From the Departments of Chemistry and Chemical
Biology, ¶ Molecular Biology and Genetics, and
** Molecular Medicine, Cornell University, Ithaca, New York
14853 and the
Department of Biochemistry and
Biophysics, Oregon State University, Corvallis, Oregon 97331
Received for publication, October 16, 2002
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ABSTRACT |
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Ezrin is a member of the ERM (ezrin,
radixin, moesin) family of proteins that
cross-link the actin cytoskeleton to the plasma membrane and also may
function in signaling cascades that regulate the assembly of actin
stress fibers. Here, we report a crystal structure for the free
(activated) FERM domain (residues 2-297) of recombinant human ezrin at
2.3 Å resolution. Structural comparison among the dormant moesin FERM
domain structure and the three known active FERM domain structures
(radixin, moesin, and now ezrin) allows the clear definition of regions
that undergo structural changes during activation. The key regions
affected are residues 135-150 and 155-180 in lobe F2 and residues
210-214 and 235-267 in lobe F3. Furthermore, we show that a large
increase in the mobilities of lobes F2 and F3 accompanies activation,
suggesting that their integrity is compromised. This leads us to
propose a new concept that we refer to as keystone interactions.
Keystone interactions occur when one protein (or protein part)
contributes residues that allow another protein to complete folding,
meaning that it becomes an integral part of the structure and would
rarely dissociate. Such interactions are well suited for long-lived
cytoskeletal protein interactions. The keystone interactions
concept leads us to predict two specific docking sites within lobes F2
and F3 that are likely to bind target proteins.
The ERM1
(ezrin (1,2)/radixin (3,4)/moesin
(5)) family of proteins serve as regulated cross-linkers between the
actin cytoskeleton and the plasma membrane (reviewed in Refs. 6 and 7).
Ezrin, radixin, and moesin are found in vertebrates as highly similar paralogs (~75% sequence identity) that differ in their primary tissue distributions but probably have a large degree of functional equivalence. In addition to binding to actin filaments and membrane proteins like CD44 (8) and ICAM2 (9), members of the ERM family appear
to function in cell signaling as they interact with several signaling
molecules, including the 85-kDa regulatory subunit of PI3-kinase
(designated p85) (10), the multifunctional regulator, RhoGDI (11),
EBP50 (12), and the tumor suppressor, hamartin (13). At least some of
these various binding activities are regulated by a masking/unmasking
phenomenon (14), and it is becoming apparent that such a mechanism is a
theme common to many cytoskeletal proteins such as vinculin (15, 16),
Wiscott-Aldrich syndrome protein (17), and formins (18). Our
structural and functional studies of the ERM proteins are aimed at
elucidating the details of this type of regulation.
The ~65-kDa ERM proteins consist of three functional domains: an
amino-terminal 300-residue FERM domain (band four-point one, ezrin, radixin, moesin homology
domains) that is responsible for binding to membrane proteins and many
signaling proteins (reviewed in Ref. 7), a central 200-residue putative
coiled-coil region that when phosphorylated on Tyr-353 contributes to
an interaction with p85 (10), and a carboxyl-terminal 100-residue
auto-inhibitory carboxyl-terminal tail domain (also known as the
C-ERMAD) that contains the F-actin binding site (19, 14). In resting
cells, ERM proteins are in a dormant state characterized by an
intramolecular association of the FERM and tail domains. This dormant
state has no known binding partners except perhaps the regulatory
subunit of protein kinase-A (20), although additional partners cannot be ruled out. One pathway of activation (i.e. release of the
FERM-tail interaction) of the ERM proteins appears to be
triggered by phosphorylation of a specific threonine in the tail domain
(Thr-558 in moesin) by the Rho-associated kinase (21) and the protein
kinase C- Our structural understanding of ERM function was given a solid
foundation with the determination of the crystal structure for a
dormant moesin FERM-tail complex (26). It revealed that the FERM domain
is a clover-shaped molecule consisting of three structural domains
(lobes F1, F2, and F3). Residues 2-82 (lobe F1) possess a
ubiquitin-like fold, residues 83-195 (lobe F2) fold into a topology
like that of acyl-CoA-binding protein (27), and residues 196-297 (lobe
F3) adopt the pleckstrin homology/phosphotyrosine binding fold found in
a broad range of signaling molecules, including dynamin, Sos, and Shc
(28-30). In the dormant molecule, the carboxyl-terminal tail binds as
an extended peptide covering a large surface of lobes F2 and F3 of the
FERM domain. It was speculated that the tail domain could cause
dormancy by either directly blocking partner protein-binding sites
and/or by inducing conformational changes in the FERM domain that alter
(and inactivate) binding sites (26).
