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New insights into interactions between the nucleotide-binding domain of CFTR and keratin 8

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The intermediate filament protein keratin 8 (K8) interacts with the nucleotide-binding domain 1 (NBD1) of the cystic fibrosis transmembrane regulator (CFTR) with phenylalanine 508 deletion (ΔF508), and this interaction hampers the biogenesis of functional ΔF508-CFTR and its insertion into the plasma membrane. Interruption of this interaction may constitute a new therapeutic target for cystic fibrosis patients bearing the ΔF508 mutation. Here we aimed to determine the binding surface between these two proteins, to facilitate the design of the interaction inhibitors. To identify the NBD1 fragments perturbed by the ΔF508 mutation, we used hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) on recombinant wild-type (wt) NBD1 and ΔF508-NBD1 of CFTR. We then performed the same analysis in the presence of a peptide from the K8 head domain, and extended this investigation using bioinformatics procedures and surface plasmon resonance, which revealed regions affected by the peptide binding in both wt-NBD1 and ΔF508-NBD1. Finally, we performed HDX-MS analysis of the NBD1 molecules and full-length K8, revealing hydrogen-bonding network changes accompanying complex formation. In conclusion, we have localized a region in the head segment of K8 that participates in its binding to NBD1. Our data also confirm the stronger binding of K8 to ΔF508-NBD1, which is supported by an additional binding site located in the vicinity of the ΔF508 mutation in NBD1.
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New insights into interactions between
the nucleotide-binding domain of CFTR
and keratin 8
Aiswarya Premchandar,
1
Anna Kupniewska,
2
Arkadiusz Bonna,
1,7
Grazyna Faure,
3
Tomasz Fraczyk,
1
Ariel Roldan,
4
Brice Hoffmann,
5
M
elanie Faria da Cunha,
2
Harald Herrmann,
6,8
Gergely L. Lukacs,
4
Aleksander Edelman,
2
* and Michał Dadlez
1
*
1
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland
2
INSERM U1151, team Canalopathies
epith
eliales : la mucoviscidose et autres maladies, Universit
e Paris Descartes, Paris, France
3
Unit
eR
ecepteurs-Canaux; Institut Pasteur, CNRS, URA 2182, Paris F-75015, France
4
Department of Physiology, McGill University, Montreal, QC, Canada
5
IMPMC, Sorbonne Universit
es, UPMC Universit
e Paris 06, UMR CNRS 7590, Museum National d’Histoire Naturelle, IRD UMR
206, IUC, Paris Cedex 05, 75005, France
6
Department of Molecular Genetics, German Cancer Research Center, Heidelberg, D-69120, Germany
7
Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom
8
Institute of Neuropathology, University Hospital Erlangen, D-91054 Erlangen, Germany
Received 6 July 2016; Accepted 16 November 2016
DOI: 10.1002/pro.3086
Published online 21 November 2016 proteinscience.org
Abstract: The intermediate filament protein keratin 8 (K8) interacts with the nucleotide-binding
domain 1 (NBD1) of the cystic fibrosis (CF) transmembrane regulator (CFTR) with phenylalanine 508
deletion (DF508), and this interaction hampers the biogenesis of functional DF508-CFTR and its
insertion into the plasma membrane. Interruption of this interaction may constitute a new thera-
peutic target for CF patients bearing the DF508 mutation. Here, we aimed to determine the binding
surface between these two proteins, to facilitate the design of the interaction inhibitors. To identify
the NBD1 fragments perturbed by the DF508 mutation, we used hydrogen–deuterium exchange
coupled with mass spectrometry (HDX-MS) on recombinant wild-type (wt) NBD1 and DF508-NBD1
of CFTR. We then performed the same analysis in the presence of a peptide from the K8 head
domain, and extended this investigation using bioinformatics procedures and surface plasmon res-
onance, which revealed regions affected by the peptide binding in both wt-NBD1 and DF508-NBD1.
Finally, we performed HDX-MS analysis of the NBD1 molecules and full-length K8, revealing
Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; HDX, hydrogen–deuterium exchange; IF, intermedi-
ate filaments; K8, keratin 8; LC, liquid chromatography; MS, mass spectrometry; NBD, nucleotide-binding domain; SPR, sur-
face plasmon resonance.
Additional Supporting Information may be found in the online version of this article.
Aiswarya Premchandar and Anna Kupniewska contributed equally to this work
The authors declare that they have no conflicting interests.
Grant sponsor: French Cystic Fibrosis foundations Vaincre la mucoviscidose and Mucoviscidose ABCF2; Grant sponsor: French Nation-
al Agency grant; Grant number: CORCF ANR-13-BSV1-0019-01; Grant sponsor: MAESTRO grant from the National Science Centre,
Poland; Grant number: 2014/14/A/NZ1/00306; Grant sponsor: Cystic Fibrosis Canada; Grant sponsor: Canadian Institute for Health
Research; Grant sponsor: National Institute of Health; Grant number: DK075302; GGL is a recipient of a Canada Research Chair.
*Correspondence to: Michał Dadlez, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Department of Biophys-
ics, Pawinskiego 5a, Warsaw, Poland. E-mail: michald@ibb.waw.pl (or) Aleksander Edelman, INSERM U1151 Universit
e Paris Des-
cartes, Paris, France. E-mail: aleksander.edelman@inserm.fr
Published by Wiley-Blackwell. V
C2016 The Protein Society PROTEIN SCIENCE 2017 VOL 26:343—354 343
hydrogen-bonding network changes accompanying complex formation. In conclusion, we have
localized a region in the head segment of K8 that participates in its binding to NBD1. Our data also
confirm the stronger binding of K8 to DF508-NBD1, which is supported by an additional binding
site located in the vicinity of the DF508 mutation in NBD1.
Keywords: cystic fibrosis; CFTR; keratin 8; NBD1; hydrogen-deuterium exchange mass spectrome-
try; protein structure
Introduction
Cystic fibrosis (CF) is the most common lethal autoso-
mal recessive disease. It is caused by mutations in the
gene encoding the CF transmembrane regulator
(CFTR), and 90% of CF patients harbor the phenylala-
nine 508 deletion (DF508) at least in one CFTR allele
(http://www.genet.sickkids.on.ca/cftr). Although CFTR
belongs to the ATP-binding cassette (ABC) transporter
family, it acts as a cAMP-dependent passive anion chan-
nel that is regulated by ATP binding and hydrolysis,
rather than as an active transporter of large molecules.
1
CFTR is a multidomain single-chain glycoprotein com-
prising two cytosolic nucleotide-binding domains
(NBD1 and NBD2), and two membrane-spanning
domains (MSD1 and MSD2) that each include six trans-
membrane a-helices linked by intracellular loops
(ICL1-4). CFTR also contains the intrinsically disor-
dered regulatory domain R, which is a primary site of
protein kinase-mediated anion channel regulation.
