Báo cáo khoa học: Deletion of Phe508 in the first nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator increases its affinity for the heat shock cognate 70 chaperone docx

Deletion of Phe508 in the first nucleotide-binding domain
of the cystic fibrosis transmembrane conductance
regulator increases its affinity for the heat shock cognate
70 chaperone
Toby S. Scott-Ward
1,2
and Margarida D. Amaral
1,2
1 Universidade de Lisboa, Faculdade de Cie
ˆ
ncias de Lisboa, BioFIG, Centre for Biodiversity, Functional and integrative Genomics, Portugal
2 Centro de Gene
´
tica Humana, Instituto Nacional de Sau
´
de Dr. Ricardo Jorge, Lisboa, Portugal
Keywords
CFTR-interacting proteins; correctors;
mechanism of disease; small molecules;
surface plasmon resonance
Correspondence
M. D. Amaral, EMBL – European Molecular
Biology Laboratory, Meyerhofstrasse 1,
69117 Heidelberg, Germany
Fax: +49 6221 387 8306
Tel: +49 6221 387 8199
E-mail:
(Received 3 August 2009, revised 29
September 2009, accepted 1 October
2009)
doi:10.1111/j.1742-4658.2009.07421.x

The primary cause of cystic fibrosis (CF), the most frequent fatal genetic
disease in Caucasians, is deletion of phenylalanine at position 508
(F508del), located in the first nucleotide-binding domain (NBD1) of the
CF transmembrane conductance regulator (CFTR) protein. F508del-CFTR
is recognized by the endoplasmic reticulum quality control (ERQC), which
targets it for proteasomal degradation, preventing this misfolded but par-
tially functional Cl
)
channel from reaching the cell membrane. We recently
proposed that the ERQC proceeds along several checkpoints, the first of
which, utilizing the chaperone heat shock cognate 70 (Hsc70), is the major
one directing F508del-CFTR for proteolysis. Therefore, a detailed charac-
terization of the interaction occurring between F508del-CFTR and Hsc70
is critical to clarify the mechanism that senses misfolded F508del-CFTR
in vivo. Here, we determined by surface plasmon resonance that: (a)
F508del-murine (m)NBD1 binds Hsc70 with higher affinity (K
D
, 2.6 nm)
than wild-type (wt) mNBD1 (13.9 nm); (b) ATP and ADP dramatically
reduce NBD1–Hsc70 binding; (c) the F508del mutation increases by
approximately six-fold the ATP concentration required to inhibit the
NBD1–Hsc70 interaction (IC
50
; wt-mNBD1, 19.7 lm ATP); and (d) the
small molecule CFTR corrector 4a (C4a), but not VRT-325 (V325; both
rescuing F508del-CFTR traffic), significantly reduces F508del-mNBD1
binding to Hsc70, by $ 30%. Altogether, these results provide a novel,
robust quantitative characterization of Hsc70–NBD1 binding, bringing
detailed insights into the molecular basis of CF. Moreover, we show how
this surface plasmon resonance assay helps to elucidate the mechanism of

action of small corrective molecules, demonstrating its potential to validate
additional therapeutic compounds for CF.
Structured digital abstract
l
MINT-7265886: mNBD1 (uniprotkb:P26361) binds (MI:0407)toHsc70 (uniprotkb:P19120)
by anti bait coimmunoprecipitation (
MI:0006)
Abbreviations
Ab, antibody; C4a, corrector 4a; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ER, endoplasmic reticulum;
ERQC, endoplasmic reticulum quality control; F508del, deletion of phenylalanine residue at position 508; h, human; Hsc70, heat shock
cognate 70; Hsp70, heat shock protein 70; I172, inhibitor CFTR
inh-172
; LA, apo-a-lactalbumin; m, murine; NBD1, first nucleotide-binding
domain; Red-LA, reduced apo-a-lactalbumin; SEM, standard error of the mean; SPR, surface plasmon resonance; V325, corrector VRT-325;
wt, wild-type.
FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS 7097
Introduction
Cystic fibrosis (CF) is a life-threatening genetic disease
caused by malfunction of CF transmembrane conduc-
tance regulator (CFTR) [1], a Cl
)
channel that plays a
central role in transepithelial ion transport [2]. A single
amino acid deletion, of phenylalanine 508 (F508del), in
the first nucleotide-binding domain (NBD1) of CFTR
accounts for approximately 70% of CF chromosomes
worldwide [2]. This mutation prevents the correct fold-
ing of CFTR, thus causing its retention by the endo-
plasmic reticulum (ER) quality control (ERQC) as an
immature intermediate that is rapidly degraded by the

ubiquitin–proteasome pathway [3]. Like that of other
secretory proteins, the proper folding of CFTR is
highly dependent on molecular chaperones, such as
heat shock protein 70 (Hsp70) and its constitutively
expressed homologue, heat shock cognate 70 (Hsc70)
[4,5]. Hsc70 and Hsp70 are single-polypeptide cytoplas-
mic proteins composed of an N-terminal ATPase
domain, a substrate-binding region, and a C-terminal
15 kDa ‘lid’ regulating binding affinity for ‘client’ pro-
teins. These chaperones bind short (approximately
seven residues) hydrophobic regions exposed in either
mutant or immature wild-type (wt) client proteins
[6,7]. The binding of Hsc70 and Hsp70 to substrates is
tightly coupled to cycles of ATP binding and hydroly-
sis followed by ADP release [8].
In our previously proposed model of the ERQC, the
folding states of CFTR are assessed within the ER at
multiple checkpoints [9,10], the first of these involving
the Hsc70 ⁄ Hsp70 machinery, the most critical one for
F508del-CFTR degradation. Biochemical data suggest
that both immature wt-CFTR and F508del-CFTR
have Hsc70-binding sites, but the Hsc70 association
with F508del-CFTR is prolonged [11]. However, there
is evidence that critical interaction sites for Hsc70
reside within the first half of CFTR, in particular
NBD1 [12,13]. Indeed, analysis of the human (h) and
murine (m) CFTR-NBD1 sequences with the online
program limbo ( performed
here predicts that they both contain at least three hep-
apeptide Hsp70-binding sites, with a high degree of

