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Yang and Mingjie Zhang
Youjun Li, Zhiyi Wei, Junyi Zhang, Zhou
Substrate Adaptor DCAF1
FERM Domain to the E3 Ubiquitin Ligase
Structural Basis of the Binding of Merlin
Developmental Biology:
published online April 4, 2014J. Biol. Chem.
10.1074/jbc.M114.551184Access the most updated version of this article at doi:
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Structure of the Merlin FERM/DCAF1 complex
1
Structural Basis of the Binding of Merlin FERM Domain to the E3 Ubiquitin Ligase Substrate
Adaptor DCAF1
Youjun Li1,4, Zhiyi Wei1,2,3,4, Junyi Zhang1, Zhou Yang1, and Mingjie Zhang1,2,*
1Division of Life Science, State Key Laboratory of Molecular Neuroscience, Hong Kong University
of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
2Center of Systems Biology and Human Health, School of Science and Institute for Advanced Study,
Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China
3Department of Biology, South University of Science and Technology of China, Shenzhen, China
Running title: Structure of the Merlin FERM/DCAF1 complex
4Contributed equally to this work
* To whom correspondence should be addressed: Mingjie Zhang, Division of Life Science, State Key
Laboratory of Molecular Neuroscience, Hong Kong University of Science and Technology, Clear
Water Bay, Kowloon, Hong Kong, China, Tel.: +852-23588709; Fax: +852-23581552; Email:
mzhang@ust.hk
Keywords: Merlin; DCAF1; FERM; Tumor suppressor gene; Hippo pathway; NF2; VPRBP
Background: Merlin controls organ size by
binding to target proteins both in cytoplasm and
nucleus.
Results: The structure of Merlin FERM domain
in complex with its binding domain of DCAF1 is
determined.
Conclusion: DCAF1 folds into a β-hairpin
structure and binds to the F3 lobe of Merlin
FERM domain.
Significance: The structure of the
Merlin/DCAF1 complex provides a template for
understanding Merlin’s interactions with its
binding partners.
ABSTRACT
The tumor suppressor gene Nf2 product,
Merlin, plays vital roles in controlling proper
development of organ sizes by specifically
binding to a large number of target proteins
localized both in cytoplasm and nuclei. The
FERM domain of Merlin is chiefly
responsible for its binding to target proteins,
although the molecular basis governing these
interactions are poorly understood due to
lack of structural information. Here we
report the crystal structure of the Merlin
FERM domain in complex with its binding
domain derived from the E3 ubiquitin ligase
substrate adaptor DCAF1 (also known as
VPRBP). Unlike target binding modes found
in ERM proteins, the Merlin-FERM binding
domain of DCAF1 folds as a β-hairpin and
binds to the α1/5-groove of the F3 lobe of
Merlin-FERM via extensive hydrophobic
interactions. In addition to providing the first
structural glimpse of a Merlin-FERM/target
complex, the structure of the Merlin/DCAF1
complex is likely to be valuable for
understanding Merlin’s interactions with its
binding partners other than DCAF1.
http://www.jbc.org/cgi/doi/10.1074/jbc.M114.551184The latest version is at
JBC Papers in Press. Published on April 4, 2014 as Manuscript M114.551184
Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
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Structure of the Merlin FERM/DCAF1 complex
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Cell-to-cell contact triggers inhibition
signals for cell growth, which is crucial for
living systems to maintain proper organ sizes by
balancing the rate of cell proliferation and
apoptosis. Disruption of the contact inhibition
gives rise to cell overgrowth and often induces
tumor formation (1). The tumor suppressor gene
Nf2, which encodes the protein 4.1, Ezrin,
Radixin, Moesin (FERM) domain-containing
protein Merlin, is an important determinant in
contact-mediated cell growth inhibition (2).
Consistent with its critical role in growth
inhibition, loss-of-function mutations of Merlin
are known to cause a series of tumor formations,
including the familial cancer syndrome
neurofibromatosis type 2 and several other
carcinomas (3–6). Mechanistically, Merlin is
known for its central roles in the Hippo pathway
in controlling organ sizes (7, 8). Recent
investigation indicated that Merlin is also
involved in a distinct cell growth regulation
mechanism by accumulating in nucleus and
inhibiting the activity of nuclear E3 ubiquitin
ligase via binding to its substrate adaptor
DCAF1 (also known as viral protein R-binding
protein, VPRBP) (9). The interaction between
Merlin and DCAF1 is mediated by the FERM
domain of Merlin (Merlin-FERM) and the
C-terminal tail of DCAF1 (DCAF1-CT) (9).
