Voltage sensor conformations in the open and closed states in ROSETTA structural models of K(+) channels.
ABSTRACT Voltage-gated ion channels control generation and propagation of action potentials in excitable cells. Significant progress has been made in understanding structure and function of the voltage-gated ion channels, highlighted by the high-resolution open-state structure of the voltage-gated potassium channel, K(v)1.2. However, because the structure of the closed state is unknown, the gating mechanism remains controversial. We adapted the rosetta membrane method to model the structures of the K(v)1.2 and KvAP channels using homology, de novo, and domain assembly methods and selected the most plausible models using a limited number of experimental constraints. Our model of K(v)1.2 in the open state is very similar in overall topology to the x-ray structure of this channel. Modeling of KvAP in the open state suggests that orientation of the voltage-sensing domain relative to the pore-forming domain is considerably different from the orientation in the K(v)1.2 open state and that the magnitude of the vertical movement of S4 is significantly greater. Structural modeling of closed state of K(v)1.2 suggests gating movement that can be viewed as a sum of two previously suggested mechanisms: translation (2-4 A) plus rotation ( approximately 180 degrees ) of the S4 segment as proposed in the original "sliding helix" or "helical screw" models coupled with a rolling motion of the S1-S3 segments around S4, similar to recent "transporter" models of gating. We propose a unified mechanism of voltage-dependent gating for K(v)1.2 and KvAP in which this major conformational change moves the gating charge across the electric field in an analogous way for both channels.
- SourceAvailable from: Julia Koehler[Show abstract] [Hide abstract]
ABSTRACT: The determination of membrane protein (MP) structures has always trailed that of soluble proteins due to difficulties in their overexpression, reconstitution into membrane mimetics, and subsequent structure determination. The percentage of MP structures in the protein databank (PDB) has been at a constant 1-2% for the last decade. In contrast, over half of all drugs target MPs, only highlighting how little we understand about drug-specific effects in the human body. To reduce this gap, researchers have attempted to predict structural features of MPs even before the first structure was experimentally elucidated. In this review, we present current computational methods to predict MP structure, starting with secondary structure prediction, prediction of trans-membrane spans, and topology. Even though these methods generate reliable predictions, challenges such as predicting kinks or precise beginnings and ends of secondary structure elements are still waiting to be addressed. We describe recent developments in the prediction of 3D structures of both α-helical MPs as well as β-barrels using comparative modeling techniques, de novo methods, and molecular dynamics (MD) simulations. The increase of MP structures has (1) facilitated comparative modeling due to availability of more and better templates, and (2) improved the statistics for knowledge-based scoring functions. Moreover, de novo methods have benefitted from the use of correlated mutations as restraints. Finally, we outline current advances that will likely shape the field in the forthcoming decade. © Proteins 2014;. © 2014 Wiley Periodicals, Inc.Proteins Structure Function and Bioinformatics 10/2014; · 3.34 Impact Factor
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ABSTRACT: Voltage-gated potassium ion channels (Kv) play an important role in a variety of cellular processes, including the functioning of excitable cells, regulation of apoptosis, cell growth and differentiation, the release of neurotransmitters and hormones, maintenance of cardiac activity, etc. Failure in the functioning of Kv channels leads to severe genetic disorders and the development of tumors, including malignant ones. Understanding the mechanisms underlying Kv channels functioning is a key factor in determining the cause of the diseases associated with mutations in the channels, and in the search for new drugs. The mechanism of activation of the channels is a topic of ongoing debate, and a consensus on the issue has not yet been reached. This review discusses the key stages in studying the mechanisms of functioning of Kv channels and describes the basic models of their activation known to date.Acta naturae. 10/2014; 6(4):10-26.