certainty (99%). Biochemical analyses of purified
NBD1 indicate that F508del reduces domain stability,
and hence promotes aggregation [14,15]. Molecular
dynamics modelling studies suggest that F508del-
NBD1 has more conformational freedom than the
wild-type (wt), thus exposing its hydrophobic interior
to the solution and impairing its interdomain contacts
[16,17]. Collectively, these data implicate NBD1 as the
most probable site for the interaction of CFTR with
Hsc70.
Several small molecules that improve CFTR folding,
biogenesis and function were recently identified in
high-throughput screens [18], such as corrector 4a
(C4a) and corrector VRT-325 (V325), which promote
trafficking of F508del-CFTR to the plasma membrane
[19,20]. However, the exact mechanism of action of
these compounds and their putative binding sites on
CFTR remain undefined.
Here, we used a novel approach, surface plasmon
resonance (SPR) [21], to quantify the interaction occur-
ring between F508del-CFTR and Hsc70, versus that of
the chaperone with wt-NBD1. Our data show that
F508del-mNBD1 binds Hsc70 with approximately five-
fold higher affinity than wt-mNBD1, and that both
ATP and ADP dramatically reduce NBD1–Hsc70
binding. Moreover, we also show that, in the presence
of a small molecule known to rescue the traffic of full-
length F508del-mCFTR to the plasma membrane, the
strength of the F508del-mNBD1–Hsc70 interaction is
reduced by $ 30%.

Results
Interaction of purified NBD1 and Hsc70 under the
SPR conditions
To confirm whether NBD1 binds specifically to Hsc70
under conditions that would subsequently be used in
SPR interaction studies, we immunoprecipitated
Hsc70–NBD1 complexes in vitro from a mixture of the
two purified proteins. Electrophoresis of the purified
F508del-mNBD1 and bovine Hsc70 prior to immuno-
precipitation (Fig. 1A) resolved single 30 and 73 kDa
l
MINT-7265906, MINT-7265964, MINT-7265981, MINT-7265951: mNBD1 (uni-
protkb:
P26361) binds (MI:0407)toHsc70 (uniprotkb:P19120)bysurface plasmon resonance
(
MI:0107)
l
MINT-7265924, MINT-7265939: hNBD1 (uniprotkb:P13569) binds ( MI:0407)toHsc70 (uni-
protkb:
P19120)bysurface plasmon resonance (MI:0107)
l
MINT-7265996: Hsc70 (uniprotkb:P19120) binds (MI:0407)toApo-alpha-lactalbumin (uni-
protkb:
P00711)bysurface plasmon resonance (MI:0107)
Interaction of Hsc70 with F508del-NBD1 T. S. Scott-Ward and M. D. Amaral
7098 FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS
protein bands (left lane, both panels), which were
confirmed to be specific by the parallel western blot
analysis with L12B4 [antibody (Ab) against NBD1 of
CFTR] and SPA-815 (Ab against Hsc70), respectively

(right lane, both panels). The data in Fig. 1B show
that purified Hsc70 is present in immunoprecipitated
complexes of either wt-mNBD1 (lanes 2, 3 and 5) or
F508del-mNBD1 (lane 4) incubated in SPR flow
buffer. Moreover, the absence of Hsc70 in immunopre-
cipitates when BSA (Fig. 1B, lane 1) was used instead
of NBD1, or in the absence of L12B4 (lane 7), demon-
strates that the binding of Hsc70 does not occur with
all protein substrates. Increasing the concentration of
wt-hNBD1 by five-fold (Fig. 1B, lane 3) did not
noticeably increase the amount of NBD1 or Hsc70
recovered (compare with lane 2), suggesting that the
L12B4-coated beads were already saturated with
NBD1. Addition of MgATP (2 mm) markedly reduced
the binding of Hsc70 to wt-mNBD1 (Fig. 1B, lane 6),
consistent with the results of previous studies with
other Hsc70 substrates [8].
Analysis of stability of NBD1 under the SPR
interaction conditions
We used intrinsic tryptophan fluorescence spectroscopy
to assess the structure and stability of NBD1 under
assay conditions that would subsequently be used in
SPR interaction studies. A comparison of emission
spectra obtained before (0 min) and after (10 min)
incubation (Fig. 2A) reveals that there was only a
small decrease in the overall intensity of the fluores-
cence emitted from F508del-mNBD1 and no shift in
the peak wavelength. Furthermore, the intensity of
fluorescence emitted at 328 nm and 343 nm from
diluted F508del-mNBD1 (Fig. 2B) displayed a minor

initial decrease, but was essentially stable during the
10 min incubation period (similar data were obtained
with wt-mNBD1; not shown; n = 2). Previous studies
indicate that, as compared with their folded forms,
denatured wt-hNBD1 and F508del-hNBD1 display a
dramatically reduced overall intensity of emitted fluo-
rescence, and peak values are red-shifted to higher
wavelengths [15]. Hence, our data suggest that: (a) the
wt-mNBD1 and F508del-mNBD1 used in this study
have a structure consistent with a native fold [15,22];
and (b) the domain is stable under the conditions used
in SPR experiments.
Assessment of optimal conditions for studying
the Hsc70–NBD1 interaction by SPR
Before determining the effect of the F508del mutation
on the NBD1–Hsc70 interaction by SPR, we performed
0
5
6
0 200 400 600 800
300 340 380 420
0
2
4
6
Fluorescence (units)
Buffer
0 min
10 min
Wavelength (nm) Time (s)

Fluorescence (units)
343 nm
328 nm
F508del-mF508del-m
343
328
A
B
Fig. 2. Spectroscopic analyses show that NBD1 is stable under the
conditions used in SPR studies. (A) Fluorescence emission spectra
(300–400 nm) of F508del-mNBD1 (1 l
M) before (0 min) and after
(10 min) incubation in buffer at 25 °C (excitation wavelength of
295 nm). (B) Change in intrinsic tryptophan fluorescence emitted at
328 and 343 nm from F508del-mNBD1 (1 l
M) during incubation in
buffer (25 °C). Data are corrected for buffer fluorescence. Similar
results were obtained with other NBD1 preparations (F508del-
mNBD1, n = 3; wt-mNBD1, not shown, n = 2).
kDa P WB
47
83
25
16
Hsc70
37
50
75
25
kDa PWB

NBD1
1
Hsc70
23 54 6 7 8 IB:
IP: NBD1
Hsc70 (µ
M)
NBD1 (µ
M)
MgATP (m
M)
1
–
–
1
1
–
1
5
–
1
1
–
1
1
–
1
1
2
1