However, the molecular basis for the
FERM-mediated Merlin/DCAF1 interaction
remains unknown.
FERM domain is a well-known protein
binding module which is composed of three
subunits, called F1, F2 and F3 lobes (10).
Numerous FERM domain-containing proteins
have been identified and classified into different
subfamilies (10). As one of the best studied
subfamily of FERM superfamily, Ezrin, Radixin,
and Moesin (ERM) are the most closely related
ones to Merlin. Similar to ERM proteins, Merlin
has ~600 residues in length and contains the
N-terminal FERM domain, a central helical
region, and a C-terminal regulatory domain
(CTD) (Fig. 1A). Despite of the high sequence
similarity with ERM proteins, especially their
FERM domains (sequence identity of ~60%),
Merlin was shown to have distinct tissue
localization and functions (11). This functional
specificity indicates that the target binding
mechanism of Merlin is different from ERM
proteins. Extensive studies have implicated a
number of potential Merlin targets (8, 9, 12, 13),
most of which (including DCAF1) were found to
interact with its FERM domain. Structural
studies confirmed that Merlin-FERM share high
structural similarity with the FERM domains of
ERM proteins (14, 15). However, due to lacking
of target-bound structures of Merlin-FERM, the
mechanisms underlying target recognitions of
Merlin were modelled largely based on the
structures of ERM proteins (especially Radixin)
in complex with their targets.
Here, we show that Merlin-FERM directly
binds to a short fragment of DCAF1 located at
the extreme C-terminal end (termed FERM
binding domain or FBD, Fig. 1A). We
determined the crystal structure of
Merlin-FERM/DCAF1-FBD complex at the 2.6
Å resolution. In addition to uncovering the
molecular basis governing the specific
Merlin-FERM/DCAF1 interaction, the complex
structure also reveals a distinct FERM-mediated
targeting mechanism in general. Unlike target
binding modes found in ERM proteins,
DCAF1-FBD folds as a β-hairpin to augment the
β-sheet of the F3 lobe of Merlin-FERM via
extensive hydrophobic interactions. Additionally,
the highly conserved, negatively charged
DCAF1 β-hairpin loop was found to contribute
to the DCAF1/Merlin interaction, likely via
binding to a positively charged cleft between the
F1 and F3 lobes of Merlin-FERM.
EXPERIMENTAL PROCEDURES
Protein expression and purification – The
Merlin constructs (Merlin-FL, residues 1-596;
Merlin-FERM, residues 1-313), and the DCAF1
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Structure of the Merlin FERM/DCAF1 complex
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constructs (DCAF1-CT, residues 1417-1507;
DCAF1-FBD, residues 1478-1507) were
amplified by PCR using the mouse and human
cDNA libraries as the template, respectively, and
individually cloned into the modified pET32a
vector. Various mutants were created using
standard two-step PCR-based methods and
confirmed by DNA sequencing. Recombinant
proteins with N-terminal Trx- and His6-tagged
were transformed to E. coli BL21(DE3) cells,
cultured at 37 °C to OD ~ 0.6 and induced
with 0.2 mM IPTG at 16 °C overnight. The
expressed proteins were purified by a Ni2+-NTA
agarose affinity chromatography followed by a
size-exclusion chromatography. During
purification, all protein samples were detected
and analyzed by SDS-PAGE coupled with
Coomassie blue staining.
Isothermal titration calorimetry (ITC) assay –
ITC was carried out on a MicroCal VP-ITC at
25°C. All proteins were dissolved in a buffer
containing 50 mM Tris pH7.5, 250 mM NaCl, 1
mM DTT, and 1 mM EDTA. The titration
processes were performed by injecting 5-10 μL
aliquots of protein samples in syringe
(concentration of 100 μM) into protein samples
in cell (concentration of 10 μM) with 27 times
and at time intervals of 120s to ensure the
titration peak returned to the baseline. The data
were analyzed using the Origin 7.0 and fitted by
the one-site binding model.