Subsequently, structures reported for the active FERM domains of
radixin with and without bound inositol (1, 4, 5)-triphosphate (IP3) (31) and of moesin (32) have given some insight into the structural aspects of activation. The structures of unmasked radixin with or without IP3 (2.8 Å resolution) were highly
similar to each other, and comparison to dormant moesin showed local
changes in three regions, 138-150, 160-178, and 243-280. The
structure of activated moesin (2.7 Å resolution) also revealed shifts
in these three regions (32). However, the moesin analysis was
complicated by crystal packing interactions that caused large shifts in
lobes F1 and -3 and concluded that consistent shifts due to activation were limited to residues 166-170 and 260-264.
Here we present the 2.3 Å resolution crystal structure of the
activated FERM domain of ezrin. This represents the first structure for
the protein ezrin. Combining this structure with the other two active
ERM structures gives us an enhanced ability to identify those
structural changes that are due to activation as opposed to sequence
differences or crystal packing interactions. Moreover, we go on to
document that mobility changes are an important aspect of activation
that has not been previously described. Furthermore, our analysis of
the active (radixin, moesin, and ezrin) and the dormant (moesin) ERM
structures reveals that the tail domain contributes residues that allow
the FERM domain to complete its folding, a concept that we refer to as
keystone interactions.
Protein Crystallization and Data Collection--
The
crystallization of and data collection for the FERM domain of human
ezrin (residues 1-297) have been described previously (33). Briefly,
the crystals were grown by the hanging drop method using a reservoir
solution containing 10-15% (w/v) monomethyl-ether PEG 2000 (Fluka),
15% glycerol (w/v), 10% 2-propanol (w/v), 0.1 M Na-Hepes,
(pH 8.1), and they belong to the space group P21 with cell
constants a = 48.5 Å, b = 112.8 Å, c = 66.3 Å,
Structure Solution and Refinement--
The crystal structure was
solved by molecular replacement and refined with the program CNS (34)
using the moesin FERM domain (residues 4-296 of PDB entry 1EF1) (26)
as a search model. Using a 10.0 to 4.0 Å resolution range, the heights
of solutions (the next highest scores in parentheses) for the rotation
and translation function were 0.23 (0.07) and 0.22 (0.09),
respectively. The crystallographic R-factor was 30.1% after
rigid body refinement using data from 25 to 2.3 Å. At this stage the
2Fo
Unusual geometries in well ordered regions of the structure include
cis-prolines at residues 75 and 297. Because of weak side chain electron density, residues Ser-148, Arg-151, and Lys-162 were
modeled as alanine in both molecules in the asymmetric unit.
Structural Comparisons--
FERM domain structures used for
comparison included ezrin chains A and B (from this work), radixin (PDB
code 1GE7), radixin bound to IP3 (PDB code 1GE6), moesin
(PDB 1E5W), and the dormant moesin bound to the carboxyl-terminal tail
domain (PDB code 1EF1). Overlays were accomplished using the programs
LSQMAN (36) and DOMOV (37). The overlays reveal that cis-Pro-75 is modeled as a trans-Pro in the radixin structures; we suspect that this
is an error and note that it has not influenced our analyses.
Structural Overview--
Crystals of the FERM domain (residues
1-297) of ezrin diffract to 2.3 Å and have two molecules in the
asymmetric unit. The structure was solved by molecular replacement, and
refinement has led to a model that consists of residues 2 to 297 for
each of two chains and 314 water molecules (Table
I). The two molecules in the asymmetric
unit A and B are in approximately equivalent environments because the
non-crystallographic symmetry is nearly a perfect centering operation.