1
––
–
–
BSA (µ
M)
33 3 3 3 3
––
NBD1
A
B
Fig. 1. In vitro interaction of NBD1 with Hsc70 confirmed by immu-
noprecipitation (IP) under conditions used in SPR studies.
(A) SDS ⁄ PAGE (P) and western blot (WB) analyses of purified
F508del-mNBD1 (F508del-NBD1; 5 lg) and bovine Hsc70 (1 lg)
with L12B4 and SPA-815, respectively, to confirm their specificity
(see Experimental procedures). (B) In vitro analysis of the inter-
action of Hsc70 with NBD1 by immunoprecipitation under condi-
tions equivalent to those used for SPR. Protein G beads coated
with L12B4–NBD1 complexes were incubated with purified Hsc70
(1 l
M; 10 min at 25 °C) and analysed by western blot using either
SPA-815 or L12B4 (see Experimental procedures). Lanes: 1, BSA
(3 l
M; without NBD1); 2, wt-hNBD1 (1 lM); 3, wt-hNBD1 (5 lM); 4,
F508del-mNBD1 (1 l
M); 5, wt-mNBD1; 6, wt-mNBD1 with MgATP
(2 m
M); 7, wt-hNBD1 with beads (without L12B4); 8, L12B4-coated
beads only (without NBD1 and Hsc70). Similar results were

obtained in three experiments (n = 3).
T. S. Scott-Ward and M. D. Amaral Interaction of Hsc70 with F508del-NBD1
FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS 7099
a series of control experiments to determine the bind-
ing specificity and optimal assay conditions. To this
end, wt-hNBD1 and BSA were immobilized on sensor
chip surfaces, and the binding of L12B4 was investi-
gated. The data in Fig. 3A show that there was a mea-
surable and time-dependent interaction of Ab (15 nm)
with wt-hNBD1, but not with BSA-coated chip
surfaces ( n = 3–5). These data indicate that even at a
low nanomolar concentration, there was potent
binding of L12B4 to immobilized NBD1 (23.7 ±
0.9 pmolÆnmol
)1
; n = 3), which is characteristic of
Ab–antigen interactions [23]. The data also indicate
that we can use SPR to assay the binding of interac-
ting proteins to NBD1. To further optimize the condi-
tions for investigating the NBD1–Hsc70 interaction,
we tested sensor chips coated with NBD1 (on-chip
NBD1). Hsc70 (0.3 lm) bound specifically to sensor
surfaces coated with wt-mNBD1, F508del-mNBD1,
and wt-hNBD1 (Fig. 3B; F508del-mNBD1, 2.5 ±
0.2 pmolÆnmol
)1
; wt-mNBD1, 2.9 ± 0.1 pmolÆnmol
)1
;
wt-hNBD1, 3.8 ± 0.2 pmolÆnmol

)1
; n = 3), with mini-
mal adsorption of Hsc70 onto BSA-coated surfaces
(< 0.2 pmolÆnmol
)1
). Hence, under these conditions,
the magnitude of Hsc70 binding to wt-mNBD1 was
substantially (approximately six-fold) lower than that
observed for L12B4.
Then, we tested the reverse situation by immobiliz-
ing Hsc70 on CM5 sensor chips (on-chip Hsc70), to
investigate its ability to bind folded or unfolded pro-
teins. The immobilized chaperone was found to
potently bind reduced apo-a-lactalbumin (Red-LA)
(Fig. 3C; 10 lm; 29.1 ± 3.8 pmolÆnmol
)1
; n = 3), an
Hsc70 substrate [13], whereas minimal binding was
detected with the nonreduced, folded form of the pro-
tein (LA) (10 lm; 1.1 ± 0.7 pmolÆnmol
)1
; n = 3). As
expected, virtually no binding of BSA (15 lm)to
Hsc70-coated surfaces could be detected (< 0.1 pmo-
lÆnmol
)1
; n = 20) under these conditions. These data
confirm that Hsc70 binding detected by SPR was only
significantly detected for unfolded protein substrates.
Interestingly, when Red-LA was applied in the pres-

ence of ATP (100 lm), the level of binding was
reduced to $ 80% of that under nucleotide-free condi-
tions (23.9 ± 1.9 pmolÆnmol
)1
; n = 3).
Next, we determined Hsc70 binding of all three NBD1
variants (0.5 lm; Fig. 3D), and found that all bound
potently to immobilized Hsc70 (F508del-mNBD1,
20.9 ± 2.0 pmolÆnmol
)1
; wt-mNBD1, 17.8 ± 2.3
pmolÆnmol
)1
; wt-hNBD1, 27.3 ± 3.8 pmolÆnmol
)1
; n =
4). Under these conditions, wt-hNBD1 displayed the
highest level of Hsc70 binding, followed by
F508del-mNBD1. Moreover, the addition of MgATP
0 500 1000
0
20
40
0
500 1000
0
3
6
B
Binding (pmol·nmol

–1
)
BSA
wt-h
wt-m
F508del-m
A
Binding (pmol·nmol
–1
)
BSA
wt-h
Applied: L12B4 Ab
Applied: Hsc70
0 500 1000
0
20
40
0 500 1000
0
20
40
D
BSA
Binding (pmol·nmol
–1
)
F508del-m
wt-h
wt-m

wt-m + ATP
Time (s)
C
Time (s)
Binding (pmol·nmol
–1
)
BSA
Red-LA
LA
On-Chip: Hsc70
On-Chip: Hsc70
Red-LA
+ATP
Time (s)
Time (s)
On-chip:
On-chip:
Applied:
Applied:
Fig. 3. Hsc70 binds specifically to NBD1
and control proteins in SPR studies. Charac-
terization of the interaction of: (A) applied
L12B4 (15 n
M) with covalently immobilized
(‘on-chip’) wt-hNBD1; and (B) applied Hsc70
(0.3 l
M) with on-chip wt-hNBD1, wt-
mNBD1, or F508del-mNBD1 (see Experi-
mental procedures). Hsc70 and L12B4

displayed minimal interaction with on-chip
BSA [
, (A) and (B)]. (C, D) The interaction
of applied Red-LA or LA (10 l
M) (C) and
wt-hNBD1, wt-mMBD1 or F508del-mNBD1
(0.5 l
M) (D) with on-chip Hsc70 (On-Chip
Hsc70; see Experimental procedures). BSA
(15 l
M) showed minimal interaction with
on-chip Hsc70 [–, (C) and (D), n = 15].
Periods of protein application (and associa-
tion) are indicated by the solid bars. After
protein application, dissociation was
measured by injecting flow buffer over the
protein-coated surface. Similar results were
obtained in additional experiments (n =3or
n = 4).
Interaction of Hsc70 with F508del-NBD1 T. S. Scott-Ward and M. D. Amaral
7100 FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS
(100 lm) dramatically reduced the binding of wt-
mNBD1 to Hsc70 (Fig. 1A; 3.7 ± 1.3 pmolÆnmol
)1
;
n = 3). These data show that we can use SPR to accu-
rately measure the specific binding of NBD1 to immobi-
lized Hsc70. The substantially higher binding of NBD1
to immobilized Hsc70 over that of Hsc70 chaperone
binding to immobilised NBD1 can be attributed to the

