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

In this study, we performed single-particle electron microscopy EM to determine the ECD dimer structures occurring in the absence of crystal contacts.. D The crystal packing of ANP–ECD s

extracellular domain in the unbound and hormone-bound states by single-particle electron microscopy

Haruo Ogawa1, Yue Qiu1, Liming Huang2, Suk-Wah Tam-Chang2, Howard S Young3

and Kunio S Misono1

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: hyoung@ualberta.ca

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: kmisono@unr.edu

(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.

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 sigsig-nal-

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.

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 ˚

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 resolureconstruc-tion 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˚2with 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).

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 [125I]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 kmax= )5 nm to k 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 emisemis-sion 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%.

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).

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 transtrans-fected, 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 (4C) 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 morota-tion in the juxtamembrane domains initiates transmembrane

signaling [9].

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 cm3Æg)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 22C

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 InnovaFounda-tion, and the Alberta Science and Research Investments Program H S Young is a Senior Scholar of the Alberta Heritage Foundation for Medical Research

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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

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