Báo cáo khoa học: Structure of the atrial natriuretic peptide receptor extracellular domain in the unbound and hormone-bound states by single-particle electron microscopy ppt

Structure of the atrial natriuretic peptide receptor
extracellular domain in the unbound and hormone-bound
states by single-particle electron microscopy
Haruo Ogawa
1
, Yue Qiu
1
, Liming Huang
2
, Suk-Wah Tam-Chang
2
, Howard S. Young
3
and Kunio S. Misono
1
1 Department of Biochemistry, University of Nevada, Reno, NV, USA
2 Department of Chemistry, University of Nevada, Reno, NV, USA
3 Department of Biochemistry, University of Alberta, Edmonton, Canada
Atrial natriuretic peptide (ANP) is a cardiac hormone
that is secreted by the atrium of the heart in response
to blood volume expansion. ANP stimulates renal salt
excretion [1] and dilates blood vessels [2,3]. Through
these activities, ANP participates in the regulation of
blood pressure and salt–fluid volume homeostasis.
ANP also has antigrowth activity on vascular cells,
through which it regulates the maintenance and
remodeling of the cardiovascular system [4–7]. These
biological activities of ANP are mediated by the cell
Keywords
fluorescence spectroscopy; natriuretic
peptide; receptor; single particle

reconstruction; transmembrane signal
transduction
Correspondence
H. S. Young, Department of Biochemistry,
University of Alberta, Edmonton, AB T6G
2H7 Canada
Fax: +1 780 492 0095
Tel: +1 780 492 3931
E-mail:
K. S. Misono, Department of Biochemistry,
University of Nevada School of Medicine,
Reno, NV 89557, USA
Fax: +1 775 784 1419
Tel: +1 775 784 4690
E-mail:
(Received 10 October 2008, revised 14
December 2008, accepted 22 December
2008)
doi:10.1111/j.1742-4658.2009.06870.x
Atrial natriuretic peptide (ANP) plays a major role in blood pressure and
volume regulation. ANP activities are mediated by a cell surface, single-
span transmembrane receptor linked to its intrinsic guanylate cyclase activ-
ity. The crystal structures of the dimerized ANP receptor extracellular
domain (ECD) with and without ANP have revealed a novel hormone-
induced rotation mechanism occurring in the juxtamembrane region that
appears to mediate signal transduction [Ogawa H, Qiu Y, Ogata CM &
Misono KS (2004) J Biol Chem 279, 28625–28631]. However, the ECD crys-
tal packing contains two major intermolecular contacts that suggest two
possible dimer pairs: ‘head-to-head’ (hh) and ‘tail-to-tail’ (tt) dimers associ-
ated via the membrane-distal and membrane-proximal subdomains, respec-

tively. The existence of these two potential dimer forms challenges the
proposed signaling mechanism. In this study, we performed single-particle
electron microscopy (EM) to determine the ECD dimer structures occurring
in the absence of crystal contacts. EM reconstruction yielded the dimer
structures with and without ANP in only the hh dimer forms. We further
performed steady-state fluorescence spectroscopy of Trp residues, one of
which (Trp74) occurs in the hh dimer interface and none of which occurs in
the tt dimer interface. ANP binding caused a time-dependent decrease in
Trp emission at 350 nm that was attributable to partially buried Trp74
in the unbound hh dimer interface becoming exposed to solvent water upon
ANP binding. Thus, the results of single-particle EM and Trp fluorescence
studies have provided direct evidence for hh dimer structures for unbound
and ANP-bound receptor. The results also support the proposed rotation
mechanism for transmembrane signaling by the ANP receptor.
Abbreviations
ANP, atrial natriuretic peptide; ANP–ECD, atrial natriuretic peptide–extracellular domain complex; apoECD, unbound extracellular domain;
CTF, contrast transfer function; ECD, extracellular domain; EM, electron microscopy; GCase, guanylate cyclase; hh, head-to-head;
tt, tail-to-tail.
FEBS Journal 276 (2009) 1347–1355 ª 2009 The Authors Journal compilation ª 2009 FEBS 1347
surface receptor for ANP, which possesses intrinsic
guanylate cyclase (GCase) activity. The ANP receptor
occurs as a homodimer of a single-transmembrane
polypeptide, each containing an extracellular ANP-
binding domain (ECD), a transmembrane domain, and
an intracellular domain consisting of an ATP-binding
regulatory domain and a GCase catalytic domain [8].
ANP binding to the ECD stimulates the intracellular
GCase domain, thereby generating the intracellular
second messenger cGMP. The mechanism of this
transmembrane signal transduction by the ANP recep-