ability of Hsc70 to better survive the acidic, low-salt SPR
immobilization conditions than NBD1 [24]. Hence, in
subsequent experiments, we decided to immobilize Hsc70
and quantify the binding of wt- and F508del-mNBD1.
Assessment of the impact of F508del on the
NBD1–Hsc70 interaction
To determine the strength of the NBD1–Hsc70 inter-
action, we thus immobilized the chaperone and
quantified the binding of increasing concentrations of
wt-mNBD1 and F508del-mNBD1 (Fig. 4A,B). Using
kinetic analyses, we determined that rates of associa-
tion (k
a
) and dissociation (Fig. 4C, k
d
) of wt-mNBD1
and Hsc70 are extremely slow [k
a
, 3660 ± 596 m
)1
Æs
)1
;
k
d
, (5.2 ± 1.4) · 10
)5
s
)1
; n = 3]. In contrast,

F508del-mNBD1 bound Hsc70 with a comparable
association rate, but had a five-fold lower dissociation
rate [k
a
, 4030 ± 655 m
)1
Æs
)1
; k
d
, (1.0 ± 0.2) ·
10
)5
s
)1
; n = 3). Using these constants, we calculated
that Hsc70 bound F508del-mNBD1 with five-fold
higher affinity than wt-mNBD1 [dissociation constant
(K
D
): F508del-mNBD1, 2.6 ± 0.5 nm; wt-mNBD1,
13.9 ± 0.8 nm; n = 3]. These data indicate that when
Phe508 is deleted, there is a significant increase in the
real-time affinity of mNBD1 for Hsc70 (P < 0.01).
Analysis of the dose–response data (Fig. 4D) revealed
that the maximum amount of F508del-mNBD1 bound
to Hsc70 at s aturating concentration appears to be lower
than that of wt-mNBD1 (B
max
app

: F508del-mNBD1,
73.1 ± 3.9 pmolÆnmol
)1
; wt-mNBD1, 106.6 ± 6.6 pmolÆ
nmol
)1
; n = 3). Moreover, apparent dissociation con-
stants determined directly from the dose–response data
(Eqn 1) indicate that F508del-mNBD1 bound Hsc70
with at least three-fold higher affinity than wt-mNBD1
(K
D
app
: F508del-mNBD1, 23.2 ± 1.7 nm; wt-mNBD1,
70.2 ± 4.7 nm; n = 3).
Analysis of the effect of adenine nucleotides on
the binding of NBD1 to Hsc70
To further explore the effect of adenine nucleotides on
the NBD1–Hsc70 interaction, we quantified the effect
of ATP and ADP on the binding of wt-mNBD1 and
F508del-mNBD1 to immobilized Hsc70. As shown by
the solid lines in Fig. 5A,B, increasing concentrations
of ATP caused a dramatic reduction in the binding of
1000 1500
96
98
100
102
1000 1500 0.01 0.1 1.0
0

25
50
75
100
0
50
100
0 1000 2000 0 1000 2000
0
50
100
Time (s)
Binding (pmol·nmol
–1
)
NBD1 (µM)
Binding (pmol·nmol
–1
)Binding (pmol·nmol
–1
)
Time (s)
[NBD1] (µ
M)
wt-m
F508del-m
0.02
0.1
0.2
0.05

0.5
wt-m
F508del-m
NBD1 (µ
M)
0.02
0.1
0.2
0.05
0.5
Normalised binding
F508del-m
wt-m
F508del-m
wt-m
0.1 µ
M
0.2 µM
Time (s)
A
B
C
D
Fig. 4. F508del increases the affinity of
NBD1 for on-chip Hsc70. (A, B) The inter-
action of increasing concentrations of
(A) wt-mNBD1 and (B) F508del-mNBD1 with
on-chip Hsc70. (C) Dissociation of
Hsc70-bound wt-mNBD1 and F508
del-mNBD1 (0.1 and 0.2 l

M) shown on a
highly expanded scale. Binding was
normalized independently for each dissocia-
tion curve to an initial maximum value (100)
at the start of each dissociation phase
(920 s). First-order fits (
) to the data
denote relative dissociation rates. (D) The
change in amount of NBD1 bound by
on-chip Hsc70 at equilibrium with increasing
concentrations of wt-mNBD1 (d) and
F508del-mNBD1 (s; mean ± SEM; n = 3).
Other details as in legend to Fig. 3.
T. S. Scott-Ward and M. D. Amaral Interaction of Hsc70 with F508del-NBD1
FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS 7101
wt-mNBD1 and F508del-mNBD1 (0.5 lm) to Hsc70.
In contrast, the introduction of a control molecule,
NADP (500 lm), did not significantly reduce the bind-
ing of either wt-mNBD1 or F508del-mNBD1 (0.5 lm)
to immobilized Hsc70 (Fig. 5C; wt-mNBD1, P = 0.45;
F508del-mNBD1, P = 0.46; n = 3). Interestingly, the
binding of wt-mNBD1 and F508del-mNBD1 to Hsc70
was also reduced by the addition of ADP (500 lm),
although, at this concentration, the degree of inhibi-
tion was less than for ATP (P = 0.01; n = 3). Fig-
ure 5C, displaying a comparison of the summarized
data for wt-mNBD1 and F508del-mNBD1, shows that
the interaction of wt-mNBD1 with Hsc70 was most
potently inhibited by increasing ATP concentration
(IC

50
, 19.7 ± 2.1 lm; n = 3). For F508del-mNBD1,
the ability of ATP to inhibit the Hsc70 interaction was
significantly reduced (IC
50
, 111 ± 4.8 lm; P = 0.001;
n = 3). Collectively, our data suggest that the NBD1–
Hsc70 interaction is inversely dependent on the ATP
concentration and that F508del stabilizes this
interaction.
The effect of small molecule compounds on the
NBD1–Hsc70 interaction
Small molecules recently identified in high-throughput
screens have been proposed to affect CFTR-NBD1
conformation [25,26]. Here, we tested by SPR whether
and how these molecules affect the NBD1–Hsc70
interaction. The data in Fig. 6A show there was a signifi-
cant reduction in the Hsc70 binding of F508del-mNBD1
(1 lm) upon acute application of C4a, which was not
Con
C4a
V325
Con
C4a
V325
0
25
50
04080
0