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ABSTRACT: Electrically excitable cells, such as neurons, exhibit tremendous diversity in their firing patterns, a consequence of the complex collection of ion channels present in any specific cell. Although numerous methods are capable of measuring cellular electrical signals, understanding which types of ion channels give rise to these signals remains a significant challenge. Here, we describe exogenous probes which use a novel mechanism to report activity of voltage-gated channels. We have synthesized chemoselective derivatives of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2-type voltage gated potassium channels. We find that voltage activation of Kv2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks Kv2 activation. We identify GxTX residues that can be replaced by thiol- or alkyne-bearing amino acids, without disrupting toxin folding or activity, and chemoselectively ligate fluorophores or affinity probes to these sites. We find that GxTX-fluorophore conjugates colocalize with Kv2.1 clusters in live cells and are released from channels activated by voltage stimuli. Kv2.1 activation can be detected with concentrations of probe that have a trivial impact on cellular currents. Chemoselective GxTX mutants conjugated to dendrimeric beads likewise bind live cells expressing Kv2.1, and the beads are released by channel activation. These optical sensors of conformational change are prototype probes that can indicate when ion channels contribute to electrical signaling.Proceedings of the National Academy of Sciences 10/2014; · 9.81 Impact Factor
Voltage sensor conformations in the open and closed
states in ROSETTA structural models of K?channels
Vladimir Yarov-Yarovoy*, David Baker†‡, and William A. Catterall*§
Departments of *Pharmacology and†Biochemistry and‡Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195
Contributed by William A. Catterall, March 22, 2006
Voltage-gated ion channels control generation and propagation of
action potentials in excitable cells. Significant progress has been
ion channels, highlighted by the high-resolution open-state struc-
ture of the voltage-gated potassium channel, Kv1.2. However,
because the structure of the closed state is unknown, the gating
mechanism remains controversial. We adapted the ROSETTA mem-
brane method to model the structures of the Kv1.2 and KvAP
channels using homology, de novo, and domain assembly methods
and selected the most plausible models using a limited number of
experimental constraints. Our model of Kv1.2 in the open state is
very similar in overall topology to the x-ray structure of this
channel. Modeling of KvAP in the open state suggests that orien-
tation of the voltage-sensing domain relative to the pore-forming
domain is considerably different from the orientation in the Kv1.2
open state and that the magnitude of the vertical movement of S4
is significantly greater. Structural modeling of closed state of Kv1.2
suggests gating movement that can be viewed as a sum of two
previously suggested mechanisms: translation (2–4 Å) plus rota-
tion (?180°) of the S4 segment as proposed in the original ‘‘sliding
helix’’ or ‘‘helical screw’’ models coupled with a rolling motion of
the S1–S3 segments around S4, similar to recent ‘‘transporter’’
models of gating. We propose a unified mechanism of voltage-
dependent gating for Kv1.2 and KvAP in which this major confor-
mational change moves the gating charge across the electric field
in an analogous way for both channels.
membrane protein ? ROSETTA method ? voltage-gated ion channel
portant for initiation and propagation of action potentials in
excitable cells. They are composed of four identical or homol-
ogous subunits, each containing six transmembrane segments:
S1–S6. Segments S1–S4 form the voltage-sensing domain
(VSD), and segments S5 and S6 connected by the P loop, which
is involved in ion selectivity, comprise the pore-forming domain
(PD). S4 has four gating-charge-carrying arginines (R1–R4)
spaced at intervals of three amino acid residues, which are highly
conserved and are thought to play a key role in coupling changes
in membrane voltage to opening and closing of the pore (3–5).
In the Kv channels ?13 electronic charges cross the membrane
electrical field per channel between the closed and open states
High-resolution structures of the bacterial potassium channel
KvAP and the mammalian potassium channel Kv1.2 recently
have been solved (9–11). Although the KvAP structures showed
the VSD in a nonnative orientation with respect to the mem-
brane and the PD, the Kv1.2 structure captured the VSD in a
conformation that is thought to represent the open state of the
subunit interacts closely with the PD of the adjacent subunit in
a clockwise direction when the channel structure is viewed from
the extracellular side of the membrane (9). However, the closed-
state structure of these channels remains unknown, and the
mechanism of action of the voltage sensor in translocating gating
charge is a subject of controversy. The original ‘‘sliding helix’’ or
oltage-gated potassium (Kv) channels are members of the
voltage-gated ion channel superfamily (1, 2), which is im-
‘‘helical screw’’ models of gating posited that S4 moves outward
along a clockwise spiral path through the protein structure upon
depolarization, making sequential interactions with negatively
charged residues (12–14). This movement was detected directly
by chemical-labeling studies (15–18). However, extensive studies
of labeling of substituted cysteine residues in S4 revealed that
only a narrow waist was protected from reaction, leading to the
concept that the gating charges of S4 move through a narrow
pathway between open internal and external vestibules (19, 20),
which we term the gating pore. This movement of the gating
charges of S4 through a narrow gating pore is supported by
experiments in which mutations of the S4 arginines were shown
to create an ion conductance pathway for protons (21, 22) or for
cations (23, 24) through the modified gating pore, indicating the
presence of a translocation pathway through the protein for S4.