Consistent with this, molecules A and B are very similar with a root
mean square deviation of C Mobility Properties of the FERM Domain--
In contrast, the
mobility properties of the FERM domain have not yet been described,
although such information is available from protein crystallography in
the form of atomic B-factors (also known as temperature factors).
Knowledge of mobility along the chain in the activated and dormant
structures is likely to provide insight into the mechanism of
activation. We first examine the mobility properties in each of the
published FERM domains, and then in the next section we compare them to
those of dormant moesin to gain insight into the activation process.
B-factors are related to the amplitude of motion of the atoms so that
in the absence of large model errors, higher B-factors indicate a
higher disorder of the atom in question (41). As seen visually in Fig.
1A and graphically in Fig. 1B, the B-factors
along the chain of ezrin vary in a complex manner. In particular, local
peaks in mobility are mostly associated with loops between secondary
structural elements and termini (42). Lobe F1 behaves as a rather tight unit, with relatively small deviations from the mean B-factor, whereas
lobes F2 and F3 appear less cohesive, having higher average mobility
(Fig. 5) and exhibiting some regions with very high mobilities. These
occur near residues 140 in lobe F2 and residues 210 and 260 in lobe F3.
In each of these three cases, the loops are not well tethered to the
core of the lobe, and the sequences have a high proportion of polar residues.
Most of the mobility features are common among the three activated ERM
protein structures (Fig. 1B). Notable differences are limited: ezrin is less mobile near residue 230, radixin is less mobile
near residue 260, and moesin is more mobile near residue 160. The first
two cases may be explained by crystal contacts because ezrin residue
230 packs against residue 52 of a symmetry mate, and the loop near
residue 260 in radixin packs against residues 116 and 234 from a
symmetry mate. In the third case, the high B-factors of moesin do not
correlate with a packing interaction or a unique sequence feature.
However, such a sharp spike in a B-factor plot is sometimes caused by a
model error (43), which is plausible in this case because it is a
mobile region in a medium resolution (2.7 Å) structure, and three
residues have rare Conformational and Mobility Changes Associated with
Activation--
To best define the conformational and mobility changes
that are associated with activation, we have compared the six FERM domain structures available for ERM proteins: dormant moesin, activated
radixin, the activated radixin-IP3 complex, activated moesin, and molecules A and B of activated ezrin presented here. These
make a total of five activated and one dormant molecule. The pair of
radixin structures are very similar, as are the pair of ezrin
structures; indeed, they are not truly independent from one another
because they are in equivalent crystallographic environments. Thus, the
radixin-IP3 complex and ezrin molecule B are left out of
the systematic analyses because they do not add significant new information.
Conformational Changes Associated with Activation--
Both visual
(Fig. 2) and quantitative (Fig.
3A) analyses of mobile regions
of all of the activated structures with the dormant moesin structure
reveal that the activated ezrin and radixin structures cluster together
but that the activated moesin structure has large differences in lobes
F1 and F3. These differences are a result of a movement of moesin lobes
F1 and F3 toward each other. This movement has been ascribed to a
crystal packing effect (32) so that the activated moesin structure is
less reliable for defining movements associated with activation.
Combining information from the ezrin- and radixin-activated structures
allows us to locate regions where the pair deviate more from the
dormant structure than from each other. This provides a striking
delineation of residues that move significantly upon activation (Fig.
3B). The regions implicated are major shifts of residues
130-150, 155-180, and 240-270 and small but significant shifts near
residues 70 and 210. The larger shifts are similar to those identified
by Hamada et al. (31) in their study of the FERM domain of
activated radixin, but the smaller shifts have not been noted
before.