25
50
0
500 1000
0
25
50
[Compound] (µ
M)
Time (s)
Binding (pmol·nmol
–1
)
C4a
Con
Binding (pmol·nmol
–1
)
Binding (pmol·nmol
–1
)
I172
Incubation
Incubation
+ ATP
C4a
V325
I172
*
*

V325
F508del-m (1.0 µ
M) F508del-m (1.0 µM)
ABC
Fig. 6. C4a alters the binding of F508del-NBD1 to on-chip Hsc70. (A) Interaction of F508del-mNBD1 (1 lM) with on-chip Hsc70 in the
absence (
, Con) and presence (50 lM) of: C4a (–), V325 ( ), or I172 (gray ). (B) Change in amount of F508del-mNBD1 bound by on-chip
Hsc70 in the presence of I172 (
;50lM ) or increasing concentrations of V325 (s ) and C4a (d; mean ± SEM; n = 3). (C) Binding of
F508del-mNBD1 to Hsc70 ( h; Con ) following incubation of NBD1 (1 l
M) with C4a ( ) or V325 ( ;50lM ) for 30 min at 16 °C in SPR buf-
fer alone (Incubation) or in SPR buffer plus 100 l
M MgATP (Incubation + ATP; mean ± SEM; n = 3). Other details as in the legend to Fig. 3.
0
10
20
30
0 500 1000 0 500 1000
0
10
20
30
1 10 100 1000
0
10
20
30
103
ATP (µ
M)

23
203
Binding (pmol·nmol
–1
)
Binding (pmol·nmol
–1
)
Binding (pmol·nmol
–1
)
Time (s) Time (s) [Adenine nucleotide] (µM)
wt-m
F508del-m
3
ATP (µ
M)
53
503
103
23
203
3
53
wt-m F508del-m
ATP
ADP
NADP
ABC
Fig. 5. Adenine nucleotides reduce the binding of NBD1 to on-chip Hsc70. The interaction of (A) wt-mNBD1 (0.5 lM) and (B) F508del-

mNBD1 (0.5 l
M) with on-chip Hsc70 in the presence of NADP (– –, 500 lM), MgADP ( , 500 lM) or increasing concentrations of MgATP (–).
(C) The change in binding of wt-mNBD1 (d) and F508del-mNBD1 (s) to on-chip Hsc70 in the presence of NADP (D,
; 500 lM), MgADP
(j,h; 500 l
M), or increasing concentrations of MgATP (d,s) (mean ± SEM; n = 3). Other details as in the legends to Figs 3 and 4.
Interaction of Hsc70 with F508del-NBD1 T. S. Scott-Ward and M. D. Amaral
7102 FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS
observed for V325 or inhibitor CFTR
inh-172
(I172;
n = 3). We quantified the Hsc70 binding of
F508del-mNBD1 in the presence of increasing concen-
trations of C4a and V325 (Fig. 6B). The data show that
only at the higher concentration tested (50 lm) could
C4a, but not V325 or I172, significantly reduce the
Hsc70 binding of F508del-mNBD1, by $ 30% (control,
32.2 ± 2.2 pmolÆnmol
)1
; C4a, 23.4 ± 1.9 pmolÆnmol
)1
;
P = 0.04; n = 3). To better understand the mechanism
of action of these correctors, we also preincubated
F508del-NBD1 with either C4a or V325 (50 lm, 30 min,
16 °C) prior to application, and then quantified NBD1
binding to Hsc70. As shown in Fig. 6C, again only C4a,
and not V325, significantly reduced the binding of
F508del-mNBD1 to Hsc70 (C4a, P = 0.04; V325,
P = 0.58; n = 3). Comparison of the data with those in

Fig. 6B indicates that the magnitude of inhibition
($ 30%) was comparable to that observed with acute
compound application. However, following preincuba-
tion of NBD1 with C4a or V325 at increased MgATP
concentration (106 lm), we observed no effect of these
corrector compounds on the NBD1–Hsc70 interaction
(Fig. 6C; P = 0.74–0.95; n = 3).
Discussion
Various components of the ERQC recognize aberrant
conformations of secretory proteins and target them
for proteasomal degradation so as to avoid clogging of
the secretory pathway. In the case of the CFTR protein
bearing F508del, the major CF-causing mutation, it has
been shown that the molecular chaperone Hsc70 plays
a major role in this disposal mechanism [11,12]. In the
present study, we have used an SPR approach to inves-
tigate how deleting F508 from NBD1 of mCFTR alters
the interaction of the domain with the molecular chap-
erone Hsc70, and whether small molecule correctors of
CFTR folding can affect this critical interaction.
Both wt-NBD1 and F508del-NBD1 bind Hsc70
Our data indicate that Hsc70 can interact with both
wt-NBD1 and F508del-NBD1, in agreement with pre-
vious studies [12] showing that both wt-CFTR and
F508del-CFTR can associate with this chaperone via
NBD1. The strength of NBD1–Hsc70 binding determined
here by SPR is substantially higher (low nanomolar K
D
)
than the majority of previous non-SPR assessments of

Hsc70 ⁄ Hsp70–substrate interactions (nanomolar to
micromolar K
D
values [27–29]). This is likely to reflect
the fact that we have used a potent Hsc70 substrate
(isolated NBD1 of CFTR). Moreover, the conditions
under which the experiments were performed favour
high-affinity interaction of Hsc70 with a client protein.
Nonetheless, although we could detect interaction of
Hsc70 with Red-LA, this was not the case for LA. In
addition, previous measurements of the affinity of
Hsc70–substrate binding have almost exclusively been
the result of peptide, and not protein, binding by
Hsp70 ⁄ Hsc70 [6–8,30].
Previous studies have used SPR to quantify the
binding of nonchaperone proteins (the cytoskeletal,
PDZ-anchoring protein NHERF ⁄ EBP50 and the
related, PDZ-based scaffold protein Shank2) to C-ter-
minal CFTR peptides and, in one case (the Ca
2+
⁄
lipid-dependent annexin A5), isolated NBD1. Although
the proteins studied are unrelated to Hsc70 ⁄ Hsp70,
they were also found to bind CFTR fragments with
high affinities (K
D
; C-terminus–EBP50, 22 nm; C-ter-
minus–Shank2, 56 nm [31]; NBD1–annexin A5, 4 nm
[32]). However, in contrast to our findings, Trouve
et al. [32] found that ATP increased the binding of