Crystallization – For crystallization, the tagged
proteins were treated by a small amount of HRV
3C protease at 4°C overnight to cleave the fusion
tags and further purified by a size-exclusion
chromatography. Crystals of the
Merlin-FERM/DCAF1-FBD complex were
obtained by hanging drop vapor diffusion
method at 16°C within 5 days. To set up a
hanging drop, 1 μL of concentrated protein
mixture (~20 mg/mL) at 1:1 stoichiometric ratio
was mixed with 1 μL of crystallization solution
with 20% Isopropanol and 5% PEG8000, pH 8.0.
Before diffraction experiments, crystals were
soaked in crystallization solution containing
30% glycerol for cryoprotection. The diffraction
data were collected at Shanghai Synchrotron
Radiation Facility and were processed and
scaled using HKL2000 (16) (Table 1).
Structure determination – The initial phase was
determined by molecular replacement using the
apo form of Merlin-FERM (PDB code: 1ISN) as
the searching model. The model was refined in
Phenix (17) against the 2.6 Å dataset. The
DCAF1-FBD peptide was built subsequently in
COOT (18). In the final stage, an additional TLS
refinement was performed in Phenix. The final
model was further validated by using
MolProbity (19). The refinement statistics are
listed in Table 1. The structural model of
DCAF1-FBD was well assigned except for the
connecting loop for the β-hairpin structure. All
structure figures were prepared using PyMOL
(http://pymol.sourceforge.net/). The sequence
alignments were prepared and presented using
ClustalW (20) and ESPript (21), respectively.
RESULTS
The FERM domain of Merlin specifically binds
to a short, C-terminal tail fragment of DCAF1
– Merlin-FERM was shown to interact with the
DCAF1 C-terminal region recently (9). We first
tried to verify this interaction using purified
recombinant proteins. Quantitative binding
assays showed that the C-terminal region of
DCAF1 (DCAF1-CT, residues 1417-1507) binds
to Merlin-FERM with a dissociation constant
(Kd) of ~3 μM (Fig. 1B). Further boundary
mapping revealed that the N-terminal part of
DCAF1-CT (residues 1417-1477) showed no
detectable binding to Merlin-FERM (Data not
shown), and the remaining C-terminal 30
residues (residues 1478-1507) displayed the
same binding affinity as the entire DCAF1-CT
does (Fig. 1C), indicating that the last 30
residues in DCAF1 contains the complete FERM
binding domain (DCAF1-FBD). The binding of
Merlin-FERM to DCAF1-FBD is mainly driven
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Structure of the Merlin FERM/DCAF1 complex
4
by enthalpy (Fig. 1B&C). Interestingly, although
sharing a high amino acid sequence identity with
Merlin, the FERM domain of Moesin had no
detectable binding to DCAF1-FBD (Fig. 1D),
indicating that the FERM domain of Merlin
encodes its intrinsic target binding specificities.
A Merlin-FERM chimera (termed as
Merlin-FERMF3/Moesin), in which the F3 lobe was
replaced by the corresponding F3 lobe of Moesin,
failed to bind DCAF1-FBD (Fig. 1E), indicating
that the F3 lobe is chiefly responsible for
Merlin-FERM to bind to DCAF1.
Overall Structure of the
Merlin-FERM/DCAF1-FBD complex – To
understand the molecular basis governing the
Merlin/DCAF1 interaction, we determined the
crystal structure of Merlin-FERM in complex
with DCAF1-FBD at 2.6 Å resolution using the
molecular replacement method (Table 1). In the
crystal structure, Merlin-FERM and
DCAF1-FBD forms a 1:1 ratio complex with
two complexes per asymmetric unit (Fig. 2A). In
the complex, Merlin-FERM adopts a typical
FERM architecture, comprised of three lobes, F1,
F2, and F3. Consistent with the biochemistry
data shown in Fig. 1, the DCAF1-FBD peptide
folds as a β-hairpin to bind to the α1/5-groove
of the F3 lobe of Merlin-FERM (Fig. 2A). The
two β-strands of DCAF1-FBD are well-defined,
whereas the loop connecting the hairpin is totally
disordered (Fig. 2A&B). The overall fold of
Merlin-FERM in the DCAF1-FBD complex is
almost identical to the apo-form structure (14)
and the FERM domain of Radixin in complex
with CD44 (22) (the overall RMSD of 0.7 Å
with 285 aligned residues and of 0.9 Å with 294
aligned residues, respectively), indicating that
the DCAF1 binding does not induce obvious
conformational changes to the F3 lobe as well as
the entire FERM domain. By analyzing the B
factor distribution of Merlin-FERM in complex
and apo forms (Fig. 2C), we found that the F3
lobe in the DCAF1-bound structure shows
similar B-factors with that in the apo-form
structure, although the overall B-factor of the
DACF1-bound structure is much higher than that
of the apo structure, suggesting that the binding
to DCAF1 stabilizes the F3 lobe of the Merlin
FERM domain.