However, even with these refinements, S4 movement alone
seems unlikely to translocate three charges per subunit because
analysis of the motions of substituted fluorescent probes using
several distinct strategies indicate that there is only a small (2–4
Å) outward translocation during gating (25–28) and that the
gating-charge-carrying arginines must move through a narrowly
focused membrane electrical field (22, 29). These findings led to
the proposal that the VSD acts like a ‘‘transporter’’ in which
accessibility of the S4 gating charges changes from internal to
external side due to the relative motions of the S1–S4 segments
(27). In contrast to all of these findings, a ‘‘paddle’’ model of
gating suggested by the x-ray structure of KvAP proposed that
the S4 moves 15–20 Å through the lipid bilayer during the gating
process (30, 31). A major goal of our structural modeling work
is to develop a gating model that reconciles these seemingly
Structural models of the voltage-gated ion channels in the
closed and open states have been proposed based on multiple
modeling strategies (10, 27, 32–37), including a specific gating
model based on a transporter mechanism (27). Laine et al. (32)
proposed that the VSD of one subunit interacts with the PD of
the neighboring subunit in the clockwise direction if viewed from
the extracellular side of the membrane, based on a combination
of chemical crosslinking and modeling, and this proposal has
been confirmed by the Kv1.2 structure (9). Recently, consider-
able progress has been made in de novo high-resolution structure
prediction of water-soluble proteins (38, 39), novel protein-fold
design (40), and modeling of protein–protein interactions (41)
using the ROSETTA method (42–44). In addition, the ROSETTA
method was adapted to de novo modeling of multipass ?-helical
transmembrane proteins, and significant parts of these proteins
were predicted with root-mean-square deviation (rmsd) ?4.0 Å
from the native structure (45).
homology and de novo modeling with a goal of modeling
Conflict of interest statement: No conflicts declared.
§To whom correspondence should be addressed at: Department of Pharmacology, Box
357280, University of Washington, Seattle, WA 98195. E-mail: firstname.lastname@example.org.
© 2006 by The National Academy of Sciences of the USA
May 9, 2006 ?
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no. 19 www.pnas.org?cgi?doi?10.1073?pnas.0602350103
structures of Kv1.2 and KvAP in open and closed conformations,
guided by limited experimental constraints derived from clear
structure–function data. We assessed the validity of the models
by evaluating their consistency with experimental data not used
in their generation. We then examined the validated models to
draw insights about the gating mechanism of Kvchannels. Our
results provide a well defined mechanism for S4 movement
through a combination of the motions proposed in the previous
sliding helix?helical screw and transporter models and poten-
on bacterial KvAP channels vs. Kv1.2 and other eukaryotic ion
Results and Discussion
Model of Kv1.2 in the Open State. The x-ray structure of the Kv1.2
channel (9) has missing electron density in segments S1 and S3
the ROSETTA membrane method to model these regions as
described in Methods. Fig. 1A shows the transmembrane region
of a single subunit of Kv1.2. Our model predicts that the R3
arginine in S4 (R300 in Kv1.2) interacts with E1 in S2 (E226 in
Kv1.2) (Fig. 1 B and C). R4 (R303 in Kv1.2) is also positioned
near E1 and may interact with it in the native protein (Fig. 1 B
and C). Several other salt-bridge interactions also form between
residues in S1, S2, S3, and S4 (see Fig. 6, which is published as
supporting information on the PNAS web site). These salt
bridges, which stabilize the open-state structure, represent in-
among eukaryotic Kv channels (46–48).