2
As in all members of the ABC transporter fami-
ly, NBD1 of CFTR comprises three main subdo-
mains: (i) ABCb, an N-terminal b-sheet subdomain
containing the ATP-binding site; (ii) ABCa,ana-
helical subdomain containing F508; and (iii) a cen-
tral a/bcore, analogous to the F1-type ATPase, that
contains a six-stranded largely parallel b-sheet.
3
The NBD1 of CFTR also contains several unique
regions, including a 32-amino-acid insert between b-
strands 1 and 2 of the ABCbsubdomain, called the
regulatory insertion (RI; residues 402–438), and a
32-amino-acid extension at the C-terminus called
the regulatory extension (RE; residues 638–676)
(Fig. 1). These unique segments each contain several
sites susceptible to PKA phosphorylation, and are
involved in CFTR trafficking and in the regulation
of its Cl
2
channel function.
3–6
Both the RI and RE
show secondary structure preferences in crystallo-
graphic structures of human NBD1 deposited in the
protein data bank (PDB IDs: 2BBO, 1XMJ, 2BBS,
and 2BBT); however, these fragments exhibit a high B
factor, suggesting that they are highly flexible. The
ABCasubdomain contains the so-called structurally
diverse region (SDR; L
526
–T
547
) that exhibits different
conformations for each protein structure found in pro-
tein data bank. The secondary structural elements
suggested in this fragment by crystallography most
likely appear only due to artificial contacts that occur
during crystal formation. Prior studies have provided
only limited information about CFTR and its domains.
We particularly lack knowledge of the complexes
formed in solution where the protein retains its native
dynamic character in selected regions that are not in
agreement with the kinetic snapshots obtained by X-
ray crystallography.
6
F508 deletion does not dramatically affect
NBD1 structure but rather interferes with CFTR
maturation and delivery to the plasma membrane.
7
This mutation decreases the intrinsically low folding
efficiency of CFTR from 20–40% to 0.4%. The
residual amount of mutated CFTR that reaches the
plasma membrane exhibits gating defects but can
still exert its function.
2
Keratins 8/18 are intermediate filament (IF) pro-
teins characteristic of simple epithelial cells, serving
as scaffolding elements and mechanical stress absorb-
ers.
8
Keratin IFs occur in nature as obligate hetero-
dimers of two distinct sequence-related proteins
distinguished as type I (keratin 18) and type II (kera-
tin 8, K8) keratins.
9
Like all other IF proteins, K8
shares a tripartite organization of non-a-helical ami-
no-terminal head and carboxy-terminal tail domains
of variable length flanking a central a-helical coil
domain. While the head and the tail domains are
known to vary considerably in size and composition,
the structural organization of the rod domain is high-
ly conserved and is 310 amino acids in length, exhib-
iting a seven-residue (heptad), and eleven-residue
(hendecad) repeat pattern.
10
The K8 rod domain is
further subdivided into four regions, coil 1A, coil 1B,
coil 2A, and coil 2B, by short unstructured linker
regions (designated L1, L12, and L2, respective-
ly).
11–1 3
Under in vitro conditions, K8 exists as a
dimer in the absence of its partner, K18.
14,15
The IFs appear to play a role in the trafficking
of both wild-type (wt)-CFTR and DF508-CFTR. The
IFs: K8 and keratin 18 (K18) prevent the delivery of
functional DF508-CFTR to the plasma mem-
brane.
16–18
K18 reportedly binds to the CFTR C-
terminus, regulating its function.
19
We recently
demonstrated that K8, but not K18, physically binds
to the NBD1 of both wt and DF508 CFTR,
20
with a
greater tendency to target the unfolded destabilized
conformation caused by F508 deletion. K8 colocal-
ized with DF508-CFTR, but not with wt-CFTR, in
primary cultured human respiratory cells from CF
patients bearing the DF508 mutation, but not from
healthy individuals. Importantly, disruption of this
interaction restored CFTR-dependent chloride
344 PROTEINSCIENCE.ORG CFTR/K8 Complex Structural Insights
transport in cell lines expressing DF508-CFTR and
in DF508/DF508 mice.
20
This study aimed to attain further insight into the
molecular structure involved in the interaction of K8
with wt-NBD1/DF508-NBD1. To this end, we used
hydrogen-deuterium exchange coupled with mass spec-
trometry (HDX-MS) and surface plasmon resonance
(SPR) to investigate the interface between these pro-
teins. The HDX-MS technique is based on the fact that
the rate of exchange of amide hydrogens for deuterons
in the solvent reflects the level of structural flexibility
or the protein fragment’s exposure to solvent. This
technique also allows examination of the site of interac-
tion between biomolecules. Due to changes in hydrogen
bond stabilities or sequestration from the solvent,
amide hydrogens in the complex interface may undergo
retarded exchange compared to the separated compo-
nents. We used SPR to map which K8 fragments bound
to either wt-NBD1 or DF508-NBD1. Our results
enabled the identification of a region of K8 that binds
to both wt-NBD1 and DF508-NBD1, and defined a sec-
ondary binding region in NBD1 that supports the
stronger binding of DF508-NBD1 to K8.
Results
HDX-MS reveals regions of stability in NBDs
In the preliminary experiments, we obtained HDX
exchange patterns for the human recombinant wt-
NBD1 and DF508-NBD1, which both contained an
additional single solubilization mutation (1S) in the
linker between the ABCband ABCasubdomains
(F494N). This 1S mutation did not stabilize the
NBD1 structure and only marginally reverted the
DF508-CFTR structural defect, justifying its use for
structural studies.21 Figure 1 depicts the subdomain
organization of the CFTR-NBD1 as previously
defined [see Fig. 1(b) in the study of Lewis et al.
6
].
Figure 2 shows the HDX patterns for wt-NBD1 and
DF508-NBD1 for 10 s (black) and 1 minute (orange)
in D
2
O. Only these shorter incubation times are
shown because DF508-NBD1 showed limited stabili-
ty/stronger aggregation tendencies over longer incu-
bation periods at room temperature. Additionally,
after 20 min of incubation, peptides of the NBDs
showed >50% exchange along almost the entire
length (data not shown), indicating a high overall
level of dynamics in both NBD1 variants.