An atypical target binding mode for FERM
domains revealed by the
Merlin-FERM/DCAF1-FBD complex –
Although other lobes are also known to
participate in binding to target proteins, the F3
lobes of FERM domains are the major target
binding sites (22–31). The F3 lobes of Merlin
and ERM proteins adopt a PTB-like fold,
consisting of a seven-stranded β-sandwich and a
C-terminal α-helix (14, 15, 24) (Fig. 2A). A
groove (termed α-groove) mainly formed by
5F3 and α1F3 is a well-characterized binding
region in FERM domains including those of
Moesin (24), Radixin (22, 25–28), Talin (29, 32,
33), Myosin-X (30, 34), and Sorting Nexin-17
(31). Most of these targets, especially those of
ERM-binding proteins, bind to the α-groove
via a β-strand or a β-strand-like extended
structures (Fig. 3B&C), and thus the contact
between the target and the F3 lobe is not
extensive (and their interactions are often very
weak; Fig. 3B). DCAF1-FBD also binds to the
α-groove of Merlin-FERM (Fig. 3A). However,
instead of using a single β-strand, DCAF1-FBD
adopts a β-hairpin structure and augments one of
the anti-parallel β-sheets of Merlin-FERM
formed by 5F3, 6F3, and 7F3. Residues from
both DCAF1-FBD β-hairpin strands participate
in the binding to Merlin-FERM, mainly via
hydrophobic interactions (Fig. 3A). The highly
conserved hydrophobic residues (V1482 and
L1484 in βA and I1501, L1503, and L1505 in
βB) from DCAF1-FBD contact with the
hydrophobic α-groove to form a hydrophobic
core (Fig. 3A and 3D), presumably stabilizing
the β-hairpin folding of DCAF1-FBD as well as
the Merlin/DCAF1 complex. Consistent with
this analysis, the ITC-based measurements
showed that the mutant with the deletion of
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Structure of the Merlin FERM/DCAF1 complex
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either A or B in DCAF1-FBD failed to bind to
Merlin-FERM (Table 2). Similarly, disruption of
the hydrophobic interaction by substituting
L1503, which is central to the hydrophobic core
formed by DCAF1-FBD and the α-groove of
the F3 lobe, with lysine also abolished the
binding of DCAF1 to Merlin (Table 2). In
addition, several conserved salt bridges formed
between negatively charged residues (E1480 and
E1481) in DCAF1-FBD and positively charged
residues (R309 and K312) in the F3 lobe also
contribute to the FBD/F3 interaction (Fig. 3A).
Fitting with this analysis, a charge-reversal
mutation of DCAF1-FBD (E1480K/E1481K)
largely impaired its binding to Merlin-FERM
(Table 2).
The negatively charged connecting loop of the
DCAF1-FBD hairpin is likely to be involved in
the binding to Merlin-FERM – We were
surprised to find that the most conserved region
in DCAF1-FBD is the structurally disordered,
β-hairpin connecting loop (Fig. 3E). This loop is
rich in negatively charged residues as well as
four serine/threonine residues, some of which
are predicted to be potential phosphorylation
sites of protein kinases such as CK2.
Interestingly, a cleft nearby the α-groove and at
the inter-face of the F1 and F3 lobes of
Merlin-FERM is highly positively charged (Fig.