Domain Assembly of Kv1.2 in the Open State. We used the x-ray
structures of the separate VSD and PD of Kv1.2 in the open state
to test whether the ROSETTA membrane domain assembly
method (see Methods) can predict the interaction of the VSD
with the PD using constraints from well established structure–
function data for the final selection of models. Amino acid side
method, and hence the accuracy of the models is limited to 2–4
Å at best (45). Among the 10 largest clusters of models (see
Methods), the third cluster agreed with experimental data dem-
onstrating proximity of residues at the extracellular ends of S4
from one subunit and S5 from the adjacent subunit (32, 35–37)
(Fig. 2 A and B). This model was very similar to the x-ray
structure of Kv1.2 in the open state (Fig. 2 C and D), with rms
deviation over 260 residues of only 2.0 Å from the native
structure (9). Other models in this cluster also were similar to the
native structure (see Methods). The success of the domain-
assembly method in predicting an open state model that closely
matches the native structure encouraged us to use this method
to construct a model of Kv1.2 in the closed state.
Model of Kv1.2 in the Closed State. We applied the ROSETTA
membrane domain assembly method to build a model of Kv1.2
in the closed state as described in Methods. Based on experi-
mental evidence that E1 in S2 (E283 in Shaker; E226 in Kv1.2)
is positioned near R1 in S4 (R362 in Shaker; R294 in Kv1.2) in
the closed state (23), we constrained the centroids of those
residues to be within 8.0 Å from each other (see Methods). Other
state. (A) Side view of the ribbon representation of the ROSETTA membrane
model of a single subunit of Kv1.2. Regions of the Kv1.2 structure modeled
using the backbone coordinates of the unidentified residues in the x-ray
structure of Kv1.2 as a template by the ROSETTA membrane homology method
the identified residues in the x-ray structure of Kv1.2 (9) as a template by the
ROSETTA membrane homology method are shown in blue. Regions of the Kv1.2
structure modeled using the ROSETTA membrane de novo method are shown in
green. Transmembrane segments are labeled from S1 to S6, the selectivity
filter helix is labeled P, and the position of the S4–S5 linker is indicated by an
arrow. N and C termini residues of the transmembrane region are labeled N
and C, respectively. (B) Side view of the VSD segments S1–S4 only (colored
individually) of the model shown in A. Side chains of gating-charge-carrying
arginines in S4 (labeled R1–R4) and E226 in S2 (labeled E1) are shown in stick
representation. Blue, red, and cyan atoms in the side chains shown represent
nitrogen, oxygen, and carbon atoms, respectively. (C) View of the model
shown in B from the extracellular side of the membrane. All structural figures
presented in this work were generated using MOLSCRIPT (61) and RASTER3D (62).
Homology model of the transmembrane region of Kv1.2 in the open
Kv1.2. (A) View of the Kv1.2 model from the extracellular side of the mem-
brane. All four subunits are colored individually. Segments S1–S6 for the
blue-colored subunit and S5–S6 for the green-colored subunit are labeled
accordingly. (B) View of the model in A from the side of the membrane.
Segments S1–S4, S6, and S4–S5 linker for blue- and yellow-colored subunits
linker is indicated by an arrow for blue- and yellow-colored subunits. Extra-
cellular and intracellular edges of the hydrocarbon core of the membrane are
the extracellular side of the membrane of the Kv1.2 structure (shown in blue)
and the best ROSETTA membrane model (shown in orange) of the open state of
to the tetramer of the PD for clarity. (D) View of the models shown in C from
the side of the membrane.
The ROSETTA membrane domain assembly model of the open state of
Yarov-Yarovoy et al.
May 9, 2006 ?
vol. 103 ?
no. 19 ?
experimental data were not used during modeling. We examined
the 10 largest clusters of models (see Methods). To account for
transfer of ?3 charge units per subunit (6–8), we selected the
third largest cluster of models in which at least two of the four
potential gating-charge-carrying arginines in S4 were exposed to
the intracellular water-accessible environment. Fig. 3 A–D shows
one of the selected models of Kv1.2 in the closed state. Other
models in this cluster were very similar except for the distance
between S2 and S3 (see Methods). Thus, the precise position of
S3 may not be accurately defined in our best models.