Comparison of the exchange patterns for wt-
NBD1 and DF508-NBD1 revealed high similarity of
fast- and slow-exchanging regions. Figure 1 shows
the correspondence between the position in the
sequence and the structural elements, which are
color-coded on the horizontal axis. The fastest levels
of exchange were registered for the regulatory sub-
domains of the NBDs: RI (E
402
–T
438
; Fig. 1, blue)
and regulatory extension (RE; P
638
–P
676
; Fig. 1,
magenta), which are positioned at the N- and C-
termini of NBD1. The N-terminus itself (residues
389–401) was also readily exchangeable, although
this region forms a b-sheet constituting the ABCb
subdomain in the crystal structure. Fast exchange
was also exhibited by three other regions within the
central part of this domain, which were intertwined
with those of greater stability. These fast-
exchanging regions included the ABCbsubdomain
(389–401, 470–490; Fig. 1, green), the central region
of the ABCasubdomain that corresponds to the
structurally diverse region (SDR; L
526
–T
547
), and the
Walker B loop region (D
572
–D
579
; Fig. 1, violet).
The highest stability was observed for the fol-
lowing regions: the N-termini (residues 495–525)
and C-termini (559–564) of the ABCasubdomain
(Fig. 1, cyan), and residues 585–630 from the F1-
like ATP-binding core subdomain (Fig. 1, red). Mod-
erate levels of exchange were noted for other
regions, including part of the F1-like ATP-binding
core subdomain (G
451
–L
475
), which also encompasses
the Walker A loop. Previous studies suggest that the
Walker A loop physically binds to ATP molecules
and the moderate protection within this region
reflects the flexibility of the ATP-binding loop. Fig-
ure 3 shows the levels of exchange from Figure 2
(corresponding to different NBD1 subdomains) over-
laid on the NBD1 model derived from Mornon et al.
(before molecular dynamics simulation, CFTRIni-
tial.pdb).
22
Color codes are used to depict different
Figure 1. Subdomain organization of the NBD1 domain in its
ATP-bound state, as previously defined by Lewis et al.
6
Premchandar et al. PROTEIN SCIENCE VOL 26:343—354 345
exchange levels at 10 s, from the most protected
(violet, slowest exchange) to the least protected (red,
fastest exchange). Figure 3 clearly illustrates that
regions of higher stability form a single common pro-
tein core, with a relatively stable region encompass-
ing the F508 mutation site.
Figure 4 shows the differences in the HDX
between wt-NBD1 and DF508-NBD1 with incubation
times of 10 seconds (black) and 1 minute (orange).
The exchange pattern was largely similar between
wt-NBD1 and DF508-NBD1 [Fig. 4(A)], although
some regions showed slight differences in exchange
level between the two variants. Differences revealed
at 10 s of incubation indicate changes in dynamic
regions, while differences revealed after 1 min of
incubation reveal changes in more structured
regions. Figure 4(B) shows an overlay of the
differences of >10% in the NBD1 model. A region
preceding the DF508 mutation site (496–504) was
more stable in the wt-NBD1 [Fig. 4(B), red; Support-
ing Information Fig. S1]. The peptides around the
DF508 mutation site (505–525) showed a different
pattern of pepsin proteolytic cleavage in DF508-
NBD1, indicating structural differences in the dele-
tion region (Supporting Information Table S1). The
first two segments of the ABCbsubdomain (389–401
and 439–450), and region 615–640 of the F1-like
core subdomain, showed slightly increased stability
in DF508-NBD1 [Fig. 4(B), blue].
Identification of K8 fragments that interact
with NBD1
To identify the NBD1–K8 binding region, we first
conducted bioinformatic and literature searches for
Figure 3. Overlay of the level of exchange after 10 s of incubation (as indicated in black in Fig. 2) on the NBD1 model
22
for wt-
NBD1 (A) and DF508-NBD1 (B). Red: >80% of exchange after 10 s (the least stable regions), Yellow: 60–80%; Green: 40–60%;
Blue: 20–40%; Violet: <20% (the most stable regions). The regions with no sequence coverage are represented in gray.
Figure 2. Relative deuterium uptake (% deuteration) in peptic peptides from wt-NBD1 (A) and DF508-NBD1 (B). Data are
shown for the selected shortest peptides that are most representative of the entire protein sequence. Each peptide’s position in
the sequence is shown on the X-axis, represented by a horizontal bar with length proportional to the peptide’s length. The
bar’s position on the vertical axis marks the fraction exchanged after 10 s (black) and 1 min (orange) of incubation. Values close
to 100% indicate fully unprotected regions, that is, regions of high flexibility. Values close to 0% indicate protected regions,
that is, more stable structures. Y-Axis error bars represent standard deviations calculated from three independent experiments.
The colored bars below the X-axis represent the subdomain organization as defined in Figure 1.
346 PROTEINSCIENCE.ORG CFTR/K8 Complex Structural Insights
possible interaction regions. Candidate K8 peptides
potentially responsible for interaction with NBD1
were selected according to the following rationale.
First, the majority of proteins that interact with IFs
target the head domain (desmoplakin,
23–25
plectin,
26
fimbrin,
27
and S100a/b
28
). Second, antibodies direct-
ed against the N-terminal fragment of K8 (but not
against its C-terminus) introduced into DF508-
CFTR-expressing HeLa cells reportedly lead to the
functional expression of mutated CFTR.
29
Third, the
dynamic properties of K8 displayed several frag-
ments with high flexibility, with the head region
among the regions of highest flexibility.
15
Based on
these observations, three peptides from the head
region and a short N-terminal fragment of coil 1A of
K8
13
were selected for further interaction studies
and synthesized:
Peptide 1: Ac-N
83
IQAVRTQEKEQIKTLNNKFA
SF
105
-NH
2
Peptide 2: Ac-T
67
VNQSLLSPLVLEVDPNIQ
85
-NH
2
Peptide 3: Ac-S
27
GPGSRISSSSFSRVGSSNFRGG
LGGG
53
-NH
2
Peptide 2 was not soluble (50 mMTris-HCl, pH
8.4; 150 mMNaCl) and was not investigated further.
Peptides 1 and 3 were mixed with NBD1 variants,
and the changes in HDX pattern were assessed. A
region covering positions 429–433 [Fig. 5(A,B); Sup-
porting Information Fig. S2(A), S2(B)], correspond-
ing to part of the RI subdomain, was commonly
stabilized by Peptide 1 in both wt-NBD1 and DF508-
NBD1. The effect was small but reproducible, and
was slightly stronger in DF508-NBD1 [Fig. 5(B);
Supporting Information Fig. S2(A), panel v] than in
wt-NBD1 [Fig. 5(A); Supporting Information Fig.
S2(A), panel iii]. In the DF508-NBD1–Peptide 1 com-
plex, we observed additional stabilization in the
regions 400–408 from RI, and in three other pepti-
des between positions 550–650 of the F1-like core
subdomain. This effect was absent in wt-NBD1.
However, wt-NBD1 alone showed slight stabilization
of the region 660–680. In contrast to Peptide 1, Pep-
tide 3 did not lead to any substantial changes when
incubated with either NBD1 variant [Fig. 5(C,D)].
These data suggest that Peptide 1 overlays with the
K8 region that interacts with NBD1.
