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

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Structure of the atrial natriuretic peptide receptorextracellular domain in the unbound and hormone-boundstates by single-particle electron microscopyHaruo Ogawa1, Yue Qiu1, Liming Huang2, Suk-Wah Tam-Chang2, Howard S. Young3and Kunio S. Misono11 Department of Biochemistry, University of Nevada, Reno, NV, USA2 Department of Chemistry, University of Nevada, Reno, NV, USA3 Department of Biochemistry, University of Alberta, Edmonton, CanadaAtrial natriuretic peptide (ANP) is a cardiac hormonethat is secreted by the atrium of the heart in responseto blood volume expansion. ANP stimulates renal saltexcretion [1] and dilates blood vessels [2,3]. Throughthese activities, ANP participates in the regulation ofblood pressure and salt–fluid volume homeostasis.ANP also has antigrowth activity on vascular cells,through which it regulates the maintenance andremodeling of the cardiovascular system [4–7]. Thesebiological activities of ANP are mediated by the cellKeywordsfluorescence spectroscopy; natriureticpeptide; receptor; single particlereconstruction; transmembrane signaltransductionCorrespondenceH. S. Young, Department of Biochemistry,University of Alberta, Edmonton, AB T6G2H7 CanadaFax: +1 780 492 0095Tel: +1 780 492 3931E-mail: hyoung@ualberta.caK. S. Misono, Department of Biochemistry,University of Nevada School of Medicine,Reno, NV 89557, USAFax: +1 775 784 1419Tel: +1 775 784 4690E-mail: kmisono@unr.edu(Received 10 October 2008, revised 14December 2008, accepted 22 December2008)doi:10.1111/j.1742-4658.2009.06870.xAtrial natriuretic peptide (ANP) plays a major role in blood pressure andvolume 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 extracellulardomain (ECD) with and without ANP have revealed a novel hormone-induced rotation mechanism occurring in the juxtamembrane region thatappears 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 twopossible 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 theproposed signaling mechanism. In this study, we performed single-particleelectron microscopy (EM) to determine the ECD dimer structures occurringin the absence of crystal contacts. EM reconstruction yielded the dimerstructures with and without ANP in only the hh dimer forms. We furtherperformed steady-state fluorescence spectroscopy of Trp residues, one ofwhich (Trp74) occurs in the hh dimer interface and none of which occurs inthe tt dimer interface. ANP binding caused a time-dependent decrease inTrp emission at 350 nm that was attributable to partially buried Trp74in the unbound hh dimer interface becoming exposed to solvent water uponANP binding. Thus, the results of single-particle EM and Trp fluorescencestudies have provided direct evidence for hh dimer structures for unboundand ANP-bound receptor. The results also support the proposed rotationmechanism for transmembrane signaling by the ANP receptor.AbbreviationsANP, 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 1347surface receptor for ANP, which possesses intrinsicguanylate cyclase (GCase) activity. The ANP receptoroccurs as a homodimer of a single-transmembranepolypeptide, each containing an extracellular ANP-binding domain (ECD), a transmembrane domain, andan intracellular domain consisting of an ATP-bindingregulatory domain and a GCase catalytic domain [8].ANP binding to the ECD stimulates the intracellularGCase domain, thereby generating the intracellularsecond messenger cGMP. The mechanism of thistransmembrane signal transduction by the ANP recep-tor is only partially understood.To understand the signaling mechanism, we earlierdetermined the crystal structures of the dimerizedECD with [9] and without [10] bound ANP. Comp-arison of the two structures has revealed that ANPbinding causes a large change in the quaternaryarrangement of the ECD dimer without significantintramolecular structure change. This change in thequaternary structure causes an alteration in the relativeangular orientation of the two juxtamembranedomains in the dimer that is equivalent to rotatingeach by 24° [9]. There is no appreciable change in thedistance between the two juxtamembrane domains. Onthe basis of this finding, we have proposed that a novelhormone-induced rotation mechanism occurring in thejuxtamembrane region may trigger transmembrane sig-nal transduction [9,11]. However, this proposed signal-ing mechanism has been questioned because ofuncertainty concerning the quaternary structure of theunbound ECD (apoECD) dimer.The crystal packing of apoECD contains two majorintermolecular contacts (Fig. 1A), which generate twopossible dimer pairs: an hh dimer associated with themembrane-distal subdomain (Fig. 1B) and a tt dimerassociated with the membrane-proximal subdomain(Fig. 1C). The buried surface areas in the hh