S4 in our closed-state model of Kv1.2 is in close proximity to
S5 from the adjacent subunit (Fig. 3 A and B), in agreement with
results suggesting this topological arrangement in the closed
state (35–37, 49). These segments packed significantly more
tightly in our closed-state model compared with the open-state
structure, which is realistic because the hydrophobic face of the
S4 helix rotated ?180° from the lipid environment in the open
state to the protein environment at the S4–S5 interface in the
closed state (Fig. 3 C and D). However, S4 is not completely
buried within the protein in the closed state because some of its
hydrophobic residues are exposed to lipid between S1 from one
subunit and S5 from the adjacent subunit (Fig. 3A).
Gating Movements of the Kv1.2 VSD. Comparison of the closed-
state model and the open-state structure of Kv1.2 suggests a
series of linked structural changes during the transition from the
closed to the open conformation (Fig. 3E; see also Fig. 7, Tables
1 and 2, and Movies 1–3, which are published as supporting
information on the PNAS web site). S4 moves ?3 Å outward
on eukaryotic Kv channels (16, 17, 22, 26–29, 50, 51), and L293
and R297 in S4 move 1–2 Å relative to the pore axis between the
closed and open states, in agreement with distances measured by
luminescence resonance energy transfer (LRET) for homolo-
gous residues (L361 and R365) in the Shaker Kv channel (28).
In addition, S4 rotates clockwise ?180° about its axis, and the
extracellular part of S4 changes its tilt angle from ?10° to ?45°
relative to the membrane normal vector. The combined outward
translocation and clockwise rotation during activation closely
resemble the spiral motion of this segment that was first pro-
posed in the sliding helix and helical screw models of gating (12,
13) and subsequently has been supported by extensive experi-
mental results (15–20, 25, 26, 50).
In the transition from the closed to open state, the S4–S5
the pore axis, and ?4 Å outward. Residues in the S4–S5 linker
remain in contact with the same set of residues in the intracel-
lular end of the S6 from the same subunit in both the closed and
open states. This continuous contact may allow the S4–S5 linker
to pull the associated S6 segment to bend and open the pore as
the S4 voltage sensor moves outward and rotates. These results
agree with experimental observations demonstrating interac-
tions between the S4–S5 linker and the intracellular end of S6
this interaction plays an important role in voltage-dependent
gating by coupling conformational changes in the VSD with
closing and opening of the pore.
The S1, S2, and S3 segments move around S4 in the clockwise
direction (Fig. 3E), so that the intracellular end of S3 moves
closer to the S4–S5 linker. Rotation of S4 around its axis and
simultaneous rolling of S2 around S4 allow sequential interac-
tions of the four gating-charge-carrying residues in S4 (R294,
R297, R300, and R303 in Kv1.2) with the conserved negatively
charged residue in S2 (E226 in Kv1.2) as proposed previously
(46–48). Importantly, S4 rotates relative to S3 during the
conformational change in contrast to the movement of these two
helices as a rigid helical hairpin as predicted in the paddle model
of gating (30). Substitutions of the gating-charge-carrying argi-
nines in voltage-gated K?and Na?channels with histidine or
glutamine creates state-dependent proton- or ion-conducting
pores within the VSD (22–24). These data support mechanisms
of gating in which the arginine side chains move through a
narrow gating pore formed between extracellular and intracel-
lular water-filled vestibules during conformational changes in
the VSD between the closed and open states. Thus, the coor-
dination of the small outward movement and clockwise rotation
of S4 with the rolling motion of the S1–S3 around S4 results in
the movement of three gating-charge-carrying arginine residues
Kv1.2. (A) View of the Kv1.2 model from the extracellular side of the mem-
brane. The model is colored and labeled as in Fig. 2A. (B) View of the model
2B. The S4–S5 linker for blue and red subunits is labeled accordingly. (C) Side
view of the VSD only of the model in A. The model is colored and labeled as
in Fig. 1B. (D) View of the model in C from the intracellular side of the
membrane. (E) Kv1.2 models of the closed and open states shown in cylinder
for clarity. Transmembrane segments S1–S6 and P-loop are colored by a
rainbow scheme from blue to red. The S4–S5 linker is in purple. Approximate
positions of the C?atoms of the first and fourth gating charge-carrying
arginines in S4 (labeled as R1 and R4 and colored in blue) and E226 in S2
(labeled E1 and colored in red) are shown in sphere representation. All
intracellular and extracellular loops, except for the S4–S5 linker, are repre-
sented by curved lines for simplicity. Vertical translation of R4 between the
plane of the membrane is indicated by arrows. S3 is represented by ribbon to
show clearly the positions of the gating-charge-carrying arginines in S4.
