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• W2131007783 abstract "Many enteroviruses bind to the complement control protein decay-accelerating factor (DAF) to facilitate cell entry. We present here a structure for echovirus (EV) type 12 bound to DAF using cryo-negative stain transmission electron microscopy and three-dimensional image reconstruction to 16-Å resolution, which we interpreted using the atomic structures of EV11 and DAF. DAF binds to a hypervariable region of the capsid close to the 2-fold symmetry axes in an interaction that involves mostly the short consensus repeat 3 domain of DAF and the capsid protein VP2. A bulge in the density for the short consensus repeat 3 domain suggests that a loop at residues 174-180 rearranges to prevent steric collision between closely packed molecules at the 2-fold symmetry axes. Detailed analysis of receptor interactions between a variety of echoviruses and DAF using surface plasmon resonance and comparison of this structure (and our previous work; Bhella, D., Goodfellow, I. G., Roversi, P., Pettigrew, D., Chaudhry, Y., Evans, D. J., and Lea, S. M. (2004) J. Biol. Chem. 279, 8325-8332) with reconstructions published for EV7 bound to DAF support major differences in receptor recognition among these viruses. However, comparison of the electron density for the two virus·receptor complexes (rather than comparisons of the pseudo-atomic models derived from fitting the coordinates into these densities) suggests that the dramatic differences in interaction affinities/specificities may arise from relatively subtle structural differences rather than from large-scale repositioning of the receptor with respect to the virus surface. Many enteroviruses bind to the complement control protein decay-accelerating factor (DAF) to facilitate cell entry. We present here a structure for echovirus (EV) type 12 bound to DAF using cryo-negative stain transmission electron microscopy and three-dimensional image reconstruction to 16-Å resolution, which we interpreted using the atomic structures of EV11 and DAF. DAF binds to a hypervariable region of the capsid close to the 2-fold symmetry axes in an interaction that involves mostly the short consensus repeat 3 domain of DAF and the capsid protein VP2. A bulge in the density for the short consensus repeat 3 domain suggests that a loop at residues 174-180 rearranges to prevent steric collision between closely packed molecules at the 2-fold symmetry axes. Detailed analysis of receptor interactions between a variety of echoviruses and DAF using surface plasmon resonance and comparison of this structure (and our previous work; Bhella, D., Goodfellow, I. G., Roversi, P., Pettigrew, D., Chaudhry, Y., Evans, D. J., and Lea, S. M. (2004) J. Biol. Chem. 279, 8325-8332) with reconstructions published for EV7 bound to DAF support major differences in receptor recognition among these viruses. However, comparison of the electron density for the two virus·receptor complexes (rather than comparisons of the pseudo-atomic models derived from fitting the coordinates into these densities) suggests that the dramatic differences in interaction affinities/specificities may arise from relatively subtle structural differences rather than from large-scale repositioning of the receptor with respect to the virus surface. Echoviruses are small (∼300 Å in diameter) non-enveloped icosahedral viruses classified in the Enterovirus genus of the Picornaviridae family. Echovirus infection is usually mild, although these viruses can cause severe diseases such as aseptic meningitis, encephalitis, and myocarditis. The ∼7.5-kb positive sense RNA genome encodes a single polyprotein that is co- and post-translationally cleaved, yielding the structural proteins, enzymes, and additional proteins necessary for virus replication. Picornavirus capsids assemble from four structural proteins (VP1-4) as a pseudo T = 3 icosahedral shell with VP1-3 occupying the three quasi-equivalent positions in the icosahedral lattice and VP4 lying beneath the pentameric apex (1Hogle J.M. Chow M. Filman D.J. Science. 