and ttcontacts in crystals are estimated to be 1100 A˚2and1680 A˚2, respectively [9]. These values are both largeand are within the range often found in physiologicalprotein–protein interactions. Thus, it is not clear fromthe crystallographic data alone whether the hh or ttdimer 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 ofapoECD in the tt dimer configuration based on thefact that the tt contact was estimated to be larger thanthe hh contact [10]. However, our subsequent site-directed mutagenesis studies of interface residues usingthe full-length ANP receptor expressed in COS cellsshowed that mutations in the hh interface, but not inthe tt interface, affected signaling (stimulation ofcGMP production by ANP) [12]. These findings havesuggested that the hh dimers, but not the tt dimers,represent the physiological structures.On the other hand, it has been proposed that the hhdimer and tt dimer structures both occur, and representthe inactive and the hormone-activated states of thereceptor, respectively [13,14]. It is hypothesized that ahormone-induced rearrangement of the ECD from thehh to the tt dimer structure brings the juxtamembraneFig. 1. Crystal packing of apoECD and ANP–ECD. (A) The crystal packing of apoECD contains two major intermolecular contacts, onebetween the membrane-distal domains of two ECD monomers and another between the membrane-proximal domains. (B, C) The formercontact yields the hh dimer model (B) and the latter yields the tt dimer model (C). (D) The crystal packing of ANP–ECD similarly containstwo 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] usingthe programO [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 apoECDand 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 FEBSdomains into proximity, thereby mediating signal trans-duction [14]. This proposed mechanism involving aligand-induced domain approximation has beendescribed in some reports as being well accepted fornatriuretic peptide receptors [15,16], and been suggestedto be similar to those of the G-protein-coupledmetabotropic glutamamate receptor [15–17] and theerythropoietin receptor [18,19]. In contrast, our pro-posed rotation mechanism, which is based on the hhdimer structures for both apoECD and ANP–ECD, ismediated by a ligand-induced rotation of the juxta-membrane domains with essentially no change in theinterdomain distance. To resolve this discrepancy overthe ANP receptor signaling mechanism, it has becomeimperative to determine the ECD dimer structures inmore physiological buffer solution conditions and inthe absence of crystal contacts.In this study, we have carried out single-particleimage reconstruction of the ECD dimer with and with-out bound ANP using electron microscopy (EM). Thismethod provides the ECD dimer structure as it occursin solution free of crystal contacts. We reasoned thatthe crystal contacts, which occur under certain arti-ficial and rather extreme sets of conditions used forprotein crystallization, will not occur under solutionconditions closer to the physiological state. Only thenaturally occurring intermolecular contacts shouldremain. The results of our single-particle EM studiesdescribed in this article support the above reasoning,and have identified the hh dimer as the only formfound in solution. The single-particle reconstructionsfor the apoECD dimer and ANP–ECD agree closelywith the respective crystal structures, suggesting thatcrystal contacts have not appreciably altered the dimerstructures. To further support our finding, we alsopresent here steady-state fluorescence studies of Trpresidues, taking advantage of the fact that Trp74occurs 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. Weobserved quenching of Trp fluorescence concomitantwith ANP binding, which is consistent with the apo-ECD being in the hh dimer structure. The implicationsof 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 DiscussionEM and single-particle reconstructionFrom electron micrographs of negatively stainedapoECD, more than 22 000 particles were selected(Fig. 2A). The particles were centered and groupedinto self-similar groups by iterative multivariate statis-tical analysis-based classification. Class averages werethen generated by iterative alignment and averaging.Among the 35 class averages generated, many showedclear two-fold symmetry, with several orientations con-sistent with the hh dimer (Fig. 2B). A set of Eulerangles was then assigned to these class averages, usingcommon lines in Fourier space (startAny command ineman), and an initial 3D model was built. The initialmodel was used for five iterations of refinement, oruntil convergence was achieved. The 3D reconstructionhad the following approximate dimensions: width,90 A˚; height, 80 A˚; and depth, 50 A˚. This volume isconsistent with an ECD dimer. The final reconstruc-tion after a minimum of five rounds of refinementexhibited clear two-fold symmetry, which was enforced(Fig. 2C). The data were not corrected for the contrastFig. 