The ROSETTA membrane domain assembly model of the closed state of
www.pnas.org?cgi?doi?10.1073?pnas.0602350103Yarov-Yarovoy et al.
from internal to external exposure in the transition to the open
state (see Fig. 3E and Movies 1–3 and also Figs. 8 and 9, which
are published as supporting information on the PNAS web site).
This rolling motion of S1–S3 is similar in concept to the
transporter models of gating-charge movement that were pro-
posed based on fluorescent imaging experiments that demon-
strated small transmembrane movement of S4 (27).
Model of KvAP in an Open?Inactivated State. The x-ray structures of
the full-length KvAP channel reported to date have their VSD
structure disrupted such that important interactions with the PD
are apparently lost because of absence of native lipid bilayer
environment (10, 11). The x-ray structure of the separate VSD
of the VSD of Kv1.2 and also has the first three gating-charge-
carrying arginines facing the extracellular side of the membrane.
It therefore may represent an open or open?inactivated confor-
mation of the VSD. We used this VSD structure and the
open-state structure of the PD of KvAP (10) to model the
transmembrane region of the KvAP (see Methods). Our largest
cluster of models generated without constraints from experi-
mental data resembled the Kv1.2 structure in that the VSD
interacts with the PD of the adjacent subunit in clockwise
direction, as viewed from the extracellular side (see Methods).
Many models in this cluster were in good agreement with EPR
spectroscopy accessibility data for the open?inactivated state of
the KvAP (57), and we used the EPR data to search for the most
favorable model among the 10 largest clusters (see Methods).
The most favorable ROSETTA membrane model of KvAP in the
open state (Fig. 4) has significant differences from the Kv1.2
structure (9) and from the previous KvAP model (11) (see Fig.
10, which is published as supporting information on the PNAS
web site). The most striking difference in predicted topology is
the position of S1 in close contact with the pore-forming S5 and
S6 in KvAP model (Fig. 4 A and B), compared with its location
on the outside edge of the VSD-PD complex in the Kv1.2
structure (9). In addition, S4 is positioned more on the periphery
of the KvAP model and does not interact directly with the PD
(Fig. 4 A and B), in contrast to the location of S4 near the PD
in the Kv1.2 structure (9). Our model fits the predictions of lipid
accessibility from EPR data for 97% of the amino acid residues
studied by Cuello et al. (57) in the VSD, but S4 in our model of
the open?inactivated state of KvAP appears to be significantly
further outward than its expected interfacial position based on
are published as supporting information on the PNAS web site).
Model of KvAP in the Closed State. To further develop the com-
parison between the Kv1.2 and KvAP models, we also modeled
the closed state of KvAP. We used the ROSETTA homology?de
novo method to construct a KvAP model in the closed state,
using the Kv1.2 closed-state model as a template (see Methods).
This approach assumes that the closed-state structures of KvAP
and Kv1.2 are similar, even though there are significant differ-
ences between the open-state structures. The center of the
largest cluster of models of KvAP in the closed state is shown in
Fig. 5. Comparison of the closed- and open?inactivated state
models of KvAP suggest substantial structural changes during
transition from the closed to open conformation (Fig. 5E and
Table 1; see also Movies 4–6, which are published as supporting
information on the PNAS web site). S4 in KvAP moves ?14 Å
outward relative to the selectivity filter, rotates clockwise ?180°
about its axis, and moves away from S5 from the adjacent
subunit. The S4–S5 linker moves away from S6 in the same
subunit, eventually breaking hydrophobic interactions with the
intracellular end of S6 (Fig. 5E). S1, S2, and S3 roll around S4
in the clockwise direction, as viewed from the extracellular side.