1985; 229: 1358-1365Crossref PubMed Scopus (878) Google Scholar). Enteroviruses have a distinctive morphology (common to many picornaviruses) consisting of a star-shaped mesa at the 5-fold symmetry axes surrounded by a deep cleft or “canyon” (2Stuart A.D. McKee T.A. Williams P.A. Harley C. Shen S. Stuart D.I. Brown T.D.K. Lea S.M. J. Virol. 2002; 76: 7694-7704Crossref PubMed Scopus (45) Google Scholar, 3Rossmann M.G. Arnold E. Erickson J.W. Frankenberger E.A. Griffith J.P. Hecht H.J. Johnson J.E. Kamer G. Luo M. Mosser A.G. Rueckert R.R. Sherry B. Vriend G. Nature. 1985; 317: 145-153Crossref PubMed Scopus (1007) Google Scholar).Substantial variations in receptor usage and cell entry mechanisms are found within the Picornaviridae family. The major receptor group rhinoviruses along with coxsackievirus type A21 use ICAM-1 (intracellular adhesion molecule 1), whereas the polioviruses bind to a protein of unknown function called the poliovirus receptor, and coxsackievirus type B3 binds to the coxsackie-adenovirus receptor. These receptors, which belong to the immunoglobulin-like family of proteins, bind to the capsid surface in the canyon (4He Y.N. Chipman P.R. Howitt J. Bator C.M. Whitt M.A. Baker T.S. Kuhn R.J. Anderson C.W. Freimuth P. Rossmann M.G. Nat. Struct. Biol. 2001; 8: 874-878Crossref PubMed Scopus (159) Google Scholar, 5He Y.N. Mueller S. Chipman P.R. Bator C.M. Peng X.Z. Bowman V.D. Mukhopadhyay S. Wimmer E. Kuhn R.J. Rossmann M.G. J. Virol. 2003; 77: 4827-4835Crossref PubMed Scopus (62) Google Scholar, 6Kolatkar P.R. Bella J. Olson N.H. Bator C.M. Baker T.S. Rossmann M.G. EMBO J. 1999; 18: 6249-6259Crossref PubMed Scopus (164) Google Scholar, 7Belnap D.M. McDermott B.M. Filman D.J. Cheng N.Q. Trus B.L. Zuccola H.J. Racaniello V.R. Hogle J.M. Steven A.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 73-78Crossref PubMed Scopus (192) Google Scholar, 8Xiao C. Bator C.M. Bowman V.D. Rieder E. He Y.N. Hebert B. Bella J. Baker T.S. Wimmer E. Kuhn R.J. Rossmann M.G. J. Virol. 2001; 75: 2444-2451Crossref PubMed Scopus (69) Google Scholar). Receptor binding in solution induces conformational changes in the capsid akin to those that occur at the cell surface, resulting in loss of VP4, while the normally buried hydrophobic N terminus of VP1 is externalized (9Hoover-Litty H. Greve J.M. J. Virol. 1993; 67: 390-397Crossref PubMed Google Scholar, 10Fenwick M.L. Cooper P.D. Virology. 1962; 18: 212-223Crossref PubMed Scopus (67) Google Scholar, 11Fricks C.E. Hogle J.M. J. Virol. 1990; 64: 1934-1945Crossref PubMed Google Scholar, 12Guttman N. Baltimore D. Virology. 1977; 82: 25-36Crossref PubMed Scopus (53) Google Scholar). It is hypothesized that this forms a pore in the cell membrane through which the genome passes to enter the cytoplasm. Recent studies of poliovirus virions bound to poliovirus receptor molecules embedded in lipid vesicles indicate that this pore may form at the 5-fold symmetry axis, although it is not clear whether a specific vertex is required to facilitate genome egress (13Bubeck D. Filman D.J. Hogle J.M. Nat. Struct. Mol. Biol. 2005; 12: 615-618Crossref PubMed Scopus (71) Google Scholar).Many enteroviruses bind to decay-accelerating factor (DAF 5The abbreviations used are: DAF, decay-accelerating factor; SCR, short consensus repeat; EV, echovirus; SPR, surface plasmon resonance; EM, electron microscopy. 5The abbreviations used are: DAF, decay-accelerating factor; SCR, short consensus repeat; EV, echovirus; SPR, surface plasmon resonance; EM, electron microscopy.; CD55), a member of the regulator of complement activation protein family (14Bergelson J.M. Chan M. Solomon K.R. Stjohn N.F. Lin H.M. Finberg R.W. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6245-6248Crossref PubMed Scopus (269) Google Scholar, 15Karnauchow T.M. Tolson D.L. Harrison B.A. Altman E. Lublin D.M. Dimock K. J. Virol. 1996; 70: 5143-5152Crossref PubMed Google Scholar, 16Powell R.M. Ward T. Goodfellow I. Almond J.W. Evans D.J. J. Gen. Virol. 1999; 80: 3145-3152Crossref PubMed Scopus (40) Google Scholar, 17Shafren D.R. Dorahy D.J. Ingham R.A. Burns G.F. Barry R.D. J. Virol. 1997; 71: 4736-4743Crossref PubMed Google Scholar, 18Ward T. Pipkin P.A. Clarkson N.A. Stone D.M. Minor P.D. Almond J.W. EMBO J. 1994; 13: 5070-5074Crossref PubMed Scopus (134) Google Scholar). DAF is a 70-kDa protein comprising four short consensus repeat (SCR) domains linked by an O-glycosylated serine-, threonine-, and proline-rich linker to a glycosylphosphatidylinositol anchor at the cell membrane. DAF is present on the surface of the majority of serum-exposed cells, where it protects them from complement-mediated lysis by accelerating the decay of the classical and alternative pathway C3 and C5 convertases (19Lublin D.M. Atkinson J.P. Annu. Rev. Immunol. 1989; 7: 35-58Crossref PubMed Scopus (394) Google Scholar). Unlike the Ig-like receptors, DAF does not induce uncoating in solution, although conformational changes in the virion do occur at the cell surface. This suggests that another molecule is recruited to induce uncoating or, alternatively, that DAF must be presented in the context of a cell membrane (20Powell R.M. Ward T. Evans D.J. Almond J.W. J. Virol. 1997; 71: 9306-9312Crossref PubMed Google Scholar).We have previously calculated a quasi-atomic resolution model of the echovirus (EV) type 12·receptor complex based on cryo-negative stain transmission electron microscopy and image reconstruction of EV12 bound to a fragment of DAF comprising SCR3 and SCR4 (DAF34) (EM Data Bank code 1057 and Protein Data Bank code 1UPN) (21Bhella D. Goodfellow I.G. Roversi P. Pettigrew D. Chaudhry Y. Evans D.J. Lea S.M. J. Biol. Chem. 2004; 279: 8325-8332Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). This model shows that EV12-DAF binding occurs predominantly between SCR3 and VP2 (residues 142-164). The interaction takes place close to the 2-fold symmetry axes rather than in the canyon, and the receptor fragment lies flat against the capsid surface. Comparison of our structure and model with those published for the EV7·DAF complex indicated major differences between these two closely related viruses (Protein Data Bank code 1M11) (22He Y.N. Lin F. Chipman P.R. Bator C.M. Baker T.S. Shoham M. Kuhn R.J. Medof M.E. Rossmann M.G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10325-10329Crossref PubMed Scopus (59) Google Scholar). Previous genetic and biochemical studies of DAF-binding enteroviruses have shown that they can be divided into two major classes: those that bind to SCR1 and those that bind to SCR3 with additional binding to SCR4 and sometimes SCR2 (15Karnauchow T.M. Tolson D.L. Harrison B.A. Altman E. Lublin D.M. Dimock K. J. Virol. 1996; 70: 5143-5152Crossref PubMed Google Scholar, 16Powell R.M. Ward T. Goodfellow I. Almond J.W. Evans D.J. J. Gen. Virol. 1999; 80: 3145-3152Crossref PubMed Scopus (40) Google Scholar, 17Shafren D.R. Dorahy D.J. Ingham R.A. Burns G.F. Barry R.D. J. Virol. 