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 wereobtained for ANP–ECD. (C, D) The 3D density maps obtained bysingle-particle EM for apoECD (C) and ANP–ECD (D). The scale barcorresponds to 10 A˚.H. Ogawa et al. Natriuretic peptide receptor signaling mechanismFEBS Journal 276 (2009) 1347–1355 ª 2009 The Authors Journal compilation ª 2009 FEBS 1349transfer function (CTF), and only data within the firstzero of the CTF were used. On the basis of thedefocus series, this effectively limited the resolution ofthe reconstruction to 22 A˚. Therefore, the reconstruc-tion was low-pass-filtered at this resolution. The hand-edness of the reconstructions was determined bycomparison with the known crystal structures of thedimers [9,10].A similar approach was utilized for ANP–ECD,where the ECD was incubated with a 1.1-fold molarexcess of ANP for 1 h before grid preparation. Visualinspection of electron micrographs of negativelystained ANP–ECD showed no apparent differences ascompared to apoECD. More than 19 000 particleswere selected, centered, and classified as describedabove. Reference-free 3D reconstruction and refine-ment resulted in a model that showed clear two-foldsymmetry, consistent with the X-ray structure ofANP–ECD (Fig. 2D).Comparison of the 3D reconstructions by EM andthe crystal structuresIn the crystal packing of apoECD, the buried surfaceareas 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 crystallographicdata alone to determine which dimer structure repre-sents the physiological state. To identify the correctapoECD dimer, the crystal structures for apoECD inthe hh dimer (Fig. 1B) and tt dimer (Fig. 1C) formswere superimposed on the 3D reconstruction of apo-ECD obtained by single-particle EM (Fig. 3A,C). Themolecular envelope of the hh dimer crystal structureagreed closely with the EM density map, whereas thatin the tt dimer form clearly showed a large structuraldiscrepancy. These results demonstrate that apoECD,in the absence of crystal contacts, assumes the hhdimer structure.In the crystal packing of ANP–ECD, two ECDmonomers form an hh dimer, with one molecule ofANP captured in between these monomers [9]. In thisstructure, ANP binding involves a very large buried sur-face area (1450 A˚2with one ECD monomer and1320 A˚2with the other monomer, for a total buried sur-face area of 2770 A˚2), which strongly supports thenotion that the hh dimer structure represents the physi-ological ANP–ECD structure. The crystal structure ofANP–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 otherhand, the tt dimer model (Fig. 1F) showed a large dis-crepancy with the EM reconstruction (Fig. 3D).We also performed reference-based single-particlereconstruction using the hh and tt dimer crystal struc-tures as initial models (Fig. S1). The reconstruction ofapoECD and ANP–ECD using the hh dimers as theinitial models quickly converged within five refinementcycles on a reconstruction that was similar to the hhdimer described above. In contrast, the refinementsusing the tt dimer as the initial model quickly divergedfrom 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 thatboth apoECD and ANP–ECD occur entirely in the hhdimer form in solution. Hence, the tt contacts in crys-tals are artificial interactions that only occur under theconditions used for crystallization and do not occur inmore physiological solution conditions. Additionally,the close agreement of the EM reconstructions withtheir respective crystal structures indicates that thecrystal contacts did not appreciably alter the quater-nary structures of the dimers.Steady-state fluorescence studies of ANP-inducedstructural changeEach ECD monomer contains 10 Trp residues. Ofthese, 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), Trp74of one monomer interacts with Trp74 of the othermonomer and contributes to the hh dimer contact [9].In ANP–ECD (Fig. 4B), these two Trp74 residues arepulled apart and are exposed to the solvent. We haveFig. 3. Superimposition of the X-ray crystallographic structures onthe density maps obtained by single-particle EM. (A, C) The X-raystructures (ribbon models) of apoECD in the hh dimer and tt dimerforms, respectively, are superimposed on the apoECD density mapobtained by single-particle EM (blue shading). (B, D) The crystalstructure of ANP–ECD [9] and the hypothetical tt dimer model forthe complex, respectively, are superimposed on the EM densitymap 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 FEBSshown previously that ANP binding causes no appre-ciable intramolecular structural change in the ECDmonomers (rmsd of Ca atoms between the apo andthe complex structures, 0.64 A˚) [9]. Furthermore, noTrp residues make contact with ANP in the