To dissect conformational changes into components due to rigid lobe
rotations and those that are local in nature, we calculated independent
overlays for each lobe. The resulting rigid lobe rotations were very
small, ranging from 0.5° to 1.5°, and the resulting plot of local
shifts was virtually unchanged from Fig. 3. An unexpected benefit of
this lobe-wise analysis is that treatment of the activated moesin
structure in this manner removes the crystal packing affects (lobe
rotations of 4-8°), so that local changes in moesin agree with those
seen in ezrin and radixin (data not shown). The following three
paragraphs summarize the conformational changes undergone by each lobe
during activation.
The rigid body movements of lobe F1 (residues 4-80) are very small,
involving a rotation ranging from 0.5-0.7° for the ezrin and radixin
structures. The only significant local structural change, at residues
73-75 (Fig. 3B), involves a turn just after strand
For lobe F2 (residues 88-199), the lobe rotation is again small,
ranging from 1.2-1.5°. However, major local changes (up to 3-Å
shifts) occur for residues 132-151 and 155-177 (Fig. 3B). As seen in Fig. 4A, these
changes involve coordinated shifts of the last turn of helix B and the
following loop and of all of helix C (maximal at its amino terminus). A
look at side chain positions reveals some clear ways that these
movements associated with activation can compensate for the removal of
helix A of the carboxyl-terminal tail. The three most notable cases are
the shift of Trp-175 to fill the pocket vacated by Leu-525, a flip of
the side chain of Arg-171 to help fill the space vacated by Leu-529, and a shift in the side chain of Trp-168, as well as the whole of
residues 160-162, to fill space vacated by residues 533 to 537 (Fig.
4A). Residues 132-151 pack onto the backside of lobe F2
helix C, and their motion appears to be due to being pulled along by
the shifting helix C. In addition to many nonspecific contacts in this
core, a key side chain connecting helix C to this chain segment is the
buried His-176 whose side chain hydrogen bonds to Tyr-146-OH and
Gly-135-O
For lobe F3, the rigid body rotation is again small, only 0.7-1.4°.
Notable local changes in the conformation of lobe F3 include the small
shifts of residues 211-213 and the extensive shifts of residues
240-267, which have a maximal shift of nearly 6 Å at residue 261. These movements can be described as mainly a collapsing of the
Mobility Changes because of Activation--
A broad comparison of
the mobilities of the activated structures with those of dormant moesin
reveals that the overall mobility of lobe F1 remains similar during
activation, but the mobilities of lobes F2 and F3 as a whole increase
significantly (Fig. 5). This results in
an inversion such that the lobe F1 goes from being the least well
ordered lobe to being the most well ordered lobe. The transition occurs
near residue 88 (see Fig. 1B), which is about halfway
through the linker between lobes F1 and F2 (26). Three regions have
very dramatic increases in mobility; these are near residues 140, 210, and 265, mentioned above as the three regions that are highly mobile in
the activated structures. For the loop near residue 140, the case is
less clear that this increase in mobility is associated with
activation, because these residues are not in direct contact with the
carboxyl-terminal tail in the dormant structure and we do not see any
structural changes that would clearly cause them to become more mobile.
One plausible explanation is that these residues are actually already
rather mobile in the dormant moesin structure but that a crystal
packing interaction in that crystal (residues 141-149 pack against
residues 560-565 in another molecule) damped their natural mobility.
We conclude that residues 135-145 are very mobile in the activated FERM domain, but we must leave open the question of how much their mobility increases upon activation.
Inspection of the dormant moesin complex structure (26) indicates that
the increases in mobility near residues 212 and 265 can be directly
traced to the removal of helix D of the carboxyl-terminal tail. In the
first case, residues 210, 211, 212, and 214 all make H-bonds to
residues 574-577 (the last four residues of the inhibitory tail), and
Leu-216 is buried by the side chain of Met-577 in the complex (see Fig.