tor is only partially understood.
To understand the signaling mechanism, we earlier
determined the crystal structures of the dimerized
ECD with [9] and without [10] bound ANP. Comp-
arison of the two structures has revealed that ANP
binding causes a large change in the quaternary
arrangement of the ECD dimer without significant
intramolecular structure change. This change in the
quaternary structure causes an alteration in the relative
angular orientation of the two juxtamembrane
domains in the dimer that is equivalent to rotating
each by 24° [9]. There is no appreciable change in the
distance between the two juxtamembrane domains. On
the basis of this finding, we have proposed that a novel
hormone-induced rotation mechanism occurring in the
juxtamembrane region may trigger transmembrane sig-
nal transduction [9,11]. However, this proposed signal-
ing mechanism has been questioned because of
uncertainty concerning the quaternary structure of the
unbound ECD (apoECD) dimer.
The crystal packing of apoECD contains two major
intermolecular contacts (Fig. 1A), which generate two
possible dimer pairs: an hh dimer associated with the
membrane-distal subdomain (Fig. 1B) and a tt dimer
associated with the membrane-proximal subdomain
(Fig. 1C). The buried surface areas in the hh and tt
contacts in crystals are estimated to be 1100 A
˚
2
and

1680 A
˚
2
, respectively [9]. These values are both large
and are within the range often found in physiological
protein–protein interactions. Thus, it is not clear from
the crystallographic data alone whether the hh or tt
dimer represents the physiological structure. Similarly,
the ANP–ECD complex (ANP–ECD) may also occur,
at least theoretically, in an hh or a tt dimer form
(Fig. 1E,F). We originally reported the structure of
apoECD in the tt dimer configuration based on the
fact that the tt contact was estimated to be larger than
the hh contact [10]. However, our subsequent site-
directed mutagenesis studies of interface residues using
the full-length ANP receptor expressed in COS cells
showed that mutations in the hh interface, but not in
the tt interface, affected signaling (stimulation of
cGMP production by ANP) [12]. These findings have
suggested that the hh dimers, but not the tt dimers,
represent the physiological structures.
On the other hand, it has been proposed that the hh
dimer and tt dimer structures both occur, and represent
the inactive and the hormone-activated states of the
receptor, respectively [13,14]. It is hypothesized that a
hormone-induced rearrangement of the ECD from the
hh to the tt dimer structure brings the juxtamembrane
Fig. 1. Crystal packing of apoECD and ANP–ECD. (A) The crystal packing of apoECD contains two major intermolecular contacts, one
between the membrane-distal domains of two ECD monomers and another between the membrane-proximal domains. (B, C) The former
contact yields the hh dimer model (B) and the latter yields the tt dimer model (C). (D) The crystal packing of ANP–ECD similarly contains

two intermolecular contacts that give the hh dimer (E) and tt dimer (F) models for the complex. The hh dimer model for apoECD was con-
structed by performing a symmetry operation based on the coordinates of the apoECD tt dimer (Protein Data Bank code: 1DP4) [10] using
the program
O [25]. The tt dimer model for ANP–ECD was similarly constructed on the basis of the structure of the complex described previ-
ously (Protein Data Bank code: 1T34) [9]. Our current results show that the hh dimer structures represent the native structures of apoECD
and ANP–ECD, whereas the tt dimer models represent artificial crystallographic pairs.
Natriuretic peptide receptor signaling mechanism H. Ogawa et al.
1348 FEBS Journal 276 (2009) 1347–1355 ª 2009 The Authors Journal compilation ª 2009 FEBS
domains into proximity, thereby mediating signal trans-
duction [14]. This proposed mechanism involving a
ligand-induced domain approximation has been
described in some reports as being well accepted for
natriuretic peptide receptors [15,16], and been suggested
to be similar to those of the G-protein-coupled
metabotropic glutamamate receptor [15–17] and the
erythropoietin receptor [18,19]. In contrast, our pro-
posed rotation mechanism, which is based on the hh
dimer structures for both apoECD and ANP–ECD, is
mediated by a ligand-induced rotation of the juxta-
membrane domains with essentially no change in the
interdomain distance. To resolve this discrepancy over
the ANP receptor signaling mechanism, it has become
imperative to determine the ECD dimer structures in
more physiological buffer solution conditions and in
the absence of crystal contacts.
In this study, we have carried out single-particle
image reconstruction of the ECD dimer with and with-
out bound ANP using electron microscopy (EM). This
method provides the ECD dimer structure as it occurs
in solution free of crystal contacts. We reasoned that