4A). A number of conserved residues from both
the F1 and F3 lobes form this positively charged
cleft (Fig. 3D and Fig. 4A). The binding of the
DCAF1-FBD hairpin to the α-groove of the F3
lobe juxtaposes the negatively charged β-hairpin
loop to the positively charged F1/F3-cleft of the
FERM domain (Fig. 4A). It is rational to
hypothesize that the charge-charge attraction
between the DCAF1-FBD hairpin loop and
Merlin-FERM F1/F3-cleft may further enhance
the Merlin/DCAF1 interaction. To test this
hypothesis, we deleted the connecting loop or
mutated five negatively charged residues in the
connecting loop to non-charged Ala
(D1490A/D1493A/D1496A/E1498A/D1499A)
and measured their binding affinity with
Merlin-FERM, and found that both of the
mutants indeed showed decreased binding (albeit
rather modestly) to Merlin-FERM (Table 2). It is
possible that addition of negative charges by
phosphorylation(s) of Ser/Thr within the loop
may further enhance the interaction between
DCAF1 and Merlin-FERM. We note with
interest that the F1/F3 cleft of the ERM proteins
(e.g. the Radixin FERM domain shown in Fig.
4B) is also positively charged. Importantly, the
positively charged F1/F3 cleft of Radixin-FERM
is known to bind to negatively charged
phosphoinositol phosphates such as InsP3, which
is involved in the activation of ERM proteins
(35).
The auto-inhibited and structurally closed form
of Merlin cannot bind to DCAF1-FBD –
Similar with ERM proteins, Merlin is believed to
adopt a closed conformation in solution, in
which the C-terminal regulatory tail binds to the
FERM domain and keeps the full-length Merlin
in an auto-inhibited conformation (36). Based on
the structure of auto-inhibited Moesin (24, 37)
and in view of the very high amino acid
sequence identity between Merlin and Moesin, it
is generally accepted that the C-terminal tail of
Merlin is likely to binding to its own FERM
domain with a mode similar to that found in the
Moesin. If this model-based analysis were true,
the auto-inhibitory tail binding site and the
DCAF1-FBD binding site on the FERM domain
partially overlap with each other. We predict that
DCAF1-FBD is not expected to bind to the
full-length, auto-inhibited Merlin, as the
intra-molecular FERM head and inhibitory tail
interaction would prevail. To test our hypothesis,
we measured the binding affinity between the
full length Merlin protein (Merlin-FL) and
DCAF1-FBD. Fully fit with our structural
analysis and prediction, Merlin-FL showed no
detectable binding to DCAF1-FBD (Fig. 1F).
This data indicate that the productive interaction
between Merlin and DCAF1 will require Merlin
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Structure of the Merlin FERM/DCAF1 complex
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to be activated (i.e. release of the tail inhibitory
domain from the FERM domain) by certain
regulatory factor(s). Our in vitro biochemical
binding data obtained using highly purified
recombinant proteins are in apparent odd with
the conclusion drawn by Li et al (9) (see
“Discussion” for details). Curiously, the
phosphorylation mimic S518D-mutant of the full
length Merlin (Merlin-FLS518D) also showed no
detectable binding to DCAF1 (Fig. 1G), a
finding that is consistent with that by Li et al.
(9).
DISCUSSIONS
Although having been studied for many
years, the target binding mechanisms for Merlin
remains elusive, partly due to the lack of the
structural data of the full-length Merlin or
Merlin-FERM in complex with its targets.
Currently, much of the mechanistic
interpretations of Merlin/target interactions are
derived from the homology-based structural
models based on the structures of ERM proteins
as the templates. The crystal structure of
Merlin-FERM in complex with DCAF1 provides
the first atomic picture of how the FERM
domain of Merlin recognizes its target.
Specifically, Merlin-FERM uses the
conventional α-groove in the F3 lobe as the
binding site to interact with DCAF1. However,
the binding mode between Merlin-FERM and
DCAF1 is distinct from those of ERM proteins
(22, 28). Merlin-FERM binds to DCAF1 with a
much more extensive hydrophobic interface. In
this novel binding mode, DCAF1 adopts a
β-hairpin conformation with residues from both
strands participating in binding to Merlin.