Rotation of S4 around its own axis and simultaneous rolling of
S2 around S4 allows for sequential interaction of the four
gating-charge-carrying residues in S4 (R117, R120, R123, and
R126 in the KvAP) with the negatively charged residue in S2
(D62 in the KvAP). Overall, these predicted gating movements
resemble those postulated for Kv1.2 (Fig. 3E), but the movement
of S4 is much larger in KvAP and results in a more outward
position in the open state.
Our modeling suggests that the difference in the magnitude of
the S4 vertical movement observed between KvAP and Kv1.2
originates mainly from the differences in the open-state struc-
tures of these channels. However, we cannot exclude the possi-
bility that S4 may also be positioned even further inward in the
closed state structure of KvAP than in Kv1.2, because our KvAP
closed-state model suggests a more outward position of S4 than
the biotin–avidin accessibility data (see Supporting Text and also
Fig. 12, which is published as supporting information on the
PNAS web site). In the x-ray structure of the VSD of KvAP, the
conserved negatively charged residue in S2 (D62) interacts with
R133, which is located seven residues downstream from the R4
arginine, the last gating charge-carrying arginine in S4 of KvAP
(10, 11) (Fig. 4 C and D). In contrast, in the x-ray structure of
Kv1.2, the homologous negatively charged residue in S2 (E226)
interacts with R300, which is R3 arginine in S4 (9) (Fig. 1 B and
C). This structural difference places the S4 residues of KvAP
?10 Å farther toward the extracellular surface in the open?
inactivated state (Figs. 3E and 5E). In addition, the S4–S5 linker
is oriented at an angle of ?45° relative to the plane of the
membrane and partially lies within the hydrocarbon core of the
membrane in the KvAP model (Fig. 5E). In contrast, the S4–S5
linker is positioned parallel to the plane of the membrane at the
interface between the polar and hydrocarbon core membrane
environments in the Kv1.2 structure (9) (Fig. 3E).
inactivated state of KvAP. (A) View of the KvAP model from the extracellular
of the model in A from the side of the membrane. The model is colored and
labeled as in Fig. 2B. Approximate position of the first residue in the S4–S5
linker is indicated by arrow for blue and yellow subunits. (C) Side view of the
are shown in stick representation. Atoms in the side chains shown are colored
as in Fig. 1B. (D) View of the model in C from the extracellular side of the
The ROSETTA membrane domain assembly model of an open?
Yarov-Yarovoy et al.
May 9, 2006 ?
vol. 103 ?
no. 19 ?
Unified Gating Mechanism for Kv1.2 and KvAP. Our modeling sug-
gests that the eukaryotic and bacterial Kv channels have
significantly different magnitude of S4 vertical movement, an
idea that was recently discussed by Tombola and coworkers
(58) based on a survey of the literature. The large movement
of S4 in KvAP is consistent with structure–function studies on
KvAP that used a biotin–avidin accessibility approach (10, 31)
but is inconsistent with many experiments on eukaryotic Kv
channels (16, 17, 22, 26–29, 50, 51). Our models provide a
natural resolution of this apparent discrepancy by showing that
the most favored conformations of the VSDs of Kv1.2 and
KvAP in the open state are quite different. These two distantly
related Kvchannels have only ?20% sequence identity in the
transmembrane region, leaving many possibilities for differ-
ences in amino acid sequence that may cause differences in
structure (see Fig. 13, which is published as supporting infor-
mation on the PNAS web site). Nevertheless, our models
suggest that KvAP may pass through an intermediate state
whose structure is similar to the Kv1.2 open state structure
during the transition from closed to open?inactivated states
(see Movies 7–9, which are published as supporting informa-
tion on the PNAS web site). In this case, the major confor-
mational change that moves the gating-charge-carrying argi-
nines in S4 through the gating pore would be analogous in
Kv1.2 and KvAP, but an additional transition would be re-
quired to reach the KvAP open?inactivated state that was
characterized structurally, in which R133 interacts with the key
negatively charged residue in S2. It is conceivable that Kv1.2 or
other eukaryotic ion channels that have a positively charged
residue analogous to R133 may potentially reach a second
open state or an inactivated state that is similar in structure to
the KvAP open?inactivated state. Thus, we propose a unified
mechanism of gating in which the major conformational
change that moves most of the gating charge across the electric
field in the transition from the closed to open state of all
voltage-gated ion channels may be similar to Kv1.2 (see Movies
1–3), but KvAP and perhaps some other members of the ion
channel superfamily may undergo a second conformational
change in which the S4 segment moves considerably farther
outward to reach a second open or inactivated state.