the crystal contacts, which occur under certain arti-
ficial and rather extreme sets of conditions used for
protein crystallization, will not occur under solution
conditions closer to the physiological state. Only the
naturally occurring intermolecular contacts should
remain. The results of our single-particle EM studies
described in this article support the above reasoning,
and have identified the hh dimer as the only form
found in solution. The single-particle reconstructions
for the apoECD dimer and ANP–ECD agree closely
with the respective crystal structures, suggesting that
crystal contacts have not appreciably altered the dimer
structures. To further support our finding, we also
present here steady-state fluorescence studies of Trp
residues, taking advantage of the fact that Trp74
occurs at the hh interface and that its local environ-
ment changes upon ANP binding, whereas the envir-
onment of other Trp residues is largely unaltered. We
observed quenching of Trp fluorescence concomitant
with ANP binding, which is consistent with the apo-
ECD being in the hh dimer structure. The implications
of the results of single-particle EM and Trp fluores-
cence studies for the transmembrane signaling mecha-
nism of the ANP receptor are discussed.
Results and Discussion
EM and single-particle reconstruction
From electron micrographs of negatively stained
apoECD, more than 22 000 particles were selected
(Fig. 2A). The particles were centered and grouped
into self-similar groups by iterative multivariate statis-

tical analysis-based classification. Class averages were
then generated by iterative alignment and averaging.
Among the 35 class averages generated, many showed
clear two-fold symmetry, with several orientations con-
sistent with the hh dimer (Fig. 2B). A set of Euler
angles was then assigned to these class averages, using
common lines in Fourier space (startAny command in
eman), and an initial 3D model was built. The initial
model was used for five iterations of refinement, or
until convergence was achieved. The 3D reconstruction
had the following approximate dimensions: width,
90 A
˚
; height, 80 A
˚
; and depth, 50 A
˚
. This volume is
consistent with an ECD dimer. The final reconstruc-
tion after a minimum of five rounds of refinement
exhibited clear two-fold symmetry, which was enforced
(Fig. 2C). The data were not corrected for the contrast
Fig. 2. Single-particle EM of apoECD and ANP–ECD. (A) Represen-
tative electron micrograph and (B) class averages obtained for apo-
ECD. Similar electron micrographs and class averages were
obtained for ANP–ECD. (C, D) The 3D density maps obtained by
single-particle EM for apoECD (C) and ANP–ECD (D). The scale bar
corresponds to 10 A
˚
.

H. Ogawa et al. Natriuretic peptide receptor signaling mechanism
FEBS Journal 276 (2009) 1347–1355 ª 2009 The Authors Journal compilation ª 2009 FEBS 1349
transfer function (CTF), and only data within the first
zero of the CTF were used. On the basis of the
defocus series, this effectively limited the resolution of
the reconstruction to 22 A
˚
. Therefore, the reconstruc-
tion was low-pass-filtered at this resolution. The hand-
edness of the reconstructions was determined by
comparison with the known crystal structures of the
dimers [9,10].
A similar approach was utilized for ANP–ECD,
where the ECD was incubated with a 1.1-fold molar
excess of ANP for 1 h before grid preparation. Visual
inspection of electron micrographs of negatively
stained ANP–ECD showed no apparent differences as
compared to apoECD. More than 19 000 particles
were selected, centered, and classified as described
above. Reference-free 3D reconstruction and refine-
ment resulted in a model that showed clear two-fold
symmetry, consistent with the X-ray structure of
ANP–ECD (Fig. 2D).
Comparison of the 3D reconstructions by EM and
the crystal structures
In the crystal packing of apoECD, the buried surface
areas in the hh and tt dimers are within the range typi-
cally found for physiological protein–protein interac-
tions. Thus, it is not possible from the crystallographic
data alone to determine which dimer structure repre-

sents the physiological state. To identify the correct
apoECD dimer, the crystal structures for apoECD in
the hh dimer (Fig. 1B) and tt dimer (Fig. 1C) forms
were superimposed on the 3D reconstruction of apo-
ECD obtained by single-particle EM (Fig. 3A,C). The
molecular envelope of the hh dimer crystal structure
agreed closely with the EM density map, whereas that
in the tt dimer form clearly showed a large structural
discrepancy. These results demonstrate that apoECD,
in the absence of crystal contacts, assumes the hh
dimer structure.
In the crystal packing of ANP–ECD, two ECD
monomers form an hh dimer, with one molecule of
ANP captured in between these monomers [9]. In this
structure, ANP binding involves a very large buried sur-
face area (1450 A
˚
2
with one ECD monomer and
1320 A
˚
2
with the other monomer, for a total buried sur-
face area of 2770 A
˚
2
), which strongly supports the
notion that the hh dimer structure represents the physi-
ological ANP–ECD structure. The crystal structure of
ANP–ECD in the hh dimer form (Fig. 1E), when super-