Additionally, the binding of the DCAF1
β-hairpin to the α-groove of the F3 lobe
positions the negatively charged β-hairpin loop
closely to the positively charged FERM F1/F3
cleft and thereby enhances the Merlin/DCAF1
interaction. Together, our structural and
biochemical analysis reveals a distinct target
binding mode of Merlin-FERM.
The FERM domain of Merlin interacts with
its own tail, and this intra-molecular interaction
is believed to keep Merlin in an auto-inhibited
conformation (36). Release of the auto-inhibition
(e.g. by truncation of a part of its C-terminal tail)
constitutively activates Merlin (8), indicating
that the FERM domain-mediated activities of
Merlin requires the release of the C-terminal
tail-mediated auto-inhibition. Interestingly,
phosphorylation of S518 or substitutions of S518
with Glu/Asp in the predicted helical region
between the FERM domain and the inhibitory
tail domain convert Merlin into a functionally
less active state (36). This functionally less
active state of Merlin is often equated to a
conformationally more closed state, although no
direct biochemical or structural evidences exist.
We show, using highly purified proteins in vitro,
that the WT C-terminal tail auto-inhibited
Merlin FERM cannot bind to DCAF1 (Fig. 1F),
and the full-length S518D-Merlin cannot bind to
DCAF1 either (Fig. 1G). The data indicate that
the biologically less active S518D-Merlin does
not necessary correspond to conformational
more closed form of Merlin FERM domain. We
hypothesize that additional factor(s) can sense
the phosphorylation status of S518 and thus
regulate the biological activity of Merlin.
Finding such Merlin regulatory factor(s) will be
an important future topic in the Hippo signaling
pathway.
The unique target binding specificities of
the FERM domains are likely to account for a
large part of functional differences between
Merlin and ERM proteins. With this in mind, we
carefully analyzed the DCAF1-FBD binding site
on Merlin-FERM. Despite of large affinity
difference in the bindings of DCAF1-FBD to the
FERM domains of Merlin and Moesin (Fig.
1C&D), the amino acid residues of the
DCAF1-FBD binding site (i.e. the α-groove) in
Merlin, are highly conserved across Merlin and
ERM proteins except for a few amino acid
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Structure of the Merlin FERM/DCAF1 complex
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residues (Fig. 3D). Paradoxically, substitution of
these few residues in the F3 lobe of
Merlin-FERM with the corresponding residues
from Moesin (Y266F, E270K, D281P, V282D,
K284V, N286Y, L295R, and Q298A) did not
result in a loss of DCAF1 binding (data not
shown). This result indicates that the differences
in other regions of the F3 lobe or even in the F1
or F2 lobes contribute to the unique target
binding specificities of FERM domains from
Merlin and ERM proteins. Additional work is
required to decode such target binding
specificities of FERM domain proteins.
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Acknowledgements – We thank the Shanghai Synchrotron Radiation Facility (SSRF) BL17U for
X-ray beam time.
FOOTNOTES
This work was supported by grants from RGC of Hong Kong to MZ (663610, 663811, 663812,
HKUST6/CRF/10, SEG_HKUST06, AoE/M09/12, and T13-607/12R). MZ is a Kerry Holdings
Professor in Science and a Senior Fellow of IAS at HKUST.
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FIGURE LEGENDS
FIGURE 1. Characterization of the Merlin-FERM/DCAF1 interaction. (A) Domain organizations of
Merlin and DCAF1 are shown as cartoon schemes. The Merlin-FERM/DCAF1-FBD interaction is
indicated by a two-way arrow. (B-G) ITC-based measurements showing the binding affinities
between DCAF1-CT and Merlin-FERM (B), and between DCAF1-FBD and Merlin-FERM (C),
Moesin-FERM (D), Merlin-FERMF3/Moesin (E), Merlin-FL (F) and Merlin-FLS518D(G).
FIGURE 2. Overall structure of the Merlin-FERM/DCAF1-FBD complex. (A) The crystal structure
is present as ribbons diagram with the three lobes of Merlin-FERM, F1 (light green), F2 (green), and
F3 (dark green), and DCAF1-FBD (pink) drawn in their specific colors. The same color code is used
throughout the rest of the figures except as otherwise indicated. The disordered loop connecting two
β-strands is indicated by a dotted line. (B) The simulated annealing omit map (green meshes) for the
DCAF1-FBD hairpin structure. The map was contoured at 3 with the structural model superimposed.