NBD1 to annexin A5, and that deleting Phe508 had
no effect on the annexin A5–NBD1 interaction. Inter-
estingly, they demonstrated that CPX, a potential
NBD1 small molecule ligand [33], inhibited binding.
Overall, the data suggest that Hsc70 interacts with
NBD1 at alternative sites to annexin A5 and with
different characteristics.
The enhanced binding of Hsc70 to wt-hNBD1 rela-
tive to wt-mNBD1 that we find here may be due to
variations in amino acids between these forms of
NBD1 that help to stabilize this domain against inter-
action with Hsc70. Interestingly, when some of the
variant residues in NBD1 of F508del-mCFTR are
substituted for residues at corresponding positions in
F508del-hCFTR, they act as revertants of the folding
and trafficking defects [14]. This is the case for Thr539
(Ile539 in humans), a so-called revertant of F508del-
hCFTR [34], and also Ser429 (Phe429 in humans),
recently shown to contribute to rescue of the traffick-
ing defect of F508del-hCFTR [35]. The presence of
these ‘profolding’ residues in mNBD1 is a probable
explanation for the recently reported attenuated
processing ⁄ trafficking defect of F508del-mCFTR in
comparison with F508del-hCFTR [36].
F508del increases the affinity of CFTR NBD1 for
Hsc70
Our data demonstrate that deleting Phe508 from
mNBD1 increases five-fold the affinity of the domain
for the Hsc70 chaperone. However, our data also indi-
cate that Hsc70 binds $ 30% more wt-mNBD1 than

F508del-mNBD1 at saturation. One explanation for
this reduction is that the deletion of Phe508 increases
T. S. Scott-Ward and M. D. Amaral Interaction of Hsc70 with F508del-NBD1
FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS 7103
the affinity of NBD1 for not only Hsc70 but also itself.
Indeed, as F508del-NBD1 is more prone to aggregation
than wt-NBD1 [14,15], the aggregated, mutant form
would thus be incapable of binding to Hsc70. This
would effectively reduce the average concentration of
the free monomeric form of F508del-mNBD1 available
to bind Hsc70 and hence the maximum binding.
In a recent study, it was found that, in vivo, both
wt-CFTR and F508del-CFTR bind equal amounts of
the Hsp70 chaperone [37]. Hence, the differences in
binding caused by the F508del mutation observed here
might be due to the in vitro nature of the SPR
approach versus in vivo complexity. Within the cell,
additional factors influence Hsc70–CFTR binding, e.g.
cobinding and competition of Hsc70 cochaperones,
such as the E3-ubiquitin ligase CHIP, which promotes
the fast dissociation of the Hsc70–CFTR complex,
targeting CFTR for proteasomal degradation [38–40].
Moreover, some Hsc70 ⁄ Hsp70 interaction sites are
likely to occur only in the context of the native confor-
mation of the full-length protein [5], as proposed in a
recent structural model of CFTR, where the surface of
NBD1 containing Phe508 mediates an interdomain
contact with intracellular cytoplasmic loop 4 of mem-
brane-spanning domain 2 [16]. Nevertheless, our find-
ings are consistent with the reported prolonged

association of Hsc70 with F508del-CFTR relative to
that with wt-CFTR [11], which appears to constitute
the first ERQC checkpoint occurring in vivo [9,12].
Our data reported here thus suggest that the absence
of Phe508 increases the accessibility of Hsc70 to one
or more binding sites on NBD1.
Binding sites on NBD1 promoting Hsc70
interaction
A critical issue in this field is the location of the func-
tionally important Hsc70-binding site(s) on NBD1; in
particular, whether removal of Phe508 creates a novel
Hsc70-binding site in NBD1. Although it is known
that Hsc70 and Hsp70 bind short, hydrophobic peptide
pockets exposed on substrate proteins, the exact pri-
mary sequence of these peptides is variable [6,30].
Accordingly, the NBD1 proteins employed in this
study (Thr389–Gly673) contain many of these short,
hydrophobic sequences, including the region around
Phe508, constituting potential Hsc70-binding sites.
Analysis of hNBD1 and mNBD1 sequences with the
limbo program, which predicts likely binding sites of
the Hsp70 ⁄ Hsc70 homologue, DnaK, identified three
novel regions (Ser466–Leu472, Leu568–Pro574, and
Asp614–Gln621), all three of which are distant from
Phe508. Qu and Thomas [14] localized a putative
Hsc70-binding site to Gly545–Ala561 [13], a hydropho-
bic pocket that is partially exposed in the crystal struc-
ture of NBD1 and that is also distant from Phe508.
However, the residue limits of the NBD1 used in this
study (Gly404–Ser589) were different from those used

in the present study.
Nevertheless, even if no additional binding sites
occur in F508del-mNDB1, they may be more accessi-
ble to Hsc70, in comparison with wt-mCFTR. Sup-
porting this notion, biochemical analyses of purified
NBD1 indicate that F508del promotes aggregation, a
property known to result from exposure of hydro-
phobic residues [14,15]. Moreover, molecular dynamics
modelling studies suggest that F508del-NBD1 exposes
its hydrophobic interior to the solution more often
than wt-NBD1 [16,17].
Effect of ATP on the Hsc70–NBD1 interaction
Adenine nucleotides are of critical importance to the
binding of Hsc70 to CFTR [8]. Consistently, our data
show that ATP dramatically reduces the ability of
mNBD1 to bind Hsc70. However, ATP can mediate
this effect in two distinct ways. First, the binding and
hydrolysis of ATP at the nucleotide-binding domain of
Hsc70 is known to accelerate its binding and release of
substrates, reducing the affinity of their interaction.
Once ADP occupies the nucleotide-binding site of
Hsc70, it converts the chaperone to a high-affinity
binding form [8]. Second, ATP, a native ligand of
CFTR-NBD1, may bind to this domain, stabilizing its
structure [14,41] and possibly hindering one (or more)
Hsc70-binding site(s). Several lines of evidence argue
that the effect of ATP on the NBD1–Hsc70 interaction
observed here is mediated predominantly via NBD1
and not by Hsc70. First, although ATP reduced
NBD1 binding to Hsc70, it did not alter the associa-