imposed on the 3D reconstruction obtained by single-
particle EM, agreed closely (Fig. 3B). On the other
hand, the tt dimer model (Fig. 1F) showed a large dis-
crepancy with the EM reconstruction (Fig. 3D).
We also performed reference-based single-particle
reconstruction using the hh and tt dimer crystal struc-
tures as initial models (Fig. S1). The reconstruction of
apoECD and ANP–ECD using the hh dimers as the
initial models quickly converged within five refinement
cycles on a reconstruction that was similar to the hh
dimer described above. In contrast, the refinements
using the tt dimer as the initial model quickly diverged
from the initial models within five cycles of refinement.
By 20 cycles, the solution converged on a reconstruc-
tion similar to the hh dimer. These results suggest that
both apoECD and ANP–ECD occur entirely in the hh
dimer form in solution. Hence, the tt contacts in crys-
tals are artificial interactions that only occur under the
conditions used for crystallization and do not occur in
more physiological solution conditions. Additionally,
the close agreement of the EM reconstructions with
their respective crystal structures indicates that the
crystal contacts did not appreciably alter the quater-
nary structures of the dimers.
Steady-state fluorescence studies of ANP-induced
structural change
Each ECD monomer contains 10 Trp residues. Of
these, one, Trp74, occurs in the hh interface
(Fig. 4A,B). No Trp residue is present in the tt inter-
face. In the apoECD hh dimer model (Fig. 4A), Trp74

of one monomer interacts with Trp74 of the other
monomer and contributes to the hh dimer contact [9].
In ANP–ECD (Fig. 4B), these two Trp74 residues are
pulled apart and are exposed to the solvent. We have
Fig. 3. Superimposition of the X-ray crystallographic structures on
the density maps obtained by single-particle EM. (A, C) The X-ray
structures (ribbon models) of apoECD in the hh dimer and tt dimer
forms, respectively, are superimposed on the apoECD density map
obtained by single-particle EM (blue shading). (B, D) The crystal
structure of ANP–ECD [9] and the hypothetical tt dimer model for
the complex, respectively, are superimposed on the EM density
map of ANP–ECD (gold shading).
Natriuretic peptide receptor signaling mechanism H. Ogawa et al.
1350 FEBS Journal 276 (2009) 1347–1355 ª 2009 The Authors Journal compilation ª 2009 FEBS
shown previously that ANP binding causes no appre-
ciable intramolecular structural change in the ECD
monomers (rmsd of Ca atoms between the apo and
the complex structures, 0.64 A
˚
) [9]. Furthermore, no
Trp residues make contact with ANP in the bound
complex. Therefore, if the ECD assumes the hh dimer
structures, only the Trp74 residue should undergo a
significant change in its environment. On the other
hand, if the ECD assumes the tt dimer structures, no
change is expected in the Trp environment in response
to ANP binding. On the basis of the above structure
analyses, we utilized Trp fluorescence to examine the
solution structures of apoECD and ANP–ECD.
The fluorescence emission spectra of apoECD and

ANP–ECD are shown in Fig. 4C. Comparison of the
spectra shows that addition of ANP causes an approxi-
mately 7% decrease in the fluorescence emission inten-
sity at the lambda maximum 350 nm. This drop in the
fluorescence intensity was time-dependent and was lar-
gely complete in about 30 min (not shown). The course
of this intensity drop matches closely the course of
ANP binding measured using [
125
I]ANP [20]. These
findings are consistent with the hh dimer structures for
both apoECD and ANP–ECD, where the two partially
buried Trp74 residues at the apoECD hh dimer inter-
face become exposed upon ANP binding [9,12] and
quenched by water. The difference spectrum obtained
by subtracting the ANP–ECD emission from the apo-
ECD emission revealed a shift to a longer wavelength
(Fig. 4C). This red shift in the emission difference
spectrum is consistent with the two Trp74 residues that
are localized at the edge of the apo dimer interface in
a partially exposed, polar environment [21]. The
decrease in Trp emission intensity from the total emis-
sion intensity from 10 Trp residues in each ECD
monomer was relatively small (7%). The quantum
yield of Trp residues is known to vary widely, depend-
ing on the environment. The relatively small decrease
may be due to quenching of the two Trp74 residues at
the apoECD hh dimer by a staggered face-to-face
interaction between the two indole rings (Fig. 4A).
To confirm that the decrease in the fluorescence