boundcomplex. Therefore, if the ECD assumes the hh dimerstructures, only the Trp74 residue should undergo asignificant change in its environment. On the otherhand, if the ECD assumes the tt dimer structures, nochange is expected in the Trp environment in responseto ANP binding. On the basis of the above structureanalyses, we utilized Trp fluorescence to examine thesolution structures of apoECD and ANP–ECD.The fluorescence emission spectra of apoECD andANP–ECD are shown in Fig. 4C. Comparison of thespectra 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 thefluorescence intensity was time-dependent and was lar-gely complete in about 30 min (not shown). The courseof this intensity drop matches closely the course ofANP binding measured using [125I]ANP [20]. Thesefindings are consistent with the hh dimer structures forboth apoECD and ANP–ECD, where the two partiallyburied Trp74 residues at the apoECD hh dimer inter-face become exposed upon ANP binding [9,12] andquenched by water. The difference spectrum obtainedby 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 differencespectrum is consistent with the two Trp74 residues thatare localized at the edge of the apo dimer interface ina partially exposed, polar environment [21]. Thedecrease in Trp emission intensity from the total emis-sion intensity from 10 Trp residues in each ECDmonomer was relatively small (7%). The quantumyield of Trp residues is known to vary widely, depend-ing on the environment. The relatively small decreasemay be due to quenching of the two Trp74 residues atthe apoECD hh dimer by a staggered face-to-faceinteraction between the two indole rings (Fig. 4A).To confirm that the decrease in the fluorescenceintensity is due to the change in Trp74 environment,we measured the fluorescence emission of an ECDFig. 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 arelabeled in red. The bound ANP (B) does not contact any of the Trp residues. (C) Fluorescence emission spectra of apoECD (solid line) andANP–ECD (dotted line). The maximum emission intensity of apoECD was calculated as the average intensity over the wavelength rangefrom kmax= )5nmtokmax= +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 W74Rmutant [12] (solid line) and the ANP–ECD-W74R complex (dotted line). The maximum emission intensity of the apoECD W74R mutant wasconsidered to be 100%.H. Ogawa et al. Natriuretic peptide receptor signaling mechanismFEBS Journal 276 (2009) 1347–1355 ª 2009 The Authors Journal compilation ª 2009 FEBS 1351mutant, W74R. We have shown previously that theW74R mutant binds ANP with an affinity similar tothat of the wild-type [12]. The fluorescence emissionspectrum of the W74R mutant was similar to that ofthe wild-type, with a peak at around 350 nm, but witha slightly reduced intensity because of the Trp to Argmutation. As shown in Fig. 4D, addition of ANP tothe W74R mutant caused no appreciable change in theemission intensity. This finding confirms that thedecrease 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 inANP–ECD.Comparison of the apoECD and ANP–ECD EMreconstructionsTo evaluate the structural change induced by ANPbinding, the 3D reconstructions of apoECD andANP–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, theANP–ECD structure is more detailed, with a shapecharacteristic of the crystal structure. Nonetheless,both EM reconstructions exhibit dimeric shape andmonomer orientations that closely agree with thoseobserved by X-ray crystallography. In the front view,there is no appreciable change in the distance betweenthe two monomers (Fig. 5). Viewed from the side,each monomer in the ANP–ECD reconstruction isdisplaced in a clockwise direction, reminiscent of thetwist motion observed by X-ray crystallography [9].Viewed from the bottom (i.e. in the direction fromthe presumed transmembrane regions; Fig. 5, bottomview), the two juxtamembrane domains are displacedin opposite directions upon binding of ANP, withoutan appreciable change in the distance between thetwo.Proposed mechanism for transmembrane signaltransductionOn the basis of the hh dimer pairs demonstratedabove, the X-ray structures of ECD with [9] and with-out [10] bound ANP show that ANP binding causes alarge change in the quaternary structure of the ECDdimer without appreciable intramolecular structuralchange. ANP binding causes each of the two ECDmonomers to undergo a twisting motion while retain-ing the two-fold symmetry in the dimeric complex [9].This twisting motion causes the two juxtamembranedomains in the dimer to undergo parallel translocationin the opposite direction, with essentially no change inthe distance between the two (Fig. 6A). This move-ment causes an alteration in the relative angular orien-tation of the two juxtamembrane domains that isequivalent to rotating each domain by 24° (Fig. 6B).We have proposed that this hormone-induced rotationmechanism occurring in the juxtamembrane regionmay trigger ANP receptor signaling [9,11]. The ANP-induced structural change observed here by single-par-ticle EM closely resembles that recognized by X-raycrystallography, thus supporting the proposed signal-ing mechanism.In summary, the 3D reconstructions by single-parti-cle EM, which were obtained in the absence of crystalFig. 