6B). In the second case, the
key seems to be that Phe-267 of The crystal structure of ezrin presented here completes the
picture, so that a structure is now available for the FERM domains of
each of the three ERM proteins. Also, the 2.3-Å resolution of this
structure provides details such as solvent structure, not visible in
the lower resolution structures of activated moesin (2.7 Å) or radixin
(2.8 Å). Because the FERM domain fold is already well characterized,
one major value of this structure derives from the opportunity to
compare all the ERM structures to differentiate which structural
changes are because of activation and which are because of crystal
packing interactions or sequence differences among the ERM proteins. A
second major value of this work is that the analyses of B-factors
reveals that, in addition to conformational changes, FERM domain
activation involves large increases in the flexibility of lobes F2 and
F3. This increase in flexibility has important bearing on the
energetics and specificity of ligand binding (see below).
The Extent and Origins of Conformational Changes--
The use of
the quantitative variation in the activated structures as a reference
for defining what changes are significant is powerful in that it allows
a quantitative, unambiguous delineation of a set of residues that show
significant motion (Fig. 3B). However, it is worth noting
that because of the limited resolution of the structures and possible
variations among activated structures due to crystal packing
interactions and intrinsic chain mobility, we do not expect the
analysis to reliably reveal changes in structure much smaller than
about 1 Å, even though such changes must exist.
Our analysis confirms the validity of the conformational changes due to
activation that were proposed based on the activated radixin structure
(31) as opposed to the much more limited set of changes highlighted in
the analysis of the activated moesin structure (32). These changes are
all local in nature, involving virtually no relative movements of the
lobes as a whole. This is in line with the expectation of Pearson
et al. (26) that the relative positions of the three FERM
domain lobes are rather well fixed. However, it must be borne in mind
that the >5° lobe rotations induced by a crystal packing interaction
in the moesin structure show that significant motions can occur upon
the binding of an appropriate ligand.
But what triggers these changes? Both the conformational and mobility
changes associated with the activation of ERM proteins appear to be
concretely related to the loss of interactions with parts of the
autoinhibitory carboxyl-terminal tail, in particular helix A and the AB
loop (Fig. 4A), helix D (Fig. 4B), and possibly strand The Role of IP3 Binding in Activation--
It was
proposed by Hamada et al.(31), based on the
radixin-IP3 complex, that IP3 binding pushes
helix A to widen the cleft between lobes F1 and F3 and that this
movement in helix A induces the other shifts in lobe F3 and leads to
activation. The similarity of activated ezrin to activated radixin
(with or without IP3) strengthens the argument presented by
Edwards and Keep (32) that the movements of lobe F3 are not a direct
outcome of IP3 binding but are simply related to the loss
of the carboxyl-terminal tail. It could be that IP3 would
also force these changes to occur (and thus stimulate activation), but
there is no evidence that that is true. At a more fundamental level,
one important outstanding question regarding the relevance of the
IP3-radixin complex is whether IP3 can
substitute for PIP2 in activation experiments. We are not
aware of experiments that answer this question, and if IP3
does not serve as an activator then results about its binding site may
not be informative about how PIP2 activates ERM proteins. In this regard, the mutagenesis results of Barret et
al. (44), though not conclusive in themselves, support the idea
that activation by PIP2 may be more complex than the
crystallographically defined IP3 binding site would imply.
Helices A and D of the Carboxyl-terminal Tail as Keystone Binding
Partners--
As we see it, the high degree of disordering that occurs
upon activation indicates that lobes F2 and F3 of the FERM domain can
be viewed as incompletely folded domains; that is, lobes F2 and F3 need
residues from the carboxyl-terminal tail (or some other source) to be
contributed to their hydrophobic cores so they can complete their
folding (Fig. 6). In the case of lobe F2, Leu-525 and Leu-529 of helix
A insert into the core to complete the fold, and in the case of lobe
F3, Ile-571, Phe-574, and Met-577 of helix D insert to do the same.
This type of interaction suggests that the carboxyl-terminal tail
residues and the associated helices that hold them serve to complete
and stabilize the structures of lobes F2 and F3 much like a keystone
completes and stabilizes the structure of an arch. Although it was not
called a keystone interaction, a classic example in which an
incompletely folded protein requires an external piece to complete its
folding is provided by bovine trypsinogen (45). As synthesized, the
inactive protease precursor trypsinogen has a highly mobile
incompletely folded "activation domain," and upon cleavage at
Ile-16, the new amino terminus tucks inside the protein, as a keystone,
to make many interactions that drive the protein to finish folding.