intensity is due to the change in Trp74 environment,
we measured the fluorescence emission of an ECD
Fig. 4. Steady-state fluorescence spectroscopy studies of ECD in the presence and absence of ANP. (A, B) Structures of the apoECD dimer
(A) and ANP–ECD (B) in the hh dimer configuration. Only Trp74 (shown in green) occurs at the dimer interface. All other Trp residues are
labeled in red. The bound ANP (B) does not contact any of the Trp residues. (C) Fluorescence emission spectra of apoECD (solid line) and
ANP–ECD (dotted line). The maximum emission intensity of apoECD was calculated as the average intensity over the wavelength range
from k
max
= )5nmtok
max
= +5 nm, and was taken as 100% intensity. The difference emission spectrum obtained by subtracting the emis-
sion intensity of ANP–ECD from that of the apoECD dimer is indicated by circles. (D) Fluorescence emission spectra of the apoECD W74R
mutant [12] (solid line) and the ANP–ECD-W74R complex (dotted line). The maximum emission intensity of the apoECD W74R mutant was
considered to be 100%.
H. Ogawa et al. Natriuretic peptide receptor signaling mechanism
FEBS Journal 276 (2009) 1347–1355 ª 2009 The Authors Journal compilation ª 2009 FEBS 1351
mutant, W74R. We have shown previously that the
W74R mutant binds ANP with an affinity similar to
that of the wild-type [12]. The fluorescence emission
spectrum of the W74R mutant was similar to that of
the wild-type, with a peak at around 350 nm, but with
a slightly reduced intensity because of the Trp to Arg
mutation. As shown in Fig. 4D, addition of ANP to
the W74R mutant caused no appreciable change in the
emission intensity. This finding confirms that the
decrease in Trp fluorescence observed upon ANP bind-
ing to the wild-type ECD was caused by solvent expo-
sure and the resulting quenching of Trp74 emission in
ANP–ECD.
Comparison of the apoECD and ANP–ECD EM

reconstructions
To evaluate the structural change induced by ANP
binding, the 3D reconstructions of apoECD and
ANP–ECD were aligned with each other for compari-
son, using the align3d command in eman (Fig. 5).
For clarity, the reconstructions are contoured at 70%
of the expected molecular volume for an ECD dimer.
Despite the low resolution of the reconstructions, the
ANP–ECD structure is more detailed, with a shape
characteristic of the crystal structure. Nonetheless,
both EM reconstructions exhibit dimeric shape and
monomer orientations that closely agree with those
observed by X-ray crystallography. In the front view,
there is no appreciable change in the distance between
the two monomers (Fig. 5). Viewed from the side,
each monomer in the ANP–ECD reconstruction is
displaced in a clockwise direction, reminiscent of the
twist motion observed by X-ray crystallography [9].
Viewed from the bottom (i.e. in the direction from
the presumed transmembrane regions; Fig. 5, bottom
view), the two juxtamembrane domains are displaced
in opposite directions upon binding of ANP, without
an appreciable change in the distance between the
two.
Proposed mechanism for transmembrane signal
transduction
On the basis of the hh dimer pairs demonstrated
above, the X-ray structures of ECD with [9] and with-
out [10] bound ANP show that ANP binding causes a
large change in the quaternary structure of the ECD

dimer without appreciable intramolecular structural
change. ANP binding causes each of the two ECD
monomers to undergo a twisting motion while retain-
ing the two-fold symmetry in the dimeric complex [9].
This twisting motion causes the two juxtamembrane
domains in the dimer to undergo parallel translocation
in the opposite direction, with essentially no change in
the distance between the two (Fig. 6A). This move-
ment causes an alteration in the relative angular orien-
tation of the two juxtamembrane domains that is
equivalent to rotating each domain by 24° (Fig. 6B).
We have proposed that this hormone-induced rotation
mechanism occurring in the juxtamembrane region
may trigger ANP receptor signaling [9,11]. The ANP-
induced structural change observed here by single-par-
ticle EM closely resembles that recognized by X-ray
crystallography, thus supporting the proposed signal-
ing mechanism.
In summary, the 3D reconstructions by single-parti-
cle EM, which were obtained in the absence of crystal
Fig. 5. Overlay of the single-particle reconstructions in the absence (blue mesh) and presence (gold surface) of ANP. The reconstructions
are rendered at 70% of the correct molecular volume for clarity. ANP binding causes each of the two ECD monomers to undergo a twist
while maintaining the two-fold symmetry axis in the dimerized complex. The orientation of each EM construction is based on the closeness
of the fit to the respective X-ray structure as shown in Fig. 3. The front and side views are oriented such that the juxtamembrane domains
are the lower lobes of the reconstructions. The bottom view is oriented such that the reconstructions are shown from the perspective of
the membrane plane (looking up at the juxtamembrane domains).
Natriuretic peptide receptor signaling mechanism H. Ogawa et al.
1352 FEBS Journal 276 (2009) 1347–1355 ª 2009 The Authors Journal compilation ª 2009 FEBS
contacts, yielded the hh dimer structures for both
apoECD and ANP–ECD. Comparison of the 3D