Analysis of the interaction of peptide 1 with
NBD1 by SPR
We also performed SPR experiments to demonstrate
the direct binding interaction between Peptide 1 and
wt-NBD1/DF508-NBD1 of CFTR. NBD1 (wt or
mutant) was covalently attached to the dextran
matrix of the CM5-sensor chip, and we examined
the binding interaction with the K8 peptide (Fig. 6).
Kinetic parameters were calculated by quantitative
SPR analysis for both complexes [Fig. 6(A,B)]. For
the Peptide 1–wtNBD1 complex, values of
k
a
573 M
21
s
21
,k
d
52.27 310
23
s
21
, and Kapp
D531
lM were determined. For the Peptide 1–DF508-
NBD1 complex, k
a
5113 M
21
s
21
,k
d
55.18 310
24
s
21
and Kapp
D54.6 lM. Thus, the K8 Peptide 1 dis-
plays a higher affinity for mutated NBD1 than for
wt-NBD1. Moreover, the dissociation rate constant
(k
d
) for the mutated NBD1 complex was 4–fold lower
than for wtNBD1, indicating that the Peptide 1–
DF508-NBD1 complex is more stable. The randomly
scrambled K8 peptide did not interact with immobi-
lized NBD1 [Fig. 6(C)], supporting the specificity of
Figure 4. A: Difference in the fraction of exchange (% deuteration) between wt-NBD1 and DF508-NBD1, after 10 s (black) and
1 min (orange) of incubation. Peptides with differences of >10% are considered more stable in DF508-NBD1, while those with
differences below 210% are considered more stable in wt-NBD1. The colored bars below the X-axis represent the subdomain
organization as defined in Figure 1. B: Overlay of the differences in the NBD1 model. Blue: regions more stable in DF508-
NBD1; Red: regions more stable in wt-NBD1. The DF508 mutation caused a shift in protease cleavage pattern between regions
505 and 525. Therefore, we could not compare the peptides from this region between wt-NBD1 and DF508-NBD1 (see Sup-
porting Information Table S1).
Premchandar et al. PROTEIN SCIENCE VOL 26:343—354 347
this interaction, and demonstrating that this bind-
ing was dependent on the peptide sequence and not
the amino-acid composition.
Binding with K8 causes allosteric changes in
DF508-NBD1
When we added K8 itself (rather than the head pep-
tides) to the NBD1 preparations, we observed a
more widespread pattern of changes. Figure 7 sum-
marizes these results for wt-NBD1 and DF508-
NBD1. Binding of full-length K8 induced changes
along the whole sequence of DF508-NBD1, indicat-
ing either an alternate binding mode with Peptide 1
region in the context of the full-length protein, or
the existence of alternative binding regions. For the
wt-NBD1–K8 complex, we observed only a small
shift in stability in the 429–433 peptide, smaller
than observed in complex with Peptide 1 [panel (iii)
in Supporting Information Fig. S2(A), S2(B)]. Inter-
estingly, certain short regions in the wt-NBD1–K8
complex underwent destabilization [Fig. 7(C), red]—
namely, positions 496–504 (i.e., the region preceding
F508) and the positions 559–568 and 585–591, which
flank the Walker B motif and contain the diacidic
motifs D565 and D567. These changes extended up
to position 620.
The DF508-NBD1–K8 complex exhibited stronger
aggregational tendencies, which might have masked
the HDX patterns. It is possible that the observed
overall delay in HDX was caused by aggregation and
was overlaid on the region-specific exchange, making
it difficult to precisely interpret these results. Never-
theless, RI regions 400–410 and 429–433 were stabi-
lized in the DF508-NBD1–K8 complex [Fig. 7(B)], like
in the DF508-NBD1–Peptide 1 complex [Fig. 5(B)].
The other most prominently stabilized regions includ-
ed a segment of the ABCbsubdomain (389–401), the
peptide containing the DF508 mutation (505–512),
the Walker B loop containing diacidic motifs and its
C-terminal flanking region (570–579), elements of the
F1-like ATP-binding core subdomain (peptide 619–
625), and the RE (peptide 650–662). The regions stabi-
lized when in complex with K8 were not localized to a
single portion of the protein structure [Fig. 7(D)].
Interestingly, the areas of DF508-NBD1 that were
destabilized in complex with K8 included the region
Figure 5. The difference in the fraction of exchange (% deuteration) in wt-NBD1 (A, C) or DF508-NBD1 (B, D) before and after
interaction with K8 Peptide 1 (A, B) or K8 Peptide 3 (C, D) after 10 s (black) and 1 min (orange) of incubation. Peptides with a
difference in exchange fraction of >10% were stabilized in the NBD1-peptide complex, and those with a difference of <210%
were destabilized in this complex. The colored bars below the X-axis represent the subdomain organization as defined in
Figure 1.
348 PROTEINSCIENCE.ORG CFTR/K8 Complex Structural Insights
preceding the mutation site (496–504) and the N-
terminal region flanking the Walker B loop (559–568),
similar to in wt-NBD1 [compare Fig. 7(C,D), Support-
ing Information Fig. S3]. Upon binding to K8, pepti-
des covering the mutation site (Supporting
Information Table S1 and Fig. S4) behave differently
in wt-NBD1 and DF508-NBD1, with this region
becoming substantially more protected in DF508-
NBD1–K8, while protection remains unchanged in
wt-NBD1–K8. Our analysis revealed that parts of the
RI and the region preceding the Walker B loop (559–
568) were affected by K8 binding similarly in both the
wt and mutant NBD1. However, only the mutant
NBD1 was affected at the N-terminus of the ABCa
subdomain (389–401), in elements of the F1-like core
domain (619–625), and at the RE (650–662; Fig. 7).
Figure 7(C,D) highlights the DF508-NBD1
regions most strongly stabilized (blue) or destabi-
lized (red) upon interaction with K8. No strong sta-
bilization was observed in wt-NBD1. However, many
regions were stabilized in DF508-NBD1, including
regions flanking the RI domain (395–400 and 429–
433) and the C-terminal RE domain (650–662), ele-
ments of the F1-like ATP-binding core subdomain
(619–626), and a portion of the ABCasubdomain
(505–512). This last region was particularly interest-
ing since it encompasses the DF508 mutation site.
The areas destabilized upon K8 binding included the
region preceding the DF508 mutation site (496–504)
in both wt-NBD1 and DF508-NBD1. In the full-
length CFTR protein, this domain is normally
involved in binding to the intracellular loop 4
(ICL4),
22
which is an extension of transmembrane
helices 10 and 11 in the cytoplasm. In contrast to
wt-NBD1, binding of K8 to DF508-NBD1 led to the
overall stabilization of a set of external loops and
helices, without affecting the protein core [Fig. 7(D),
blue]. This effect may be due to allosteric changes
arising from the stronger binding of K8 to DF508-
NBD1. Such allosteric changes may help to discrimi-
nate wt-NBD1 and DF508-NBD1, and provide a
means for selecting the mutated form into the degra-
dation pathway.