The idea of a keystone interaction is quite different from the common
experience and expectation that protein-protein interactions will
involve the interactions of preformed surfaces of fully folded proteins. This more intimate mode of interaction brings with it a much
greater potential for high affinity binding with very low rates of
dissociation, as is seen for the dormant ERM proteins (26). It is easy
to see how such high affinity binding interactions would be highly
desirable for cytoskeletal proteins; they serve a structural role where
it is desirable for them to not be in a dynamic on-off equilibrium but
securely anchored to their partner, not releasing even once until a
signal for reorganization is received. Thus, such keystone interactions
may be a theme that will also be seen in other cytoskeletal proteins
that are regulated by masking (15-18).
Although no work has yet been published that defines the sites at which
the FERM domain will interact with its partner proteins, our
observation brings with it the clear prediction that the sites to which
carboxyl-terminal tail helices A and D bind will be high affinity sites
for partner proteins. Although the flexibility of the activated lobes
F2 and F3 makes it conceivable that a variety of structures might bind
as keystone molecules, the most natural prediction is that some partner
protein will have a recognition helix that closely mimics helix A and
binds to lobe F2 and some will have a recognition helix that closely
mimics helix D and binds to lobe F3. In the latter case, given the
manifold interactions of the 210 turn with the C terminus of helix D
(Fig. 6B), this pocket may even be specific for a
carboxyl-terminal helix. Although these are clear predictions for where
some partner proteins will bind, these need not be the only recognition
sites on the FERM domain. Indeed, the loss of the other parts of the
carboxyl-terminal tail do not appear to influence the integrity of the
structure as much, although they still may make significant
contributions to binding affinity.
Regulation by Phosphorylation--
Another possible contributor to
activation that has not received much attention in structural studies
is the phosphorylation of Tyr-146. This residue has been
reported to be phosphorylated in ezrin during epidermal growth factor
stimulation of cells (46), and it may also occur in radixin because
radixin is reported to be phosphorylated by the platelet-derived growth
factor receptor (47) at a site that has not been mapped. Whether
the phosphorylation occurs in the dormant or activated state and what
physiological role it has are as yet unanswered questions (48). In the
original structure of dormant moesin, the phosphorylation Tyr-146 was a mystery because Tyr-146 was fully buried in a well ordered part of the
structure. The very high mobility of this region in the activated FERM
proteins suggests that the phosphorylation of Tyr-146 could readily
occur because the loop containing Tyr-146 probably undergoes rapid
local unfolding to become accessible to a kinase. With regard to its
role in activation, it is clear that if Tyr-146 is phosphorylated, the
folding of the 130-150 loop that surrounds His-176 will be disrupted.
Lobe F2 will not be able to adopt its normal structure and probably
will not interact as effectively with the carboxyl-terminal tail or
other partner proteins that bind at this site. In this way,
phosphorylation at Tyr-146 of the dormant protein could lead to
dissociation of the carboxyl-terminal tail to activate the rest of the
FERM domain. However, phosphorylation at Tyr-146 of the active protein
would serve to differentially deactivate the putative binding site on
lobe F2. Another attractive possibility is that the phosphorylation of
Tyr-146 could lead to alternate signaling pathways via association with
proteins containing SH2 (src-homology 2) or
phosphotyrosine binding domains and thus may serve as a scaffold to
recruit such proteins to the plasma membrane. This provides the first
concrete proposal for how the FERM domain may be activated/deactivated
with respect to specific partner proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(22, 23) and/or the association with the
phosphotidylinositol lipid, PIP2 (24, 25).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
= 102.3. Two molecules occupy the asymmetric unit. The
diffraction data were collected at
150 °C at the Cornell High
Energy Synchrotron Source (CHESS) and processed with the MOSFLM and
SCALA programs (CCP4, 1994). Statistics on data collection and
refinement are provided in Table I.