reconstructions with and without ANP showed the
ANP-induced structural change in the dimer that was
surprisingly close to that observed by X-ray crystal-
lography. The quenching of Trp74 fluorescence emis-
sion concomitant with ANP binding is also in
agreement with apoECD and ANP–ECD in hh dimer
structures. Thus, the results of our complementary
approaches, single-particle EM, fluorescence spectros-
copy and X-ray crystallography, together demonstrate
a novel hormone-induced structural change in the
ECD dimer that generates a rotation mechanism in
the juxtamembrane regions and possibly mediates
transmembrane signal transduction.
Experimental procedures
Preparation of ECD and ANP–ECD
ECD consisting of residues 1–435 of the rat ANP receptor
was expressed by slight modification of the method
described previously [22], as follows. CHO cells were trans-
fected with pcDNA3–NPRA, and stably transfected, high-
producer cells were cloned by selection with G-418. The
cloned cells were cultured in roller bottles, and the condi-
tioned medium containing the expressed ECD was collected
every 2 days. The ECD was purified by ANP affinity chro-
matography as previously described [22]. ANP–ECD was
prepared by incubating ECD (1 mgÆmL
)1
) with a 1.1-fold
molar excess of a truncated ANP peptide, Cys-Phe-Gly-
Gly-Arg-Ile-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-
Asn-Ser-Phe-Arg, representing residues 7–27, in 5 mm

Hepes buffer (pH 7.0) containing 20 mm NaCl at room
temperature for 60 min.
Single-particle EM
Aliquots (3 lL) of ECD at 0.03 mgÆmL
)1
in the absence
(apoECD) and presence (ANP–ECD) of ANP were
applied to glow-discharged, carbon-coated grids. The grid
was washed with two drops of 2% uranyl acetate, and
then a third drop of 2% uranyl acetate was allowed to
sit on the grid for 1 min (4 °C). The excess stain was
removed by blotting with filter paper, and the sample
was allowed to air dry. Data were collected on a Tec-
nai F20 (FEI Company) located in the Microscopy and
Imaging Facility at the University of Calgary (Calgary,
Canada). The microscope was operated at 200 keV, and
images were recorded on Kodak SO-163 film under low-
dose conditions at a magnification of ·50 000, with a
defocus ranging from )1.5 to )2.5 lm. Micrographs were
digitized with a Nikon Super Coolscan 9000 with a scan-
ning resolution of 6.35 lmÆpixel
)1
, and this was followed
by pixel averaging to achieve a final resolution of
3.81 A
˚
Æpixel
)1
.
Image processing and reconstruction were performed

with the eman program package [23]. Seventeen micro-
graphs with minimal drift and astigmatism were selected
for reconstruction of apoECD. Similarly, 20 micrographs
were used for ANP–ECD. Particles were selected semiauto-
matically and extracted as 40 · 40 pixel images (boxer). In
total, 22 778 and 19 600 particle images were selected for
apoECD and ANP–ECD, respectively. No correction for
the CTF was applied. Reference-free classification was per-
formed to generate 35 class averages (refine2d.py), and an
initial set of Euler angles was then assigned to these class
averages (startAny). The initial three-dimensional models
built using common lines in Fourier space were then refined
in eman for up to 20 cycles of refinement (refine). The
assignment of Eulerian angles from class averages by
Fig. 6. ANP-induced structural change in the ANP receptor juxta-
membrane domains and proposed rotation mechanism for trans-
membrane signaling. (A) The X-ray structures of the
juxtamembrane domains in apoECD (blue) and ANP–ECD (orange)
are shown as viewed from the membrane [9]. ANP binding causes
a parallel translocation of the two juxtamembrane domains in the
opposite direction without an appreciable change in the interdomain
distance. (B) Schematic presentation of the movement of the juxta-
membrane domains in response to ANP binding. Looking down-
wards toward the cell membrane, ANP binding causes a translation
of the juxtamembrane domains from the apo position (depicted by
blue circles) to the complex positions (orange circles). The arrows
depict this parallel translocation. This movement causes a change
in the relative orientation between the two juxtamembrane
domains in the dimer that is equivalent to rotating each by 24°
counterclockwise (inset). We propose that this ligand-induced rota-