5. Overlay of the single-particle reconstructions in the absence (blue mesh) and presence (gold surface) of ANP. The reconstructionsare rendered at 70% of the correct molecular volume for clarity. ANP binding causes each of the two ECD monomers to undergo a twistwhile maintaining the two-fold symmetry axis in the dimerized complex. The orientation of each EM construction is based on the closenessof the fit to the respective X-ray structure as shown in Fig. 3. The front and side views are oriented such that the juxtamembrane domainsare the lower lobes of the reconstructions. The bottom view is oriented such that the reconstructions are shown from the perspective ofthe 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 FEBScontacts, yielded the hh dimer structures for bothapoECD and ANP–ECD. Comparison of the 3Dreconstructions with and without ANP showed theANP-induced structural change in the dimer that wassurprisingly close to that observed by X-ray crystal-lography. The quenching of Trp74 fluorescence emis-sion concomitant with ANP binding is also inagreement with apoECD and ANP–ECD in hh dimerstructures. Thus, the results of our complementaryapproaches, single-particle EM, fluorescence spectros-copy and X-ray crystallography, together demonstratea novel hormone-induced structural change in theECD dimer that generates a rotation mechanism inthe juxtamembrane regions and possibly mediatestransmembrane signal transduction.Experimental proceduresPreparation of ECD and ANP–ECDECD consisting of residues 1–435 of the rat ANP receptorwas expressed by slight modification of the methoddescribed 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. Thecloned cells were cultured in roller bottles, and the condi-tioned medium containing the expressed ECD was collectedevery 2 days. The ECD was purified by ANP affinity chro-matography as previously described [22]. ANP–ECD wasprepared by incubating ECD (1 mgÆmL)1) with a 1.1-foldmolar 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 mmHepes buffer (pH 7.0) containing 20 mm NaCl at roomtemperature for 60 min.Single-particle EMAliquots (3 lL) of ECD at 0.03 mgÆmL)1in the absence(apoECD) and presence (ANP–ECD) of ANP wereapplied to glow-discharged, carbon-coated grids. The gridwas washed with two drops of 2% uranyl acetate, andthen a third drop of 2% uranyl acetate was allowed tosit on the grid for 1 min (4 °C). The excess stain wasremoved by blotting with filter paper, and the samplewas allowed to air dry. Data were collected on a Tec-nai F20 (FEI Company) located in the Microscopy andImaging Facility at the University of Calgary (Calgary,Canada). The microscope was operated at 200 keV, andimages were recorded on Kodak SO-163 film under low-dose conditions at a magnification of ·50 000, with adefocus ranging from )1.5 to )2.5 lm. Micrographs weredigitized with a Nikon Super Coolscan 9000 with a scan-ning resolution of 6.35 lmÆpixel)1, and this was followedby pixel averaging to achieve a final resolution of3.81 A˚Æpixel)1.Image processing and reconstruction were performedwith the eman program package [23]. Seventeen micro-graphs with minimal drift and astigmatism were selectedfor reconstruction of apoECD. Similarly, 20 micrographswere used for ANP–ECD. Particles were selected semiauto-matically and extracted as 40 · 40 pixel images (boxer). Intotal, 22 778 and 19 600 particle images were selected forapoECD and ANP–ECD, respectively. No correction forthe CTF was applied. Reference-free classification was per-formed to generate 35 class averages (refine2d.py), and aninitial set of Euler angles was then assigned to these classaverages (startAny). The initial three-dimensional modelsbuilt using common lines in Fourier space were then refinedin eman for up to 20 cycles of refinement (refine). Theassignment of Eulerian angles from class averages byFig. 