NBD1 binding mainly affects the K8 head
domain
Using data from the same experiment, we compared
the HDX patterns for K8 in the presence and
absence of wt-NBD1 and DF508-NBD1 [Fig. 8(A,B)].
Interestingly, the structural consequences on K8
were generally similar after binding to wt-NBD1
and DF508-NBD1. The head region underwent the
most widespread stabilization, which was more pro-
nounced in DF508-NBD1, confirming that this
domain is the region that binds NBD1. Substantial
stabilization was observed in the head-coil 1A pep-
tide E
79
VDPNIQAVRTQEKEQIKTLNNKFASF
105
(Fig. 9) that is nearly identical to Peptide 1, support-
ing that Peptide 1 is indeed a part of the binding
site. We also detected increased stability in part of
the flexible L12 linker region, which might arrest
the mobility between the two coiled-coil rods of K8.
Binding to both versions of NBD1 also destabilized
the termini of coil 1B, coil 2A-L2, and the N-
terminal coil 2B region—which are involved in the
intermolecular interactions of K8 dimers, and in its
heterocomplexes with K18.
15
Since K8 was expected
to be dimeric under our experimental conditions, the
noted HDX pattern changes could be caused by an
equilibrium shift toward K8 monomers. In this
interpretation, interaction between the head
domains and the NBD1s would pull apart the inter-
actions at the N- and C-termini of the coiled coils
that bind the K8 dimer together.
Figure 6. SPR analyses showed the interactions of K8 Pep-
tide 1 with wt-NBD1 or DF508-NBD1 of CFTR that were
covalently immobilized on the Biacore sensor chip. Peptide 1
was injected for 60 s for the association phase, followed by
injection of running buffer alone at the same flow rate to sup-
port the dissociation phase. The response in resonance units
(RUs) is plotted as a function of time (in seconds). Peptide 1
at concentrations of 370 lM (black), 277 lM (red), 185 lM
(green), 138 lM (purple), and 69 lM (orange) was injected on
a flow-cell with immobilized wt-NBD1 (A) or immobilized
DF508-NBD1 (B). C: Randomly scrambled K8 peptide at a
concentration of 280 lM (blue) was injected on a flow-cell
with immobilized wt-NBD1. Injection of running buffer alone
is shown in black.
Premchandar et al. PROTEIN SCIENCE VOL 26:343—354 349
Discussion
In the present study, we identified the amino-acid
sequence localized in the head region of K8 as the
site of interaction with wt-NBD1 and DF508-NBD1
of CFTR. This region may play a major role in the
pathogenic interaction between K8 and DF508-CFTR
that has previously been demonstrated in DF508-
CFTR-expressing human cells.
20,30
Figure 7. Differences in the fraction of exchange (% deuteration) in wt-NBD1 (A) and DF508-NBD1 (B) before and after interac-
tion with K8 after 10 s (black) and 1 min (orange) of incubation. Peptides with a difference in exchange fraction of >10% were
stabilized upon complex formation, while those with a difference of <210% were more stable in the unbound state. The col-
ored bars below the X-axis represent the subdomain organization as defined in Figure 1. C, D: Overlay of differences >20% on
the NBD1 model for wt-NBD1 (C) and DF508-NBD1 (D), indicating the regions that were strongly stabilized (blue) or destabilized
(red) upon interaction with K8.
Figure 8. Differences in the fraction of exchange (% deuteration) in K8 peptides before and after interaction with wt-NBD1 (A)
and DF508-NBD1 (B).
350 PROTEINSCIENCE.ORG CFTR/K8 Complex Structural Insights
The global structural dynamic features of
NBD1s revealed by HDX-MS showed good agree-
ment with the crystallographic data. Essentially all
protein fragments with an elevated B-factor also
showed a fast HDX rate, with some as high as >80%
after 10 s of HDX reaction (Fig. 2).
6
This fast
exchange was especially observed in relation to the
regulatory extension, RI, and SDR. Compared with
the HDX-MS data reported by Lewis et al.,
6
we not-
ed elevated HDX rates in the regions of the Walker
A and B motifs, which engage in ATP binding. The
dilution of the final ATP concentration in our experi-
mental conditions might cause the enhanced flexibil-
ity in the ATP-binding Walker loop regions. Some
flexibility in these regions could be expected, as
these motifs form flexible loops (PDB: 1XMJ, 2BBO)
and are required for docking and hydrolysis of ATP.
6
However, the presently observed general HDX rates
in wt-NBD1 and DF508-NBD1 were higher than in
the pioneering HDX data reported by Lewis et al.
6
One possible explanation for this discrepancy is that
the recombinant NBD1s used in our study contained
only one solubilization mutation (F494N), while the
proteins used by Lewis et al. contained three solubi-
lization mutations. The so-called, “team mutation,”
comprising of 3 mutations more strongly stabilized
the NBD1s, almost fully restoring DF508-CFTR to
the plasma membrane.
21
In consistence with crystal structures, our pre-
sent results showed that the global HDX rate pat-
tern of wt-NBD1 was preserved in DF508-NBD1
[Fig. 3(A,B)], indicating that their structures are
similar.
3,6,31–33
Moreover, the HDX rates of almost
all identified peptides of DF508-NBD1 were similar
to those obtained for peptides of wt-NBD1. The
exceptions included peptides in direct proximity to
the DF508 mutation, and the first two segments of
the ABCbsubdomain (389–401 and 439–450). The
first fragment, which directly precedes the mutation,
showed a higher HDX rate in the presence of DF508,
while the other regions showed slightly increased
stability in DF508-NBD1 (Fig. 4).
Dynamic changes and allostery in NBD1 upon
binding to K8
We used the HDX-MS technique to investigate the
interactions between the NBD1 domain and K8 Pep-
tide 1, K8 Peptide 3, and full-length K8. No substan-
tial changes were observed after incubation with
Peptide 3 [Fig. 5(C,D)]. In contrast, incubation with
Peptide 1 caused small but important differences,
which were stronger with DF508-NBD1 [Fig. 5(B)]
than with wt-NBD1 [Fig. 5(A)]. In complex with
Peptide 1, both wt-NBD1 and DF508-NBD1 showed
decreased HDX at positions 429–433 (the RI region),
suggesting that K8 may bind non-specifically to both
NBD1s via this region. However, SPR revealed a
higher affinity of Peptide 1 for DF508-NBD1,
(Kapp
D54.6 lM) and a 4-fold lower dissociation rate
constant, indicating stronger stabilization with the
mutant NBD1 [Fig. 6(A,B)]. In line with this reason-
ing, the region 496–504 preceding the mutation was
strongly destabilized upon binding to both wt-NBD1
and DF508-NBD1. Moreover, full-length K8 binds
more strongly (Kapp
D529 nM)toDF508-NBD1,
20
and
our present data indicated that this stronger binding
was accompanied by widespread allosteric changes
in DF508-NBD1 that were not observed for wt-
NBD1. This further justifies the additional binding
sites we observed in the HDX experiments, indicat-
ing the higher stability of the DF508-NBD1–K8 com-
plex. These mutant-specific changes also
encompassed the mutation site. Thus, it is possible
that the K8 head contains a non-specific binding site
for both NBD1s, along with an additional binding
site for DF508-NBD1.