tion motion in the juxtamembrane domains initiates transmembrane
signaling [9].
H. Ogawa et al. Natriuretic peptide receptor signaling mechanism
FEBS Journal 276 (2009) 1347–1355 ª 2009 The Authors Journal compilation ª 2009 FEBS 1353
common lines results in two possible enantiomeric solu-
tions. The X-ray crystallographic structures were used to
determine the handedness of the reconstructions. Because
the expected two-fold symmetry for the two ECD mono-
mers in apoECD and ANP–ECD was observed, C2 symme-
try was applied throughout the refinement procedure. The
first zero of the CTF for the lowest defocus images effec-
tively limited the resolution of the final reconstruction to
 22 A
˚
. This resolution limit was confirmed by calculating
the Fourier shell correlation between two independent half
datasets (eotest command in eman; 0.5 FSC criterion).
Therefore, the final density maps were low-pass-filtered to
22 A
˚
resolution. The final 3D maps were visualized and
analyzed, and figures were created using the UCSF chi-
mera package [24]. A protein partial specific volume of
0.73 cm
3
Æg
)1
was used to set the isosurface threshold that
corresponded to the correct molecular volume.
Because of the availability of apoECD and ANP–ECD

crystal structures, we also performed reference-based
refinement (eman) as a means of evaluating agreement of
the single-particle data with the X-ray crystallographic
data. The crystal structures of apoECD (Protein Data
Bank code: 1DP4) and ANP–ECD (Protein Data Bank
code: 1T34) each contain tt dimer and hh dimer pairs.
Density maps were created from the hh and tt dimer pairs
at a resolution comparable to the EM data (pdb2mrc;
22 A
˚
resolution) for each of apoECD and ANP–ECD.
These density maps were then used as starting models for
the refine command in eman. Up to 20 cycles of refine-
ment were performed. Depending on whether the hh or tt
dimer map was used as the starting model, the refinement
quickly diverged from an incorrect solution, and it con-
verged on the correct solution within 20 cycles of refine-
ment. Finally, fitting of the atomic coordinates of the hh
or tt dimer pairs to the EM reconstructions was performed
with eman (foldhunterp). Calculated density maps from
each atomic model were used as reference structures for
the calculation.
Steady-state fluorescence spectroscopic studies
of Trp residues
Fluorescence emission spectra were acquired in a Fluoro-
log-222 fluorescence spectrometer using fluorescence soft-
ware over the wavelength range from 305 to 500 nm with
excitation at 291 nm and an emission slit width of 2 nm.
All experiments were carried out at 22 °C.
ECD or mutated ECD W74R [12], in which Trp74 was

replaced by Arg, at 1 mgÆmL
)1
concentration in 5 mm
Hepes buffer (pH 7.0), containing 20 mm NaCl was used in
the experiments. Fluorescence emission spectra of ECD or
ECD W74R were acquired before and after the addition of
a 1.1-fold molar excess of the truncated ANP peptide. The
change in the emission spectrum was followed at 2 min
intervals over a period of 60 min.
Acknowledgements
The work was supported by HL54329 to K. S. Misono
and by grants to H. S. Young from the Canadian
Institutes for Health Research, the Canada Founda-
tion for Innovation, and the Alberta Science and
Research Investments Program. H. S. Young is a
Senior Scholar of the Alberta Heritage Foundation for
Medical Research.
References
1 de Bold AJ, Borenstein HB, Veress AT & Sonnenberg
H (1981) A rapid and potent natriuretic response to
intravenous injection of atrial myocardial extract in
rats. Life Sci 28, 89–94.
2 Currie MG, Geller DM, Cole BR, Boylan JG, YuSheng
W, Holmberg SW & Needleman P (1983) Bioactive car-
diac substances: potent vasorelaxant activity in mamma-
lian atria. Science 221, 71–73.
3 Grammer RT, Fukumi H, Inagami T & Misono KS
(1983) Rat atrial natriuretic factor. Purification and
vasorelaxant activity. Biochem Biophys Res Commun
116, 696–703.

4 Itoh H, Pratt RE & Dzau VJ (1990) Atrial natriuretic
polypeptide inhibits hypertrophy of vascular smooth
muscle cells. J Clin Invest 86, 1690–1697.
5 Chrisman TD & Garbers DL (1999) Reciprocal antago-
nism coordinates C-type natriuretic peptide and mito-
gen-signaling pathways in fibroblasts. J Biol Chem 274,
4293–4299.
6 Suenobu N, Shichiri M, Iwashina M, Marumo F &
Hirata Y (1999) Natriuretic peptides and nitric oxide
induce endothelial apoptosis via a cGMP-dependent
mechanism. Arterioscler Thromb Vasc Biol 19, 140–146.
7 Wu CF, Bishopric NH & Pratt RE (1997) Atrial natri-
uretic peptide induces apoptosis in neonatal rat cardiac
myocytes. J Biol Chem 272, 14860–14866.
8 Chinkers M, Garbers DL, Chang MS, Lowe DG, Chin
HM, Goeddel DV & Schulz S (1989) A membrane form
of guanylate cyclase is an atrial natriuretic peptide
receptor. Nature 338, 78–83.
9 Ogawa H, Qiu Y, Ogata CM & Misono KS (2004)
Crystal structure of hormone-bound atrial natriuretic
peptide receptor extracellular domain: rotation mecha-
nism for transmembrane signal transduction. J Biol
Chem 279, 28625–28631.
10 van den Akker F, Zhang X, Miyagi M, Huo X, Misono
KS & Yee VC (2000) Structure of the dimerized hor-
mone-binding domain of a guanylyl-cyclase-coupled
receptor. Nature 406, 101–104.
11 Misono KS, Ogawa H, Qiu Y & Ogata CM (2005)
Structural studies of the natriuretic peptide receptor: a
novel hormone-induced rotation mechanism for trans-