Fc electron density
was of sufficient quality to guide the construction of the ezrin FERM
molecule using the program O (35). Refinement using amplitudes as the
maximum likelihood target involved manual rebuilding, simulated
annealing, and conjugated gradient minimization without NCS restraints.
An overall anisotropic B-factor correction with a low resolution cutoff
of 6 Å yielded B11 = 15 Å2, B22 =
22 Å2, B33 = 6 Å2, and
B13 = 8 Å2. Waters were placed initially using
the CNS water pick program and later by manual inspection. Simulated
annealing omit maps were also used occasionally to check the quality of
the model. The final model includes 2 chains of ezrin (A and B), each
with residues 2-297 of ezrin, and water molecules. The
crystallographic R-factor is 22.3%, and R-free
is 27.6% (Table I).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-atoms of 0.4 Å, and all
descriptions will refer to molecule A unless specifically noted. The
FERM domain is a clover-shaped molecule (Fig.
1A) consisting of three
distinct lobes and is globally similar to structures reported
previously for ERM proteins (26, 31, 32), merlin (38, 39), and the
band-4.1 protein (40). This basic structure has already been well
described and will not be elaborated here.
Statistics for x-ray structure determination
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Fig. 1.
Overall structure and mobility of ezrin.
A, a ribbon diagram of the ezrin FERM domain and
a semitransparent molecular surface illustrates the compact globular
cloverleaf-shaped structure possessing three lobes: lobe F1(residues
2-82), lobe F2 (residues 83-195), and lobe F3 (residues 196-297).
The ribbon is colored with respect to temperature factor ramped from
cold-blue to hot-red for B-factors from 20 to
80 Å2, respectively. B, temperature-factor plot as
a function of residue number for activated FERM domains of ezrin
(red), radixin (cyan), and moesin
(green), and the dormant FERM domain of moesin
(violet). All ribbon figures were prepared with the
programs Bobscript and Raster3D (49-51).
,
-angles, (
72,
169) for Leu-158, (77,
19)
for Glu-159, and (
147,28) for Lys-161.
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Fig. 2.
Overlay of the FERM domains of the ERM family
members. Superposition of the FERM domains of ezrin
(red), radixin (cyan), and active moesin
(green) onto dormant moesin (violet) bound to the
inhibitory carboxyl-terminal tail (ghostly yellow). To be
most informative, these overlays have been done as described in Fig.
3B.
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Fig. 3.
Quantitation of the structural shifts
associated with activation. A, C- root mean
square deviation of each activated FERM domain compared with the
dormant moesin domain. Ezrin (red), radixin
(cyan), and moesin (green). The ezrin and radixin
plots are highly similar, but the moesin plot has many large unique
features. B, the average mobility differences between two
activated structures (ezrin and radixin, violet) compared
with the average mobility between activated and dormant structures
(activated ezrin/dormant moesin and activated radixin/dormant moesin,
blue) is shown. Regions where the blue line rises above the
violet line define regions that appear to have a significant
conformational change upon activation. For the superimpositions used to
generate panel B, residues clearly involved in significant
local movements (residues 131-174 and 241-269) were removed from the
calculations. Overlays were calculated using LSQMAN (36).
5.
This turn is far from all other changes and does not appear to be
related to activation. Also, because activated moesin follows dormant
moesin more closely, we suspect that this shift is due to sequence
changes, because ezrin and radixin have Asn-74 and Gln-77, whereas
moesin has Ser-74 and Leu-77.
View larger version (74K):
[in a new window]
Fig. 4.
Close-ups of structural shifts associated
with activation and their relation to the loss of the carboxyl-terminal
tail. A, lobe F2 of ezrin (red) compared
with the dormant moesin (violet)-carboxyl-terminal tail
(ghostly yellow) complex. Large local shifts of the B-C loop
and helix C are visible, as these residues collapse to fill in space
that had been occupied by carboxyl-terminal tail residues.
B, like panel A, but for Lobe F3. Visible is the
collapsing of the 1
2 hairpin and the
5
6
7
subdomain into the space vacated by helix D of the carboxyl-terminal
tail.