1997; 71: 4736-4743Crossref PubMed Google Scholar). Although EV7 and EV12 both bind primarily to SCR3, there is considerable variation in receptor affinity in this class of viruses, and mutagenesis data suggest that individual virus serotypes may interact with different faces of the receptor (23Williams P. Chaudhry Y. Goodfellow I.G. Billington J. Powell R. Spiller O.B. Evans D.J. Lea S. J. Biol. Chem. 2003; 278: 10691-10696Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). To test our earlier structural hypotheses and to cast further light on the nature of receptor binding in the echoviruses, we have calculated a three-dimensional reconstruction of EV12 bound to a four-domain receptor fragment (DAF1234) and dissected virus-receptor interactions using a variety of receptor fragments and surface plasmon resonance (SPR).EXPERIMENTAL PROCEDURESProduction of Virus and Receptor—EV12 was cultivated in rhabdomyosarcoma cells and purified as described previously (21Bhella D. Goodfellow I.G. Roversi P. Pettigrew D. Chaudhry Y. Evans D.J. Lea S.M. J. Biol. Chem. 2004; 279: 8325-8332Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Soluble DAF comprising SCR1-4 for electron microscopy (EM) analysis was expressed and purified in Pichia pastoris as described previously (20Powell R.M. Ward T. Evans D.J. Almond J.W. J. Virol. 1997; 71: 9306-9312Crossref PubMed Google Scholar, 24Powell R.M. Schmitt V. Ward T. Goodfellow I. Evans D.J. Almond J.W. J. Gen. Virol. 1998; 79: 1707-1713Crossref PubMed Scopus (58) Google Scholar).For Biacore analysis, DAF3, DAF23, and DAF34 were subcloned from the DAF1234 expression vector into the pQE-30 plasmid (Qiagen Inc). All constructs were expressed as hexahistidine fusions in M15[pREP4] (Qiagen Inc.). These constructs, along with DAF1234, were purified from inclusion bodies and refolded using established SCR refolding protocols (25White J. Lukacik P. Esser D. Steward M. Giddings N. Bright J.R. Fritchley S.J. Morgan B.P. Lea S.M. Smith G.P. Smith R.A. Protein Sci. 2004; 13: 2406-2415Crossref PubMed Scopus (35) Google Scholar).Electron Microscopy—Preparations of EV12 were labeled with DAF by incubation overnight at 4 °C and prepared for microscopy by cryo-negative staining. 5 μl of virus at an concentration of ∼0.2 mg/ml was loaded onto a freshly glow-discharged QUANTIFOIL holey carbon support film (Quantifoil Micro Tools GmbH, Jena, Germany) for ∼10 s. The grid was then transferred to a droplet of 20% (w/v) ammonium molybdate solution (pH 7.4) for ∼10 s, blotted for 2-3 s, and plunged into a bath of liquid nitrogen-cooled ethane slush. Grids were stored under liquid nitrogen or imaged directly in the transmission electron microscope.Prepared specimens were imaged in a Jeol 1200 EX II transmission electron microscope equipped with an Oxford Instruments CT3500 cryo-stage (Gatan, Inc., Oxford, UK) at an accelerating voltage of 120 kV. To facilitate correction of the microscope’s contrast transfer function, each field of view was imaged at two levels of defocus. Typically, the first micrograph was recorded between 500 and 1500 nm under focus and the second between 1500 and 2500 nm under focus. Defocus paired images were recorded under low electron dose conditions at a nominal magnification of ×30,000 on Kodak SO163 film.Image Processing—Micrographs were digitized on a Nikon Super Coolscan 9000 ED CCD scanner at 4000-dpi resolution, corresponding to a raster step size of 2.18 Å in the specimen. Micrographs were converted to PIF format with the BSOFT image processing package (26Heymann J.B. J. Struct. Biol. 2001; 133: 156-169Crossref PubMed Scopus (191) Google Scholar). Particles were selected and cut out in X3D, and deconvolution of the contrast transfer function was performed using CTFMIX; at this point, defocus paired images of individual particles were merged (27Conway J.F. Steven A.C. J. Struct. Biol. 1999; 128: 106-118Crossref PubMed Scopus (146) Google Scholar). The orientations and origins of particle images were determined by a modified version of the polar Fourier transform method (PFT2) (28Bubeck D. Filman D.J. Cheng N. Steven A.C. Hogle J.M. Belnap D.M. J. Virol. 