membrane signal transduction. Peptides 26, 957–968.
Natriuretic peptide receptor signaling mechanism H. Ogawa et al.
1354 FEBS Journal 276 (2009) 1347–1355 ª 2009 The Authors Journal compilation ª 2009 FEBS
12 Qiu Y, Ogawa H, Miyagi M & Misono KS (2004)
Constitutive activation and uncoupling of the atrial
natriuretic peptide receptor by mutations at the dimer
interface. Role of the dimer structure in signalling.
J Biol Chem 279, 6115–6123.
13 De Lean A, McNicoll N & Labrecque J (2003) Natri-
uretic peptide receptor A activation stabilizes a
membrane-distal dimer interface. J Biol Chem 278,
11159–11166.
14 van den Akker F (2001) Structural insights into the
ligand binding domains of membrane bound guanylyl
cyclases and natriuretic peptide receptors. J Mol Biol
311, 923–937.
15 Pin JP, Neubig R, Bouvier M, Devi L, Filizola M,
Javitch JA, Lohse MJ, Milligan G, Palczewski K,
Parmentier M et al. (2007) International Union of Basic
and Clinical Pharmacology. LXVII. Recommendations
for the recognition and nomenclature of G protein-cou-
pled receptor heteromultimers. Pharmacol Rev 59, 5–13.
16 Brock C, Oueslati N, Soler S, Boudier L, Rondard P &
Pin JP (2007) Activation of a dimeric metabotropic
glutamate receptor by intersubunit rearrangement.
J Biol Chem 282, 33000–33008.
17 Rondard P, Huang S, Monnier C, Tu H, Blanchard B,
Oueslati N, Malhaire F, Li Y, Trinquet E, Labesse G
et al. (2008) Functioning of the dimeric GABA(B)
receptor extracellular domain revealed by glycan wedge

scanning. EMBO J 27, 1321–1332.
18 Livnah O, Stura EA, Middleton SA, Johnson DL,
Jolliffe LK & Wilson IA (1999) Crystallographic evi-
dence for preformed dimers of erythropoietin receptor
before ligand activation. Science 283, 987–990.
19 Remy I, Wilson IA & Michnick SW (1999) Erythropoi-
etin receptor activation by a ligand-induced conforma-
tion change. Science 283, 990–993.
20 Misono KS, Grammer RT, Rigby JW & Inagami T
(1985) Photoaffinity labeling of atrial natriuretic factor
receptor in bovine and rat adrenal cortical membranes.
Biochem Biophys Res Commun 130, 994–1001.
21 Lakowicz JR (2006) General features of protein fluores-
cence. In Principles of Fluorescence Spectroscopy
(Lakowicz JR, ed.), pp. 535–538. Springer, New York.
22 Misono KS, Sivasubramanian N, Berkner K & Zhang
X (1999) Expression and purification of the extracellular
ligand-binding domain of the atrial natriuretic peptide
(ANP) receptor. Biochemistry 38, 516–523.
23 Ludtke SJ, Baldwin PR & Chiu W (1999) EMAN: semi-
automated software for high-resolution single-particle
reconstructions. J Struct Biol 128, 82–97.
24 Pettersen EF, Goddard TG, Huang CC, Couch GS,
Greenblatt DM, Meng EC & Ferrin TE (2004) UCSF
Chimera – a visualization system for exploratory
research and analysis. J Comput Chem 25, 1605–1612.
25 Jones TA, Zou JY, Cowan SW & Kjeldgaard M (1991)
Improved methods for building protein models in elec-
tron density maps and the location of errors in these
models. Acta Crystallogr A47, 110–119.

Supporting information
The following supplementary material is available:
Fig. S1. Reference-based refinement of the single-parti-
cle EM data against the crystallographic structures.
Doc. S1. Reference-based reconstructions converge to
the hh dimer structures for both apoECD and
ANP-ECD.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell are not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
H. Ogawa et al. Natriuretic peptide receptor signaling mechanism
FEBS Journal 276 (2009) 1347–1355 ª 2009 The Authors Journal compilation ª 2009 FEBS 1355