Two types of experiments, analyzing the binding
of NBD1s to Peptide 1 and to full-length K8, sug-
gested that one such DF508-NBD1-specific binding
site may be located near F508. The 505–520 region
was stabilized upon K8 binding only in DF508-
NBD1. On the other hand, the preceding region
496–504 became strongly destabilized upon binding
Figure 9. Isotopic envelopes after 10 s of exchange for the
peptides, E
79
VD...ASF
105
from the K8 head domain before
binding with NBDs (ii), and after binding with wt-NBD1 (iii) or
with DF508-NBD1 (iv). These isotopic envelopes are com-
pared with the minimum possible exchange (IN, 0 s) (i) and
the maximum possible exchange (MAX, 24 h) (v). The center
of mass (m/z) is marked in red, and the values are flagged.
Premchandar et al. PROTEIN SCIENCE VOL 26:343—354 351
to both wt-NBD1 and DF508-NBD1. If K8 binds
directly to this region, the binding may involve inter-
actions with side chains, with simultaneous weaken-
ing of the H-bonding networks of amide protons in
this region. In DF508-NBD1, this effect may be
enhanced by the preferential binding of K8 to the
region of the mutation (505–520). The enhanced bind-
ing to an alternative binding site led to allosteric sta-
bilization/destabilization of several other subdomains
in DF508-NBD1, including the F1-like ATP-binding
domain, the central strand of the ABCbsubdomain,
and the NBD1–NBD2 interface [Fig. 7(C,D)].
These allosteric changes may further alter the
availability of potential binding sites for other currently
unknown factors, allowing the cell to discriminate
DF508-NBD1 from wt-NBD1 and to direct only the
mutated protein into a degradation pathway. For
instance, the 3D model shows that the region 491–504 is
involved in NBD1 binding to the ICL4 in the case of full-
length CFTR.
22
Increased affinity of the mutated NBD1
to K8 may thus lead to competitive shielding of the 491–
504 region against binding to the ICL4 loop during
CFTR protein folding, which in the case of DF508 would
preclude escape from degradation pathway and inhibit
biosynthesis of the fully matured protein.
Conclusions
In conclusion, we identified a K8 head peptide that
participates in the binding to NBD1 and showed
preferential binding to DF508-NBD1. Our data sug-
gest that K8 may contain an additional NBD-
binding region that provides an additional mutant-
specific contact site in the vicinity of the DF508
mutation. This additional contact increases the sta-
bility of the K8-DF508-NBD1 complex, causing allo-
steric changes across the entire molecule, and may
provide a trigger for its release from the ER and
direction to the degradation system.
Materials and Methods
HDX-MS experiments
The HDX experiments were performed as described
previously
15
with minor modifications. Briefly, we
used stock solutions of human NBD1 (with solubili-
zation mutation F494N, wt-NBD1, and DF508-
NBD1) at a concentration of 4 mg/ml dissolved in
reaction buffer and supplemented with 10% glycerol
and 2 mMATP. We diluted 5 mL of NBD1 103by
addition 45 mL of reaction solution with D
2
O (50 mM
Tris-HCl, pH 8.4; 150 mMNaCl). After 10 s or 1
min, the samples were acidified by addition of 10 mL
D
2
O Stop Buffer (2Mglycine buffer, pH 2.5). We
then added 2 mL of the K8 peptide solution to obtain
a 10-fold excess of peptide over the protein. To sam-
ples without peptide, we added 2 mL of the K8 pep-
tide solvent alone. K8 exists predominantly as a
dimer in 5 mMTris-HCl (pH 8.4),
15
and this buffer
condition was chosen as most conducive to our anal-
ysis of the protein system because K8 heavily aggre-
gates and precipitates out in high ionic strength
buffers. The samples were then acidified by addition
of 10 mLH
2
O stop buffer.
The samples were digested using a 2.1 330 mm-
immobilized pepsin resin column (Porozyme, ABI,
Foster City, CA) with 0.07% formic acid in water as
the mobile phase and a flow rate of 200 mL/min. The
peptides passed directly to the 2.1 35mm
2
C18 trap-
ping column (ACQUITY BEH C18 VanGuard precol-
umn, 1.7-mm resin; Waters, Milford, MA). Trapped
peptides were then eluted onto a reversed-phase col-
umn (Acquity UPLC BEH C18 column, 1.0 3100 mm,
1.7-mm resin; Waters, Milford, MA) using a 8–40%
gradient of acetonitrile in 0.1% formic acid at 40 mL/
min, controlled by the nanoACQUITY Binary Solvent
Manager. A single run lasted a total of 13.5 min. All
fluidics, valves, and columns were maintained at
0.58C using the HDX Manager (Waters, Milford,
MA)—except the pepsin digestion column, which was
maintained at 138C inside the temperature-controlled
digestion column compartment of the HDX Manager.
The C18 column outlet was coupled directly to the ion
source of a SYNAPT G2 HDMS mass spectrometer
(Waters, Milford, MA) operating in ion mobility mode.
Lock mass was activated and performed using
leucine-enkephalin (Sigma). For protein identifica-
tion, mass spectra were acquired in MS
E
mode over
the m/zrange of 50–2000. The spectrometer parame-
ters were as follows: ESI positive mode; capillary volt-
age, 3 kV; sampling cone voltage, 35 V; extraction
cone voltage, 3 V; source temperature, 808C; desolva-
tion temperature, 1758C; and desolvation gas flow,
800 L/h. The spectrometer was calibrated using stan-
dard calibrating solutions.
Peptides were identified using ProteinLynx
Global Server (PLGS) software (Waters, Milford,
MA). We used a randomized database with the fol-
lowing PLGS parameters: minimum fragment ions
per peptide, 4; false–positive rate threshold, 4%. The
identified peptides—along with peptide m/z, charge,
retention time, and ion mobility/drift time—were
passed to the DynamX 2.0 hydrogen–deuterium data
analysis program (Waters, Milford, MA).