tion–dissociation profile, suggesting that it did not
alter the kinetics of the interaction. Second, ADP,
rather than enhancing NBD1–Hsc70 binding, as pre-
dicted for an Hsc70-mediated effect, substantially
reduced binding. Third, the IC
50
values for ATP inhi-
bition of wt-mNBD1 and F508del-mNBD1 binding to
Hsc70 ($ 20 and $ 110 lm, respectively, this study)
are comparable to the previously reported apparent
dissociation constants for ATP and wt-hNBD1 and
F508del-hNBD1 ($ 90 lm [42]) but substantially
higher than the dissociation constant for ATP and
Hsc70, which is in the order of $ 0.7 lm [43]. Finally,
ATP caused a reduction of only $ 20% in the Hsc70
binding of denatured lactalbumin, itself not predicted
to interact with ATP, whereas Hsc70 binding to
wt-mNBD1 was reduced by $ 80%. Our data also
Interaction of Hsc70 with F508del-NBD1 T. S. Scott-Ward and M. D. Amaral
7104 FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS
show that the ATP concentration required to inhibit
the mNBD1–Hsc70 interaction is dramatically
increased by deleting Phe508. Less optimal ATP bind-
ing to F508del-NBD1 would be consistent with the
gating defect of F508del-CFTR, characterized by long
interburst intervals [44], according to the current
model of CFTR channel gating [45]. However, regard-
ing the isolated domain, ATP binds wt-NBD1 and
F508del-NBD1 with equivalent affinity, arguing
against such an effect. However, it remains plausible

that the F508del-induced exposure of Hsc70-binding
sites on NBD1 diminishes the ability of ATP binding
to stabilize this domain against its Hsc70 interaction.
Altogether, interpretation of these data would be con-
sistent with ATP promoting ordered homodimerization
of NBD1, a process that conceivably occludes the
chaperone-binding site to reduce Hsc70 binding. It
has, in fact, been reported that wt-NBD1 can form
homodimers under certain conditions [46,47]. Thus, a
reduced dimerization ability of F508del-NBD1 relative
to wt-NBD1 would explain why higher ATP concen-
trations are required to inhibit its binding to Hsc70.
Effect of small molecules on the interaction of
Hsc70 with F508del-NBD1
Here, we also used SPR to determine whether C4a and
V325 have an effect on the NBD1–Hsc70 interaction.
Our data demonstrate that C4a did indeed reduce
F508del-mNBD1 binding to Hsc70. This suggests that
C4a binds directly to F508del-mNBD1, either at the
same binding site, or by allosteric stabilization of
NBD1, to influence its folding and thus decrease the
affinity of the domain for the Hsc70 chaperone. This
finding is consistent with the results of recent in vivo
studies on full-length F508del-hCFTR [20,26].
However, it should be noted that substantially higher
concentrations of C4a were required to affect the
NBD1–Hsc70 interaction than those needed to correct
cell surface expression of CFTR, although, in vivo,a
more complex mechanism of action may occur than
the simple disruption of an NBD1–Hsc70 complex. A

plausible explanation, nevertheless, is that C4a binds
directly to a site on monomeric NBD1 that stabilizes
homodimerization, similarly to the ATP effect (see
above). This could likewise occlude the Hsc70-binding
site(s) and thus reduce Hsc70 binding. Strikingly, we
observed a significantly reduced effect of C4a at high
ATP concentrations.
Our data demonstrate that SPR provides a powerful
approach to quantifying the Hsc70–NBD1 interaction
and the impact of F508del (or other NBD1 mutations)
and corrector compounds on this folding-sensitive
association. In particular, our data show that ATP dra-
matically reduces the ability of mNBD1 to bind Hsc70,
and indicate that it is mostly this ability of ATP to
displace Hsc70 from NBD1 binding that is impaired by
F508del. C4a significantly inhibits the F508del-mNBD1
interaction with Hsp70, suggesting direct binding. As
this effect is abolished at high ATP concentrations, ATP
and C4a may compete for the same F508del-mNBD1
binding site ⁄ surface. We conclude that this SPR
approach constitutes a useful assay for the determina-
tion of whether and how correctors affect the Hsc70–
F508del-NBD1 interaction, which will improve our
understanding of the mechanism of action of small
molecules with therapeutic potential for CF, a critical
step in bringing them to the clinical setting. Future stud-
ies should focus on the quantification of the effect of
these correctors on the NBD1–Hsc70 interaction in the
presence of other relevant CFTR domains (e.g. intra-
cellular cytoplasmic loop 4 and ⁄ or nucleotide-binding

domain 2) at controlled MgATP concentrations.
Experimental procedures
Reagents
All three forms of NBD1 used in this study (wt-hNBD1,
wt-mNBD1, and F508del-mNBD1; Thr389–Gly673) were
prepared using the same protocol as previously described
[22]. Briefly, the proteins were expressed in BL21 (DE3
strain) Escherichia coli from pSmt3 vectors, purified by
nickel-affinity and size-exclusion chromatography, and
stored at )80 °C [10 mgÆmL
)1
NBD1, 20 mm Tris, 150 mm
NaCl, 5 mm MgCl
2
,2mm ATP, 2 mm b-mercaptoethanol,
12.5% (v ⁄ v) glycerol, pH 7.6]. Postpurification, each
preparation of NBD1 protein (wt-hNBD1, wt-mNBD1, and
F508del-mNBD1) was analysed by intrinsic tryptophan
fluorescence and CD spectroscopy to confirm that it carried
a native fold (personal communication: data at http://www.
cftrfolding.org/reagentRequestshWT.asp). Because F508del-
hNBD1 CFTR is highly insoluble and prone to aggregation
during purification [14], it was not available for use in this
study. Hence, wt-mNBD1 and F508del-mNBD1 were used
to characterize the impact of the F508del mutation on
CFTR–Hsc70 interactions. Purified bovine Hsc70 protein
(SPA-751) and monoclonal rat Ab against Hsc70 (SPA-
815) were obtained from Assay Designs (Ann Arbor, MI,
USA). Monoclonal mouse Ab against NBD1 (L12B4) was
obtained from Chemicon (Temecula, CA, USA). Biacore

materials were obtained from GE Healthcare (Milwaukee,
WI, USA). All other chemicals, proteins (BSA and bovine
apo-a-lactalbumin) and reagents were purchased from
Sigma Aldrich (St Louis, MO, USA) or BDH (Poole, UK),
and were of research grade or higher (‡ 99% purity).
T. S. Scott-Ward and M. D. Amaral Interaction of Hsc70 with F508del-NBD1
FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS 7105
Biochemical analysis of purified NBD1 and Hsc70
Coimmunoprecipitation of purified NBD1 (1 lm, unless
otherwise stated) and Hsc70 (1 lm) in SPR flow buffer
[150 mm KCl, 2 mm MgCl
2
, 0.1% (v ⁄ v) Triton X-100,
0.1% (v ⁄ v) dimethylsulfoxide, 1 mm b-mercaptoethanol,
20 mm Hepes, pH 7.0] was performed using L12B4, as pre-
viously described [39]. SDS ⁄ PAGE and western blot using
SPA-815 (Hsc70) and L12B4 (NBD1) were also performed
as previously described [39]. Protein quantification was
performed with a modified Lowry method.
Spectroscopic analysis of NBD1
Wild-type and F508del-mNBD1 (1 lm) were diluted in
NaCl ⁄ P
i
(150 mm NaCl, 20 mm Na
2
PO
4
,1mm b-mercap-
toethanol, pH 7.4) and, following excitation at 295 nm, the
emitted intrinsic tryptophan fluorescence was measured at

