CFTR (ABCC7), unique among ABC exporters as an ion channel, regulates ion and fluid transport in epithelial tissues. Loss of function due to mutations in the cftr gene causes cystic fibrosis (CF). The most common CF-causing mutation, the deletion of F508 (ΔF508) from the first nucleotide binding domain (NBD1) of CFTR, results in misfolding of the protein and clearance by cellular quality control systems. The ΔF508 mutation has two major impacts on CFTR: reduced thermal stability of NBD1 and disruption of its interface with membrane-spanning domains (MSDs). It is unknown if these two defects are independent and need to be targeted separately. To address this question we varied the extent of stabilization of NBD1 using different second site mutations and NBD1 binding small molecules with or without NBD1/MSD interface mutation. Combinations of different NBD1 changes had additive corrective effects on ΔF508 maturation that correlated with their ability to increase NBD1 thermostability. These effects were much larger than those caused by interface modification alone and accounted for most of the correction achieved by modifying both the domain and the interface. Thus, NBD1 stabilization plays a dominant role in overcoming the ΔF508 defect. Furthermore, the dual target approach resulted in a locked-open ion channel that was constitutively active in the absence of the normally obligatory dependence on phosphorylation by protein kinase A. Thus, simultaneous targeting of both the domain and the interface, as well as being non-essential for correction of biogenesis, may disrupt normal regulation of channel function.
Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate Abbreviations: CD, circular dichroism; CFTR, cystic fibrosis transmembrane conductance regulator; DSC, differential scanning calorimetry; ESD, extramembranous soluble domain; IMAC, immobilized metal-affinity chromatography; IMP, integral membrane protein; NBD1, first nucleotide binding domain; POPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; POPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; SLS, static light scattering; TM, transmembrane. Detergent abbreviations (i.e. short names) are listed in Table I. Additional Supporting Information may be found in the online version of this article. detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification.
Regulation of cardiac muscle function is initiated by binding of Ca 2+ to troponin C (cTnC) which induces a series of structural changes in cTnC and other thin filament proteins. These structural changes are further modulated by crossbridge formation and fine tuned by phosphorylation of cTnI. The objective of the present study is to use a new Förster Resonance Energy Transfer-based structural marker to distinguish structural and kinetic effects of Ca 2+ binding, crossbridge interaction and protein kinase A phosphorylation of cTnI on the conformational changes of the cTnC N-domain. The FRET-based structural marker was generated by attaching AEDANS to one cysteine of a doublecysteine mutant cTnC(13C/51C) as a FRET donor and attaching DDPM to the other cysteine as the acceptor. The doubly labeled cTnC mutant was reconstituted into the thin filament by adding cTnI, cTnT, tropomyosin and actin. Changes in the distance between Cys13 and Cys51 induced by Ca 2+ binding/dissociation were determined by FRET-sensed Ca 2+ titration and stopped-flow studies, and time-resolved fluorescence measurements. The results showed that the presence of both Ca 2+ and strong binding of myosin head to actin was required to achieve a fully open structure of the cTnC N-domain in regulated thin filaments. Equilibrium and stopped-flow studies suggested that strongly bound myosin head significantly increased the Ca 2+ sensitivity and changed the kinetics of the structural transition of the cTnC N-domain. PKA phosphorylation of cTnI impacted the Ca 2+ sensitivity and kinetics of the structural transition of the cTnC N-domain but showed no global structural effect on cTnC opening. These results provide an insight into the modulation mechanism of strong crossbridge and cTnI phosphorylation in cardiac thin filament activation/relaxation processes. Keywordsphosphorylation; cardiac troponin C; thin filament; FRET; Ca 2+ activation; kinetics Force development during cardiac muscle contraction is dependent upon the strong interactions between myosin and the actin filament. These interactions are governed by the regulatory
The key events in regulating cardiac muscle contraction involve Ca 2؉ binding to and release from cTnC (troponin C) and structural changes in cTnC and other thin filament proteins triggered by Ca 2؉ movement. Single mutations L29Q and G159D in human cTnC have been reported to associate with familial hypertrophic and dilated cardiomyopathy, respectively. We have examined the effects of these individual mutations on structural transitions in the regulatory N-domain of cTnC triggered by Ca 2؉ binding and dissociation. This study was carried out with a double mutant or triple mutants of cTnC, reconstituted into troponin with tryptophanless cTnI and cTnT. The double mutant, cTnC(L12W/N51C) labeled with 1,5-IAEDANS at Cys-51, served as a control to monitor Ca 2؉ -induced opening and closing of the N-domain by Förster resonance energy transfer (FRET). The triple mutants contained both L12W and N51C labeled with 1,5-IAEDANS, and either L29Q or G159D. Both mutations had minimal effects on the equilibrium distance between Trp-12 and Cys-51-AEDANS in the absence or presence of bound Ca 2؉ . L29Q had no effect on the closing rate of the N-domain triggered by release of Ca 2؉ , but reduced the Ca 2؉ -induced opening rate. G159D reduced both the closing and opening rates. Previous results showed that the closing rate of cTnC N-domain triggered by Ca 2؉ dissociation was substantially enhanced by PKA phosphorylation of cTnI. This rate enhancement was abolished by L29Q or G159D. These mutations alter the kinetics of structural transitions in the regulatory N-domain of cTnC that are involved in either activation (L29Q) or deactivation (G159D). Both mutations appear to be antagonistic toward phosphorylation signaling between cTnI and cTnC.Contraction of cardiac muscle is activated by the binding of Ca 2ϩ to the Ca 2ϩ -binding subunit troponin C (cTnC) 2 of the trimeric troponin complex. cTnC, the other two troponin subunits (troponin I (cTnI) and troponin T (cTnT)) and tropomyosin form the regulatory system of the contractile apparatus. These proteins are located in the thin filament. Contraction occurs when the myosin head in the thick filament interacts with actin causing the two filaments to slide past each other. The troponin complex in the thin filament regulates the actinmyosin interaction. Ca 2ϩ binding to the single site in the N-domain of cTnC initiates the activation process. cTnC has two globular regions, an N-terminal domain and a C-terminal domain. The N-domain has only one Ca 2ϩ binding site (site II) and the C-domain contains two binding site (binding sites III and IV). Ca 2ϩ binding to site II in the presence of cTnI induces an open conformation of the N-domain to expose a hydrophobic patch in the domain (1-3). Once exposed, the hydrophobic patch binds strongly with the regulatory region of cTnI. This strong interaction pulls cTnI away from actin and relieves the inhibition of actomyosin ATPase and muscle contraction. This regulation process is further fine-tuned with phosphorylation of cTnI serine 23 and serine 24...
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