HDX experiments were performed as described
for nondeuterated samples, with the reaction buffer
prepared using D
2
O (99.8%; Cambridge Isotope Lab-
oratories), and pH (uncorrected meter reading)
adjusted using DCl (Sigma). After mixing 5 mL pro-
tein stock with 45 mLD
2
O reaction buffer, the
exchange reactions were conducted at room temper-
ature for varying times, as specified in the text. The
exchange was quenched by addition of stop buffer
(2Mglycine buffer, pH 2.5) and cooling on ice. Imme-
diately after quenching, the sample was manually
injected into the nanoACQUITY UPLC system
(Waters, Milford, MA). Subsequently, pepsin
352 PROTEINSCIENCE.ORG CFTR/K8 Complex Structural Insights
digestion and liquid chromatography (LC) and MS
analyses were performed exactly as described above
for non-deuterated samples.
Two control experiments were performed to
account for in- and out-exchange artifacts, as
described previously.
15
Briefly, to assess minimum
exchange (in-exchange control), D
2
O reaction buffer
was added to stop buffer that had been cooled on ice
before protein stock addition, and this mixture was
immediately subjected to pepsin digestion and LC-
MS analysis as described above. The deuteration lev-
el in the in-exchange experiment was calculated (as
described below) and denoted as 0% exchange
(M
ex0
). For out-exchange analysis, 5 mL of protein
stock was mixed with 45 mLofD
2
O reaction buffer,
incubated for 24 h, mixed with stop buffer, and ana-
lyzed as described above. The deuteration level in
an out-exchange experiment was calculated and
denoted as 100% exchange (M100
ex ).
The above-described experiments enabled us to
obtain the same set of fragments from the control
and HDX experiments. Each experiment was repeat-
ed three times, and the results are presented as the
mean of these replicates.
HDX data analysis
For each peptide resulting from the exchange, the
deuteration level was automatically calculated using
DynamX 2.0 software. These calculations were per-
formed using the peptide list obtained from the PLGS
program, with further filtering in the DynamX 2.0
program using the following acceptance criteria: mini-
mum intensity threshold, 3000; minimum products
per amino acids, 0.3. Post-exchange isotopic envelopes
were analyzed in DynamX 2.0 with the following
parameters: RT deviation, 615 s; m/zdeviation,
612.5 ppm; drift time deviation, 62-time bins. The
calculated average masses of the peptides in the
exchange experiment (M
ex
) and in the two control
experiments (M
ex0
and M100
ex ) were then verified by
visual inspection. Ambiguous or overlapping isotopic
envelopes were discarded from further analysis.
When a split isotopic envelope was observed, separate
M
ex
values corresponding to each envelope were cal-
culated using the MassLynx program.
Final data were exported to an Excel (Microsoft)
spreadsheet for calculation of HDX mass shifts and
fractions of exchange. The percentage of relative
deuterium uptake (% Deuteration) for a given pep-
tide was calculated by accounting for both control
values using the formula:
% Deuteration5Mex2M0
ex

M100
ex 2M0
ex

3100
Exchange fraction (f) error bars were calculated as
standard deviations of three independent experiments.
Thedifferenceinexchange(DHDX) between two condi-
tions of interest was determined by subtracting the
fraction of exchange measured in these conditions.
Errors for DHDX values were calculated as the square
root of the sum of variances of the subtracted numbers.
Final figures were plotted using OriginPro 8.0 (Origin-
Lab) software.
Surface plasmon resonance
To investigate the interaction of Peptide 1 with the
wt and DF508 NBD1, we used real-time SPR techni-
ques with the Biacore 2000 system (Biacore AB; GE
Healthcare). The wt- and DF508- NBD1 molecules
were covalently immobilized via their primary amino
groups on a CM5 sensor chip (two independent flow-
cell) as described elsewhere.
34
Blank control meas-
urements were performed using one independent
flow-cell on the same sensor chip. Binding experi-
ments were run in TBS buffer (50 mMTris-HCl, pH
7.4; 150 mMNaCl; 5 mMMgCl
2
;1mMDTT) con-
taining 0.005% (w/v) surfactant P20. The association
between Peptide 1 and immobilized NBD1 was mon-
itored by injecting different concentrations of Pep-
tide 1 (70–370 mM) at 208C, with a flow rate of 30 mL
min
21
over 60 s. Between injections, the sensor chip
was regenerated by a single wash with 10 mLof
5mMNaOH, followed by two washes with 10 mLof
10 mMHCl. To correct curves for nonspecific bind-
ing, we subtracted control curves obtained by injec-
tion of the different peptide concentrations through
the blank flow channel treated with the same immo-
bilization procedure but without NBD1.
Acknowledgments
AK and MFdC were supported by Vaincre la
mucoviscidose.
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354 PROTEINSCIENCE.ORG CFTR/K8 Complex Structural Insights
... HDX-MS also demonstrated that the dynamics of this N-terminal region of NBD1 is directly coupled to the presence of the RI, as we observed very limited exchange in the 392-399 peptide in ΔRI-NBD1 compared to 2PT-NBD1 (Extended Data Fig. 6j). These findings are in agreement with previous HDX studies that identified the RI, the N-terminus and the S4 strand as fast-exchanging regions 31 . ...
... As the unfolding intermediates accumulate, the canonical population is decreased (Fig. 6a,b) and transition to a fully misfolded state promoted (Fig. 6c). The idea that F508del leads to unfolding in the β-subdomain is supported by previous HDX studies of NBD1 featuring only one stabilizing mutation 31 . In these studies, removal of F508 led to substantially different exchange rates only in the region around F508 itself, the N terminus (the S1 strand) and the S2 strand 31 . ...
... The idea that F508del leads to unfolding in the β-subdomain is supported by previous HDX studies of NBD1 featuring only one stabilizing mutation 31 . In these studies, removal of F508 led to substantially different exchange rates only in the region around F508 itself, the N terminus (the S1 strand) and the S2 strand 31 . ...
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The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs. Functional impairment of CFTR due to mutations in the cftr gene leads to cystic fibrosis. Here, we show that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternate conformation that departs from the canonical NBD fold previously observed. Crystallography reveals that this conformation involves a topological reorganization of NBD1. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by adenosine triphosphate binding. However, under destabilizing conditions, such as the disease-causing mutation F508del, this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that, in wild-type CFTR, this conformational transition of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.
... HDX-MS also demonstrated that the dynamics of this region is directly coupled to the presence of the RI as we observed very limited exchange in the 392-399 peptide in ΔRI-NBD1 compared to 2PT-NBD1 (Fig. S6I). These findings are in agreement with previous HDX studies by Premchandar et al. who identified the RI, the N-terminus and the S4 strand as fast-exchanging regions (Premchandar et al., 2017). ...
