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. 2010 Oct;19(10):1932-47.
doi: 10.1002/pro.480.

Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis

Chi Wang  1 , Irina Protasevich, Zhengrong Yang, Derek Seehausen, Timothy Skalak, Xun Zhao, Shane Atwell, J Spencer Emtage, Diana R Wetmore, Christie G Brouillette, John F Hunt
Affiliations

Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis

Chi Wang et al. Protein Sci. 2010 Oct.

Abstract

The lethal genetic disease cystic fibrosis is caused predominantly by in-frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide-binding domain (NBD1) of CFTR, which functions as an ATP-gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light-scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg-ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second-site mutations that increase the solubility of isolated F508del-NBD1 in vitro and suppress the trafficking defect of intact F508del-CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del-CFTR.

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Figures

Figure 1
Figure 1
Simultaneous monitoring of CD, trp fluorescence, and SLS during isothermal urea denaturation at 25°C of hNBD1-Δ(RI,RE) ± F508del. Protein samples (2 μM) in the absence or presence of 8 M urea were progressively mixed every 800 seconds to achieve the indicated concentration of urea, using an autotitrator for CD measurements or an equivalent kinetic protocol for fluorescence and SLS measurements. The buffer contained 150 mM NaCl, 0.5 mM MgCl2 10% glycerol, 10% ethylene glycol, 0.8 mM TCEP, 20 mM Na-HEPES, pH 7.5. Data for F508 and F508del constructs are shown in black and gray, respectively. The buffer used to purify and store the protein, which is catalytically inactive,– introduces 30 μM Mg-ATP. Additional Mg-ATP was added to the indicated final concentration. CD (top) was monitored at 230 nm instead of 222 nm to enable use of a higher concentration of Mg-ATP without interference from nucleotide absorbance. Intrinsic trp fluorescence (middle) was excited at 297 nm and monitored at 340 nm. SLS at 297 nm (bottom) was measured at a 90° angle simultaneously with trp fluorescence in a T-format fluorimeter (see Methods). SLS counts are background-subtracted for scattering from the protein-free 0 M urea buffer for measurements in the fluorimeter, so the ratio of the SLS signal at a given urea concentration to that at the start of the titration gives a rough estimate of weight-averaged aggregation state. Note, however, that this calculation underestimates the aggregation state at higher urea concentrations because it ignores the effect of urea in reducing protein contrast.
Figure 2
Figure 2
Kinetics of isothermal urea denaturation at 25°C of hNBD1-Δ(RI,RE) ± F508del. CD (top), trp fluorescence (middle), and SLS (bottom) signals were monitored using identical methods to Fig. 1 except that fluorescence emission at 340 nm and 90° SLS were measured in the Jasco spectropolarimeter using 230 nm incident light. At ∼100 seconds, 2 μM hNBD1-Δ(RI,RE) with (black) or without (gray) F508del was added from urea-free storage buffer to the Standard Stabilizing Buffer containing 30 μM Mg-ATP and the indicated concentration of urea.
Figure 3
Figure 3
Effects of Mg-ADP versus Mg-ATP on isothermal urea denaturation of hNBD1-Δ(RI,RE). Denaturation of the F508 (left) or F508del (right) domain was conducted at 25°C under conditions identical to Fig. 1 and monitored using CD (top) and trp fluorescence (bottom, with 230 nm excitation and 340 nm emission). Proteins were diluted into Standard Stabilizing Buffer containing 400 μM Mg-ADP (crosses), 400 μM Mg-ATP (gray diamonds), or no additional nucleotide (gray closed circles). Because purified proteins are stored in Standard Stabilizing Buffer containing Mg-ATP, they introduce 30 μM Mg-ATP into every experimental sample in addition to any nucleotide present in the measurement buffer. The final Mg-ATP concentration in each experiment is indicated in the legends.
Figure 4
Figure 4
Trafficking suppressor mutations stabilize hNBD1-Δ(RI,RE)-F508del against the initial unfolding transition. Urea denaturation at 25°C in the presence of 30 μM Mg-ATP was conducted under conditions identical to Fig. 1 and monitored using CD spectroscopy (top), trp fluorescence spectroscopy (middle, with 297 nm excitation and 340 nm emission), and SLS (bottom, at 297 nm). The denaturation of hNBD1-Δ(RI,RE)-F508del is compared to that of the same construct containing in addition the Teem suppressor triplet (left), V510D (center), or F494N/Q637R (right).
Figure 5
Figure 5
Schematic model of isothermal unfolding pathway of hNBD1. The crystal structure of hNBD1 (2PZE) is shown at the bottom either with (right) or without (left) bound Mg-ATP. The F1- like core subdomain is shown in orange, the ABCβ subdomain in green, and the ABCα subdomain in blue., A(C) represents the aggregation-prone intermediate produced by the initial chemical unfolding transition, while U(C) represents the fully denatured state produced by urea. The conformation and biophysical properties of the chemical unfolding intermediate A(C) are likely to be very similar to those of the thermal unfolding intermediate A(T) described in Ref. . Unfolding at high urea concentration may still proceed through A(C) as a kinetic intermediate, but it does not accumulate to appreciable concentration under these conditions. See Lewis et al. for a detailed description of the subdomain organization of hNBD1 and the stereochemical effects of the F508del mutation, the Teem suppressor mutation triplet, and the F494N/Q637R solubilizing mutations.
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