Structural modeling of the Kv1.2 and KvAP channels using
ROSETTA suggests that the open state conformations of the
voltage sensors of these channels are significantly different,
whereas the voltage sensors may be similar in conformation in
their closed states. This finding potentially explains the differ-
ence in magnitude of the S4 translational movement observed
experimentally for these channels. We propose a mechanism of
conformational changes of the VSD during channel gating that
sliding helix or helical screw models coupled with a rolling
motion of S1–S3 around S4 similar to recent transporter models
of gating. Extension of our ROSETTA models by homology
modeling raises the possibility that the major conformational
change in the VSD may be similar in these two distantly related
Kv channels, thereby unifying the structural basis of their gating.
The combination of structure-prediction methods with limited
experimental data should become an increasingly powerful
approach to complex problems in structural biology as structure
prediction methodology matures. We close with the thought that
our models, although in better agreement with a wide range of
structure–function data than previous ones, require detailed
work for validation. These models make many specific predic-
tions that will catalyze further research and will serve as rigorous
tests of their validity.
ROSETTA Membrane Method. We modified the homology (59) and
domain assembly (A. M. Wollacott, A. Zanghellini, and D.B.,
unpublished data) modes of ROSETTA (42) to use the membrane
environment-specific score function developed for modeling of
?-helical transmembrane proteins (45) for modeling of the Kv
channels. Multiple sequence alignment information based score
derived from the ConSeq server (http:??conseq.bioinfo-
.tau.ac.il) (60) for the Kv1.2 and KvAP sequences was added to
the ROSETTA membrane score function and improved sampling
of native-like structures (V.Y.-Y. and D.B., unpublished data).
Further details of the modeling procedures and illustrations of
additional sequence alignments, starting models, and final clus-
ters are presented in Figs. 14–23, which are published as
supporting information on the PNAS web site. Coordinates of all
structural models presented in this work are available from
V.Y.-Y. upon request (e-mail: email@example.com).
We thank Benoit Roux for critical comments on the manuscript and for
Shaker Kv channel models; Roderick Mackinnon for the KvAP channel
model; Eduardo Perozo for EPR spectroscopy accessibility data; Todd
Scheuer, Jack Schonbrun, and Alexandre Zanghellini for helpful dis-
View of the KvAP model from the extracellular side of the membrane. The
side of the membrane. The model is colored and labeled as in Fig. 2B. The
S4–S5 linker for blue and red subunits is labeled accordingly. (C) Side view of
the VSD only of the model in A. The model is colored and labeled as in Fig. 4C.
(D) View of the model in C from the intracellular side of the membrane. (E)
KvAP models of the closed and open?inactivated (labeled as ‘‘open’’) states
shown in cylinder representation and as described in Fig. 3E. Approximate
positions of the C?atoms of the first and fourth gating-charge-carrying
arginines in S4 (labeled R1 and R4), R133 in S4, and D62 in S2 (labeled D1) are
shown in sphere representation.
The ROSETTA membrane homology model of KvAP in closed state. (A)
www.pnas.org?cgi?doi?10.1073?pnas.0602350103Yarov-Yarovoy et al.
cussion; Keith Laidig for excellent administration of computational
resources; and Laura Gonzalez for making movies. This work was
supported by National Institute of Mental Health Career Development
Research Grant K01 MH67625 (to V.Y.-Y.), a Howard Hughes Medical
Institute grant (to D.B.), and National Institutes of Health Grant R01
NS15751 (to W.A.C.).
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