... As the unfolding intermediates accumulate, the canonical population is decreased (Fig. 5) and transition to a fully misfolded state is promoted (Fig. 7C). The idea that F508del leads to unfolding in the β-subdomain is supported by previous HDX studies of NBD1 featuring only one stabilizing mutation (Premchandar et al., 2017). In these studies, Premchandar et al. showed that removal of F508 led to substantially different exchange rates only in the region around F508 itself, the N-terminus and the S2 strand (Premchandar et al., 2017). ...
... The idea that F508del leads to unfolding in the β-subdomain is supported by previous HDX studies of NBD1 featuring only one stabilizing mutation (Premchandar et al., 2017). In these studies, Premchandar et al. showed that removal of F508 led to substantially different exchange rates only in the region around F508 itself, the N-terminus and the S2 strand (Premchandar et al., 2017). ...
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Cystic Fibrosis (CF) is a common lethal genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. Misfolding and degradation of CFTR are the hallmarks of the predominant mutation, F508del, located in the first nucleotide binding domain (NBD1). While the mutation is known to affect the thermal stability of NBD1 and assembly of CFTR domains, the molecular events that lead to misfolding of F508del-CFTR remain elusive. Here, we demonstrate that NBD1 of CFTR can adopt an alternative conformation that departs from the canonical NBD fold previously observed for CFTR and other ATP-binding cassette (ABC) transporter proteins. Crystallography studies reveal that this conformation involves a topological reorganization of the β-subdomain of NBD1. This alternative state is adopted by wild-type CFTR in cells and enhances channel activity. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by ATP binding. Under destabilizing conditions, however, this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that in wild-type CFTR switching to this topologically-swapped conformation of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.
... Cytokeratin 8 (K8), that binds F508del-NBD1 with a higher affinity that the WT, suggesting that K8 contributes to F508del-CFTR retention and pointing to destruction of K8/F508del-CFTR interaction as a therapeutic target [63]. K8 fragments have been also used in SPR to map the binding interface of the K8/CFTR complex to design of specific inhibitors [68]. Similarly, also heat shock cognate 70 (HSC70) binds F508del-CFTR with higher affinity in respect to WT CFTR [14]. ...
... (iv) As already mentioned above, the binding of mutated CFTR to chaperones responsible for its retention is considered a therapeutic target to rescue CFTR activity. Relevantly, when tested in SPR competition assays, Corr-4a, VRT-325 and CFTRinh-172 effectively inhibit the binding of HSC70 to sensorchip immobilized F508del-CFTR [14,68]. ...
... iv) As already mentioned above, the binding of mutated CFTR to chaperones responsible for its retention is considered a therapeutic target to rescue CFTR activity. Relevantly, when tested in SPR competition assays, Corr-4a, VRT-325 and CFTRinh-172 effectively inhibit the binding of HSC70 to sensorchip immobilized F508del-CFTR [14,68]. ...
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Cystic fibrosis transmembrane conductance regulator (CFTR)-rescuing drugs have already transformed cystic fibrosis (CF) from a fatal disease to a treatable chronic condition. However, new-generation drugs able to bind CFTR with higher specificity/affinity and to exert stronger therapeutic benefits and fewer side effects are still awaited. Computational methods and biosensors have become indispensable tools in the process of drug discovery for many important human pathologies. Instead, they have been used only piecemeal in CF so far, calling for their appropriate integration with well-tried CF biochemical and cell-based models to speed up the discovery of new CFTR-rescuing drugs. This review will give an overview of the available structures and computational models of CFTR and of the biosensors, biochemical and cell-based assays already used in CF-oriented studies. It will also give the reader some insights about how to integrate these tools as to improve the efficiency of the drug discovery process targeted to CFTR.
... HDX-MS also demonstrated that the dynamics of this region is directly coupled to the presence of the RI as we observed very limited exchange in the 392-399 peptide in ΔRI-NBD1 compared to 2PT-NBD1 (Fig. S6I). These findings are in agreement with previous HDX studies which identified the RI, the N-terminus and the S4 strand as fast-exchanging regions 36 . ...
... As the unfolding intermediates accumulate, the canonical population is decreased (Fig. 5) and transition to a fully misfolded state is promoted (Fig. 7C). The idea that F508del leads to unfolding in the b-subdomain is supported by previous HDX studies of NBD1 featuring only one stabilizing mutation 36 . In these studies, removal of F508 led to substantially different exchange rates only in the region around F508 itself, the N-terminus (the S1 strand) and the S2 strand 36 . ...
Preprint
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The cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is essential to maintain fluid homeostasis in key organs such as the lungs or the digestive systems. Functional impairment of CFTR due to mutation in the cftr gene lead to Cystic Fibrosis (CF) the most common lethal genetic disorder. Here we observe that the first nucleotide-binding domain (NBD1) of CFTR can spontaneously adopt an alternative conformation that departs from the canonical NBD fold previously observed for CFTR and other ATP-binding cassette (ABC) transporter proteins. Crystallography studies reveal that this conformation involves a topological reorganization of the β-subdomain of NBD1. This alternative state is adopted by wild-type CFTR in cells, where it leads to enhanced channel activity. Single-molecule fluorescence resonance energy transfer microscopy shows that the equilibrium between the conformations is regulated by ATP binding. However, under destabilizing conditions, such as a the prominent disease-causing mutation F508el , this conformational flexibility enables unfolding of the β-subdomain. Our data indicate that in wild-type CFTR switching to this topologically-swapped conformation of NBD1 regulates channel function, but, in the presence of the F508del mutation, it allows domain misfolding and subsequent protein degradation. Our work provides a framework to design conformation-specific therapeutics to prevent noxious transitions.
... Having said this, KRTs 8, 18, and 19 have all been reported to functionally interact with the cystic fibrosis transmembrane regulator (CFTR), a multispan transmembrane receptor. The interaction of KRT8 with the CFTR∆F508 mutant, the most common mutation in patients with cystic fibrosis, at nucleotide-binding domain 1 (NBD1) inhibits the translocation of CFTR to the cell surface [35,36]. A deletion of KRT8 rescued CFTR cell surface localization [37]. ...
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Cystic Fibrosis Transmembrane conductance Regulator, CFTR, is a membrane protein expressed in epithelia. A protein kinase A (PKA)-regulated Cl− channel, it is a rate-limiting factor in fluid transport. Mutations in CFTR are responsible for cystic fibrosis, CF, an autosomal recessive disease. The most frequent mutation is deletion of phenylalanine at position 508, delF508. The regulation of trafficking and degradation of CFTR/delF508CFTR as well as its function(s) is a complex process which involves a number of proteins including chaperones and adaptors. It is now known that cytoskeletal proteins, previously considered only as structural proteins, are also important factors in the regulation of cellular processes and functions. The aim of the present review is to focus on how microfilaments, microtubules and intermediary filaments form a dynamic interactome with CFTR to participate in the regulation of CFTR-dependent transepithelial ion transport, CFTR trafficking and degradation.