 221-241) similarly substituted at position 232. Both libraries were analyzed to measure mutant-dependent variations in mobility on SDS-PAGE; size and shape on size exclusion chromatography; retention times on reverse phase HPLC; and helical content by circular dichroism spectroscopy. Analysis of a scatter plot between TM3/4 hairpin and TM4 peptide retention times showed a strong correlation (r=0.94, p<0.05), with retention times largely a function of residue hydrophobicity. In contrast, while the hairpin library migrated over a significant range on SDS-PAGE, migration rates for TM4 hydrophobic residues at position 232 converged at a single value, suggesting that residue-dependent re-orientations of hairpin van der Waals interfaces may expose varying faces of the TM3 and/or TM4 helices to the SDS detergent. The overall results suggest that mutant-mediated variations are a principal determinant of tertiary interhelical folding interactions in membranes.
Subject(s)
Amino Acids/chemistry , Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Cystic Fibrosis/metabolism , Peptides/chemical synthesis , Amino Acid Sequence , Amino Acids/genetics , Amino Acids/metabolism , Chromatography, Gel , Chromatography, Reverse-Phase , Circular Dichroism , Cystic Fibrosis/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Electrophoresis, Polyacrylamide Gel , Genomic Library , Helix-Turn-Helix Motifs , Humans , Hydrophobic and Hydrophilic Interactions , Ion Transport , Models, Molecular , Molecular Sequence Data , Mutation , Peptides/genetics , Peptides/metabolism , Protein Folding , Protein Structure, Tertiary , Sodium Dodecyl Sulfate/chemistryABSTRACT
Developing a greater understanding of the function of the translocon-and the source of its selectivity for transmembrane helix insertion-are important steps toward deciphering the role of disease-causing mutations in membrane regions. To address these phenomena, we have prepared a library of helix-loop-helix ("hairpin") constructs derived from helices 3 and 4 of the first membrane domain of CFTR, in which position 232 was mutated individually to each of the 20 commonly-occurring amino acids. Using retention times on a reverse phase-HPLC C18 column to mimic the process of hairpin partitioning, we have quantitatively determined a hydropathy scale in the context of a bona fide membrane protein fragment that correlates to an in vivo hydropathy scale with r=-0.78-a value that rises to r=-0.92 when Asp and Glu are excluded due to protonation effects. Our results provide evidence that the translocon may act as a facilitator in the insertion selection process, effectively allowing the bilayer to "decide" through favorable non-polar solvation whether or not to allow a translocating helix to enter the membrane.
Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/chemistry , Mutation , Peptides/chemistry , Protein Structure, Secondary , Amino Acid Sequence , Chromatography, High Pressure Liquid/methods , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Humans , Hydrophobic and Hydrophilic Interactions , Lipid Bilayers/chemistry , Membrane Proteins/chemistry , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed/methods , Peptides/genetics , Protein FoldingABSTRACT
The [NiFe]-hydrogenase protein produced by many types of bacteria contains a dinuclear metal center that is required for enzymatic activity. Assembly of this metal cluster involves the coordinated activity of a number of helper proteins including the accessory protein, HypB, which is necessary for Ni(II) incorporation into the hydrogenase proteins. The HypB protein from Escherichia coli has two metal-binding sites, a high-affinity Ni(II) site that includes ligands from the N-terminal domain and a low-affinity metal site located within the C-terminal GTPase domain. In order to determine the physiological relevance of the two separate sites, hydrogenase production was assessed in strains of E. coli expressing wild-type HypB, the isolated GTPase domain, or site-directed mutants of metal-binding residues. These experiments demonstrate that both metal sites of HypB are critical for the maturation of the hydrogenase enzymes in E. coli. X-ray absorption spectroscopy of purified proteins was used to examine the detailed coordination spheres of each nickel-loaded site. In addition, because the low-affinity metal site has a stronger preference for Zn(II) than Ni(II), the ligands and geometry for this metal were also resolved. The results from these experiments are discussed in the context of a mechanism for Ni(II) insertion into the hydrogenase protein.
Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , GTP-Binding Proteins/chemistry , Metalloproteins/chemistry , Nickel/chemistry , Absorptiometry, Photon , Amino Acid Substitution , Binding Sites/physiology , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Hydrogenase/chemistry , Hydrogenase/genetics , Hydrogenase/metabolism , Ligands , Metalloproteins/genetics , Metalloproteins/metabolism , Mutagenesis, Site-Directed , Mutation, Missense , Nickel/metabolism , Protein Structure, Tertiary/physiology , Zinc/chemistry , Zinc/metabolismABSTRACT
N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.