2005; 79: 7745-7755Crossref PubMed Scopus (125) Google Scholar, 29Baker T.S. Cheng R.H. J. Struct. Biol. 1996; 116: 120-130Crossref PubMed Scopus (323) Google Scholar) using an unlabeled low resolution reconstruction of EV12 (21Bhella D. Goodfellow I.G. Roversi P. Pettigrew D. Chaudhry Y. Evans D.J. Lea S.M. J. Biol. Chem. 2004; 279: 8325-8332Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) as the starting model. Successive iterations of polar Fourier transform refinement and three-dimensional reconstruction (EM3DR2) led to the calculation of final reconstructions for EV12·DAF1234 and EV12 at 14-Å resolution. Resolution assessment was accomplished by randomly dividing the data set into equal subsets, which were used to generate two independent reconstructions. A number of measures of agreement between these reconstructions were calculated, including the Fourier shell correlation and the spectral signal-to-noise ratio. Reconstructions were visualized in UCSF Chimera using the EMAN radial depth cueing plug-in Isosurface Colorizer (30Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. J. Comput. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (26737) Google Scholar, 31Ludtke S.J. Baldwin P.R. Chiu W. J. Struct. Biol. 1999; 128: 82-97Crossref PubMed Scopus (2099) Google Scholar).Docking of Crystallographic Coordinates—The crystallographic coordinates for EV11 (2Stuart A.D. McKee T.A. Williams P.A. Harley C. Shen S. Stuart D.I. Brown T.D.K. Lea S.M. J. Virol. 2002; 76: 7694-7704Crossref PubMed Scopus (45) Google Scholar) and DAF1234 (32Lukacik P. Roversi P. White J. Esser D. Smith G.P. Billington J. Williams P.A. Rudd P.M. Wormald M.R. Harvey D.J. Crispin M.D. Radcliffe C.M. Dwek R.A. Evans D.J. Morgan B.P. Smith R.A. Lea S.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1279-1284Crossref PubMed Scopus (94) Google Scholar) were fitted to the EV12·DAF1234 reconstruction to construct a quasi-atomic resolution model of the virus·receptor complex. The reconstructed density for EV12·DAF1234 was converted to CCP4 format and placed at the origin of a unit cell in space group P23 with a cubic cell edge of 555.9 Å such that the icosahedral 2- and 3-fold symmetry axes coincided with the crystallographic symmetry axes. A pentameric asymmetric unit of the EV11 crystal structure was placed within the unit cell in the same orientation, confirming the hand assignment and scale of the EM density. The main chain atom correlation coefficient for the virus capsid was calculated as 55% between experimental and model densities.A hybrid DAF structure was calculated to create an optimal fit for each SCR domain in the experimentally derived receptor density as follows. SCR3 and SCR4 were constrained to the previously determined positions. As no further points of contact were found between the virus and receptor, we assumed that the presence of SCR1 and SCR2 would not alter the fundamental interaction. Furthermore, merged densities from SCR2 and SCR3 in symmetry-related molecules across the 2-fold axes precluded further refinement of the previous fit. For SCR3 and SCR4, a real space correlation coefficient of 55.4% was calculated using a 14-Å tagged density map with B-factors set to 100.To achieve an optimal fit for SCR2, 14 different models of DAF1234 (based on the seven different crystal structures deposited in the Protein Data Bank) were fitted to the existing model as well as 43 models derived from NMR data (32Lukacik P. Roversi P. White J. Esser D. Smith G.P. Billington J. Williams P.A. Rudd P.M. Wormald M.R. Harvey D.J. Crispin M.D. Radcliffe C.M. Dwek R.A. Evans D.J. Morgan B.P. Smith R.A. Lea S.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1279-1284Crossref PubMed Scopus (94) Google Scholar, 33Uhrinova S. Lin F. Ball G. Bromek K. Uhrin D. Medof M.E. Barlow P.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4718-4723Crossref PubMed Scopus (40) Google Scholar). lsqkab was used to superimpose SCR3 of each model on the capsid-docked SCR3 (34Kabsch W. Acta Crystallogr. Sect. A. 1976; 32: 922-923Crossref Scopus (2308) Google Scholar, 35Bailey S. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (41) Google Scholar). The real space main chain correlation coefficient for SCR2 was used as a scoring function to determine the best interdomain orientation. Fitting of SCR1 was accomplished in the same manner once a satisfactory fit for SCR2 was attained using crystallographic data only, as no NMR data exist for the SCR1-SCR2 linker region. Molecular models were visualized using PyMOL (DeLano Scientific, San Carlos, CA), MolScript (36Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar), and Raster3D (37Merritt E.A. Bacon D.J. Methods Enzymol. 1997; 277: 505-524Crossref PubMed Scopus (3869) Google Scholar).SPR Studies—All SPR measurements were performed on a Biacore 2000 instrument (Biacore AB, Uppsala, Sweden) using HBS-EP (10 mm HEPES, pH 7.4, containing 150 mm NaCl, 3 mm EDTA and 0.005% (v/v) surfactant P20) as the running buffer. EV12, EV7, and EV6 were covalently coupled to the surface of a CM-5 sensor chip (Biacore AB) by primary amine coupling. After the activation step, 5-10 μl of virus (diluted to 50 μg/ml in 10 mm sodium formate (pH 3.0)) was injected repeatedly over a single flow channel until the signal was 3000-6000 response units above the (uncoupled) base line. It has been previously shown that low pH does not significantly alter the infectivity of the viruses studied (38Lea S.M. Powell R.M. McKee T. Evans D.J. Brown D. Stuart D.I. van der Merwe P.A. J. Biol. Chem. 1998; 273: 30443-30447Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). To prevent further coupling reactions, unreacted succinimide esters on the chip surface were displaced by saturating the surface with 40 μl of 1 m ethanolamine (pH 8.5). The immobilization procedure was repeated for two different viruses on each of the other two channels. To provide a mock-coupled (no virus) control channel, the fourth channel was inactivated immediately after the activation step.Experimental runs were based on previous protocols for SPR studies of EV11-207 (38Lea S.M. Powell R.M. McKee T. Evans D.J. Brown D. Stuart D.I. van der Merwe P.A. J. Biol. Chem. 1998; 273: 30443-30447Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). After coupling the virus to the chip, the system was primed several times to ensure a stable base-line response before the experiment began. To avoid mass transfer effects, all experiments were performed at a high flow rate (25-30 μl/min) using HBS-EP as the running buffer.Binding of recombinant DAF1234 was measured by sequentially injecting 30 μl of a 3/2-fold dilution series in HBS-EP from 8.25 μm (high to low concentration) and also from 30 μm. The injection was performed using the KINJECT command, with a dissociation time of 1000-1400 s, followed by two 20-μl 4 m NaCl wash steps. The experiment was then repeated using the same dilution series except from low to high concentration. This was to confirm that the equilibrium signal for a given ligand concentration did not change with time.Binding of recombinant DAF3, DAF34, and DAF23 was measured by sequentially injecting a 2/3-fold dilution series from 25 μm and from 100 μm (high to low concentration and then low to high concentration). Otherwise, the protocol was identical to the one used for DAF1234.To analyze the equilibrium constant for the dissociation (KD) of the ligand virus, the corresponding FC4 mock response signal was aligned and subtracted from each measurement using BIAevaluation (Biacore AB). The average equilibrium response (Req) was determined for each ligand concentration (C) and plotted on a graph of Req (response units) versus C (molar). A nonlinear fit of the 1:1 Langmuir binding model to the data yielded an estimate for the equilibrium dissociation constant (KD).RESULTSThree-dimensional Reconstruction of EV12 Bound to DAF1234—42 defocus paired micrographs of unlabeled EV12 and 96 paired micrographs of DAF1234-labeled EV12 were selected for image processing on the basis of ice thickness, virion numbers, defocus, and astigmatism. 