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: 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. 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. 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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 . Structure of the atrial natriuretic peptide receptor extracellular domain in the unbound and hormone-bound states by single-particle electron microscopy Haruo. containing an extracellular ANP- binding domain (ECD), a transmembrane domain, and an intracellular domain consisting of an ATP-binding regulatory domain and