(C) The B-factor (red curve) and real-space (blue curve) correlation plot of Merlin-FERM in our
crystal structure. The B-factor distribution (grey dashed curve) of the apo Merlin-FERM structure
(PDB id: 1ISN) was shown for comparison.
FIGURE 3. Structural and sequence analysis of the Merlin-FERM/DCAF1-FBD interaction. (A)
Molecular details of the Merlin-FERM/DCAF1-FBD interaction. The residues involved in forming
the FERM/FBD interface are shown in the stick mode. Hydrogen bonds and salt bridges are indicated
by dashed lines. (B) and (C) Two examples showing that CD44 (PDB id: 2ZPY) and ICAM-2 (1J19)
bind to the α-groove in the F3 lobe of Radixin (an ERM protein) as the single β-strand structures.
We also measured the binding between the CD44 peptide and Moesin-FERM by ITC and found that
their interaction is very weak (Kd >100 µM). (D) Sequence alignment of the α-groove region in
the FERM domains from Merlin and ERM family members. (E) Sequence alignment of the FBD
regions from DCAF1 proteins across different species. In these two alignments, residues that are
absolutely conserved and highly conserved are highlighted in red and yellow, respectively. The
secondary structural elements are indicated above the alignments. The residues shown in Fig. 3A are
indicated by triangles in (D) and (E), respectively. The disordered region in DCAF1-FBD is indicated
by a dashed line in (E).
FIGURE 4. The negatively charged connecting loop of the DCAF1-FBD β-hairpin contributes to the
binding to Merlin-FERM. (A) The model of the connecting loop interacting with the highly positively
charged F1/F3 cleft on Merlin-FERM. The figure is drawn using electrostatic surface charge potential
map with blue for the positive charge potential and red for the negative charge potential. The
positively charged residues involved in forming the cleft are labeled. (B) The structure of the FERM
domain of Radixin in complex with the headgroup of phosphatidylinositol 4,5-bisphosphate (PDB id:
1GC6) was shown in the same presentation mode as (A) for comparison. The headgroup structure is
highlighted by a yellow circle.
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TABLES
Table 1. Statistics of data collection and model refinement
Data collection
Space group P3121
Unit cell parameters (Å) a = 97.1, c = 224.3
Resolution range (Å) 50 – 2.6 (2.64 – 2.6)
No. of unique reflections 37815
Redundancy 4.6 (4.8)
I/σ 20.11 (2.22)
Completeness (%) 98.16 (99.05)
Rmerge (%)a 7.09 (92.5)
Structure refinement
Rcryst / Rfree (%)b 22.4 (31.9) / 25.6 (35.1)
r.m.s.d bonds (Å) / angles (º) 0.003 / 0.7
Average B factor 96.2
No. of atoms
protein atoms
water molecules
other molecules
5070
31
6
No. of reflections
working set
test set
53440
1994
Ramachandran plotc
favored regions (%)
allowed regions (%)
outliner (%)
97.5
2.2
0.3
a Rmerge = ∑|Ii - Im|/∑Ii, where Ii is the intensity of the measured reflection and Im is the mean intensity
of all symmetry related reflections.
b Rcryst = Σ||Fobs| - |Fcalc||/Σ|Fobs|, where Fobs and Fcalc are observed and calculated structure factors.
Rfree = ΣT||Fobs| - |Fcalc||/ΣT|Fobs|, where T is a test data set of about 5% of the total reflections randomly
chosen and set aside prior to refinement.
c Defined by MolProbity.
Numbers in parentheses represent the value for the highest resolution shell.
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Table 2. ITC-based analysis of the interactions between Merlin-FERM and various
DCAF1-FBD mutants.
DCAF1-FBD Kd (μM)
WT 2.5 ± 0.2
Δ1478-1487 undetectable
Δ1501-1507 undetectable
Δ1490-1499 7.0 ± 2.1
D1490A/D1493A/D1496A/E1498A/D1499A 9.0 ± 1.8
E1480K/E1481K 12 ± 2
L1484K 22 ± 2
L1503K undetectable
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Figure 1.
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Figure 2.
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Figure 3.
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Figure 4.
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