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 thejuxtamembrane domains in apoECD (blue) and ANP–ECD (orange)are shown as viewed from the membrane [9]. ANP binding causesa parallel translocation of the two juxtamembrane domains in theopposite direction without an appreciable change in the interdomaindistance. (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 translationof the juxtamembrane domains from the apo position (depicted byblue circles) to the complex positions (orange circles). The arrowsdepict this parallel translocation. This movement causes a changein the relative orientation between the two juxtamembranedomains 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 transmembranesignaling [9].H. Ogawa et al. Natriuretic peptide receptor signaling mechanismFEBS Journal 276 (2009) 1347–1355 ª 2009 The Authors Journal compilation ª 2009 FEBS 1353common lines results in two possible enantiomeric solu-tions. The X-ray crystallographic structures were used todetermine the handedness of the reconstructions. Becausethe 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. Thefirst 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 calculatingthe Fourier shell correlation between two independent halfdatasets (eotest command in eman; 0.5 FSC criterion).Therefore, the final density maps were low-pass-filtered to22 A˚resolution. The final 3D maps were visualized andanalyzed, and figures were created using the UCSF chi-mera package [24]. A protein partial specific volume of0.73 cm3Æg)1was used to set the isosurface threshold thatcorresponded to the correct molecular volume.Because of the availability of apoECD and ANP–ECDcrystal structures, we also performed reference-basedrefinement (eman) as a means of evaluating agreement ofthe single-particle data with the X-ray crystallographicdata. The crystal structures of apoECD (Protein DataBank code: 1DP4) and ANP–ECD (Protein Data Bankcode: 1T34) each contain tt dimer and hh dimer pairs.Density maps were created from the hh and tt dimer pairsat 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 forthe refine command in eman. Up to 20 cycles of refine-ment were performed. Depending on whether the hh or ttdimer map was used as the starting model, the refinementquickly 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 hhor tt dimer pairs to the EM reconstructions was performedwith eman (foldhunterp). Calculated density maps fromeach atomic model were used as reference structures forthe calculation.Steady-state fluorescence spectroscopic studiesof Trp residuesFluorescence emission spectra were acquired in a Fluoro-log-222 fluorescence spectrometer using fluorescence soft-ware over the wavelength range from 305 to 500 nm withexcitation 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 wasreplaced by Arg, at 1 mgÆmL)1concentration in 5 mmHepes buffer (pH 7.0), containing 20 mm NaCl was used inthe experiments. Fluorescence emission spectra of ECD orECD W74R were acquired before and after the addition ofa 1.1-fold molar excess of the truncated ANP peptide. Thechange in the emission spectrum was followed at 2 minintervals over a period of 60 min.AcknowledgementsThe work was supported by HL54329 to K. S. Misonoand by grants to H. S. Young from the CanadianInstitutes for Health Research, the Canada Founda-tion for Innovation, and the Alberta Science andResearch Investments Program. H. S. Young is aSenior Scholar of the Alberta Heritage Foundation forMedical Research.References1 de Bold AJ, Borenstein HB, Veress AT & SonnenbergH (1981) A rapid and potent natriuretic response tointravenous injection of atrial myocardial extract inrats. Life Sci 28, 89–94.2 Currie MG, Geller DM, Cole BR, Boylan JG, YuShengW, 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 andvasorelaxant activity. Biochem Biophys Res Commun116, 696–703.4 Itoh H, Pratt RE & Dzau VJ (1990) Atrial natriureticpolypeptide inhibits hypertrophy of vascular smoothmuscle cells. 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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 receptorbefore 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 factorreceptor 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 & ZhangX (1999) Expression and purification of the extracellularligand-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-particlereconstructions. J Struct Biol 128, 82–97.24 Pettersen EF, Goddard TG, Huang CC, Couch GS,Greenblatt DM, Meng EC & Ferrin TE (2004) UCSFChimera – a visualization system for exploratoryresearch 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 thesemodels. Acta Crystallogr A47, 110–119.Supporting informationThe 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 tothe hh dimer structures for both apoECD andANP-ECD.This supplementary material can be found in theonline version of this article.Please note: Wiley-Blackwell are not responsible forthe content or functionality of any supplementarymaterials supplied by the authors. Any queries (otherthan missing material) should be directed to the corre-sponding author for the article.H. Ogawa et al. Natriuretic peptide receptor signaling mechanismFEBS 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-bindingregulatory domain and
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Xem thêm: 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, 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, 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