1-
2 hairpin and the
-meander subdomain (
5, -6, -7) into the
space occupied by helix D of the carboxyl-terminal tail (Fig.
4B). As with lobe F2, it is possible to describe some clear ways that these movements can be seen to compensate for the removal of
helix D of the carboxyl-terminal tail. The movement of the
-meander
seems tied to a shift of Phe-267 to fill the space that had been
occupied by Phe-574 and so Pro-265 and Asp-266 can fill the space
occupied by Ile-571. Similarly, the side chain of Lys-237 flips
directions so that it fills the space occupied by Met-577. Interestingly, the change of Lys-237 seems correlated with flips of the
side chains of Glu-229 and Asn-231 (Fig. 4B).
View larger version (19K):
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Fig. 5.
Significant disordering upon activation.
The average B-factors for each lobe are shown for the dormant FERM
domain structure (dash, square) and the three
activated FERM domains: ezrin (thin, diamond), radixin
(thick, square), and moesin (thickest, triangle).
Details of the B-factor variations within each lobe for each of the
proteins are shown in Fig. 1B.
-strand 7 was well buried by helix
D, but with helix D absent Phe-267 is no longer pinned in place, which
increases the lever arm around which the loop near residue 260 can
fluctuate.
View larger version (50K):
[in a new window]
Fig. 6.
The keystone view of carboxyl-terminal tail
interactions with lobes F2 and F3. Both panels show the structure
of the dormant moesin-carboxyl-terminal tail complex. A,
stereoview of lobe F2 (violet) with helix A of the
carboxyl-terminal tail (yellow). B, stereoview of
lobe F3 (violet) with the helix D of the carboxyl-terminal
tail (yellow). For context, the complete backbone trace of
the FERM domain lobe is shown as a ribbon, and selected side
chains lining the binding pocket are shown. Selected hydrogen bonds are
shown as dashed lines. Note in both cases how the
carboxyl-terminal tail contributes intimately to the hydrophobic core
of the domain.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 (see Fig. 2). The effect of the loss of strand
1 of the
carboxyl-terminal tail is not clear because the residues in contact
with that strand (e.g. Ile-245, Ile-248) do move but their movement can already be related to the loss of helix D. The loss of
other parts of the carboxyl-terminal tail are not visibly associated with conformational changes even though they cover about half the
surface area buried by the carboxyl-terminal tail. Indeed, of 57 FERM
domain residues highlighted by Pearson et al. (Fig. 4 of
Ref. 26) as involved in carboxyl-terminal tail binding, only 28 of them
are in the regions that change upon activation.
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ACKNOWLEDGEMENTS |
---|
We thank Matt Pearson for use of coordinates before publication for solving the molecular replacement problem. We also thank Dan Thiel, Marian Szebenyi, and Lana Walsh for beam time at CHESS and the entire CHESS staff for assistance during data collection. We thank Cindy Westmiller for excellent secretarial help.
![]() |
FOOTNOTES |
---|
* This work is based upon research conducted at the Cornell High Energy Synchrotron Source (CHESS), which is supported by the National Science Foundation and NIGMS, National Institutes of Health under award DMR 9713424. This work was supported by National Institutes of Health Grants GM36652 (to A. P. B.) and 2 R01 GM40654 (to R. A. C.) and NIH Training Grant GM07273 (to W. J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1NI2) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Present address: Dept. of Physiology and Biophysics, SUNY, HSC, Stony Brook, NY 11794-8116.
To whom correspondence may be addressed. Tel.: 607-255-5713;
Fax: 607-255-6249; E-mail: apb5@cornell.edu.
§§ To whom correspondence may be addressed. Tel.: 541-737-3200; Fax: 541-737-0481; E-mail: karplusp@ucs.orst.edu.
Published, JBC Papers in Press, November 11, 2002, DOI 10.1074/jbc.M210601200
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ABBREVIATIONS |
---|
The abbreviations used are: ERM, ezrin, radizin, moesin; IP3, inositol-triphosphate; PIP2, phosphatidylinositol(4,5)-bisphosphate.
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