25 °C as previously described [15]. The intensity of fluores-
cence emitted from NBD1 at 328 and 343 nm was corrected
for background fluorescence from buffer at these two wave-
lengths.
SPR
Interaction analyses were performed in SPR flow buffer
at constant temperature (25 °C), using a Biacore 2000
system (GE Healthcare) as previously described [6].
Ligand proteins (20 lgÆmL
)1
in sodium acetate; 10 mm,
pH 5.0) were covalently immobilized (‘on-chip’) on the
surface of carboxymethyl-dextran (CM5) sensor chips,
according to the manufacturer’s instructions (estimated
final concentrations of immobilized proteins on sensor
chip surface: 0.8–1.5 mm). The binding and dissociation
of free analyte (‘applied’) proteins at a constant flow rate
(30 lLÆmin
)1
, 180 lL) was then measured. The surface of
the chip was regenerated between sample applications
with sequential injections of HCl (10 mm,20lL) and
NaOH (10 mm,20lL). LA (20 mgÆmL
)1
) was reduced by
incubating in SPR flow buffer containing dithiothreitol
(20 mm, 60 min, 4 °C) as previously described [48]. Red-
LA was then diluted to 10 lm (145 lgÆmL
)1
) and applied

immediately.
The effect of small molecules (C4a, V325, I172; 10 mm,
100% dimethylsulfoxide) was determined by diluting these
compounds to different concentrations (as indicated in
the figure legends) in flow buffer containing BSA
(0.2 mgÆmL
)1
), and applying them either immediately (acute
effect) or after 30 min of incubation at 16 °C (incubated
effect). The final dimethylsulfoxide concentration was
adjusted to 0.5%. Protein-coated CM5 chips were used for
2 weeks, or until nonspecific binding increased (‡ 5%). All
experiments were performed in parallel with an inactivated,
or blank, flow cell not coated with protein.
Data analysis
All SPR sensograms were corrected for buffer-induced
refractive index changes at an uncoated reference surface,
analysed using biaevaluation software (biaeval; v. 3.2;
GE Healthcare), and displayed in sigmaplot (v. 10; Systat,
San Jose, CA, USA) as pmoles of interacting protein (e.g.
NBD1) bound per nmole of immobilized protein (e.g.
Hsc70). Molar concentrations of the proteins were calcu-
lated from their measured concentration (mgÆmL
)1
), using
their molecular masses as determined from amino acid com-
position (wt-NBD1, 31 976 Da; F508del-NBD1, 31 829 Da;
wt-hNBD1, 31 969 Da; bovine Hsc70, 71 241 Da).
The kinetics of interaction (association, k
a

, and dissocia-
tion, k
d
, rates) were determined from each set of dose–
response data by global fitting of the association and
dissociation phases of all binding curves in that dataset
(biaeval). The dissociation constant (K
D
) for each dose–
response set was then determined (k
d
⁄ k
a
), and the values
were averaged (mean K
D
). To determine the apparent
maximal binding (B
max
app
, pmolÆnmol
)1
) and dissociation
constant (K
D
app
, lm) of mNBD1 directly from the data
(Eqn 1), the amount of mNBD1 bound at equilibrium (B
eq
,

pmolÆnmol
)1
) was determined from kinetic analysis and
plotted against [mNBD1] (sigmaplot). The [MgATP]
required to inhibit binding by 50% [IC
50
, lm; Eqn (2)] was
determined by plotting the amount of mNBD1 bound at
320 s against [MgATP]
B
eq
¼
B
max
app
½NBD1
K
D
app
þ½NBD1
ð1Þ
B ¼ B
min
þ
B
max
À B
min
1 þ 10
ðIC

50
À½ATPÞ
ð2Þ
where: B
eq
is binding at equilibrium; B
max
app
and K
D
app
are
the apparent maximum binding and dissociation constants,
respectively; B is binding (at 320 s); B
min
and B
max
are
minimum and maximum binding, respectively; and [NBD1]
and [ATP] are the concentrations of NBD1 and ATP,
respectively.
Statistical analysis
Unless otherwise stated, data are presented as the
mean ± standard error of the mean (SEM) (n ‡ 3). Mean
data for NBD1 dose responses were calculated as follows:
(a) global kinetic analysis of each dose–response set
generated a k
d
and k
a

value for each [NBD1]; (b) these
values were averaged; and (c) the averaged values were
used to calculate a single K
D
(k
d
⁄ k
a
). The mean K
D
was
then determined by averaging K
D
values from repeat dose–
response experiments (n = 3). For mean K
D
app
and B
max
app
values, the K
D
app
and B
max
app
were determined directly
for each dose–response dataset as described, and the
Interaction of Hsc70 with F508del-NBD1 T. S. Scott-Ward and M. D. Amaral
7106 FEBS Journal 276 (2009) 7097–7109 ª 2009 The Authors Journal compilation ª 2009 FEBS

values were averaged (n = 3). Owing to the small size of
datasets (n =3 or n = 4), an unpaired, two-tailed t-test
was used to determine whether two mean values were
statistically different (sigmastat, v. 3.5; Systat). Differ-
ences were considered to be statistically significant when
the P-value (probability of the mean values being equal)
was less than 0.05 (a = 0.05). In all cases, data were
tested to ensure normality (Kolmogorov–Smirnov test)
and equal variance (Levene median test) prior to apply-
ing the t-test.
Acknowledgements
We are grateful to P. Thomas (University of Texas
South Western) and the CF Folding Consortium
() for generously providing
the purified NBD1 proteins, to the Cystic Fibrosis
Foundation (Bethesda, MD, USA), B. Bridges (Rosa-
lind Franklin University of Medicine and Science,
Chicago, IL, USA) for access to small molecule com-
pounds, and to J. Findlay (Leeds University, UK) for
SPR training of T. S. Scott-Ward. This work was
supported by grants TargetScreen (European Union,
FP6-2005-LH-037365) and POCTI ⁄ SAU⁄ MMO ⁄ 58425 ⁄
2004 (FCT, Portugal), and pluriannual funding from
CIGMH (FCT, Portugal). T. S. Scott-Ward is the reci-
pient of postdoctoral fellowship SFRH ⁄ BPD ⁄ 18989 ⁄
2004 (FCT, Portugal).
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