1796 unlabeled and 1742 labeled particle image pairs were selected and corrected for the effects of the microscope’s contrast transfer function. Several rounds of iterative orientation refinement and three-dimensional reconstruction were performed until stable orientations and origins were achieved for each data set. In total, 1501 images of unlabeled and 1339 images of labeled virions were used to calculate final reconstructions (Fig. 1). The resolution assessment for these reconstructions gave values of 14 and 16 Å, respectively (Electron Microscopy Data Bank accession numbers 1182 and 1183).We have previously calculated a structure for EV12 bound to a two-domain fragment of DAF (DAF34) (21Bhella D. Goodfellow I.G. Roversi P. Pettigrew D. Chaudhry Y. Evans D.J. Lea S.M. J. Biol. Chem. 2004; 279: 8325-8332Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). This structure has clear regions of contiguous density, and two domains could be readily defined (Fig. 1B). The rod-shaped fragment was found to bind to the capsid surface close to the 2-fold symmetry axes, oriented such that it lay approximately equidistant between the 3- and 5-fold axes, pointing to two neighboring 2-fold symmetry axes. When viewed along the 2-fold axes, two DAF34 molecules appear as hands on a clock in a “ten past eight” orientation. The crystallographic structure was readily docked into the reconstruction, unambiguously identifying the domain lying closest to the 2-fold axes and in contact with the capsid surface as SCR3. Our structure for EV12·DAF1234 (Fig. 1C) is consistent with our previous reconstruction and model, containing density that overlaps with that previously found in the EV12·DAF34 structure. Further density is present lying across the 2-fold symmetry axes, extending out of SCR3, in a “six o’clock” orientation. This density bends sharply away from the capsid surface, close to the 3-fold axis. As this globular region (which appears to consist of two distinct lobes) is farthest from SCR3 and SCR4 and is consistent with our previous quasi-atomic resolution model for EV12·DAF1234, we attribute it to SCR1. We consider that the larger region of density lying across the 2-fold axis most likely comprises SCR2 and the SCR2-proximal region of SCR3 from two symmetry-related molecules. SCR1 and SCR2 do not make additional contacts with the capsid surface; therefore, our previous description of contact residues involved in this interaction remains valid and unchanged.Construction of a Quasi-atomic Model for EV12·DAF1234, Fitting SCR2—Starting from our previous model for EV12·DAF34, a hybrid EV12·DAF1234 structure was constructed by successively fitting SCR2 and SCR1 into the reconstructed density for the virus·receptor complex (Fig. 2) (Protein Data Bank code 2C8I). SCR3 and SCR4 were constrained to their positions in our previous model, as we consider this fit to be robust, and there is no evidence at this resolution for a change in the orientation of the molecule. SCR3 from each of 14 different crystallographic (Fig. 2A) and 43 different NMR (Fig. 2B) models was overlaid on the capsid-docked SCR3, and the SCR2 fit was scored according to correlation with the EM density. Of the crystallographic models, chain B from Protein Data Bank deposition 1OK3 was found to have the SCR2-SCR3 interdomain angle giving the highest correlation with the EM density (55%) (Fig. 2C) (32Lukacik P. Roversi P. White J. Esser D. Smith G.P. Billington J. Williams P.A. Rudd P.M. Wormald M.R. Harvey D.J. Crispin M.D. Radcliffe C.M. Dwek R.A. Evans D.J. Morgan B.P. Smith R.A. Lea S.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 1279-1284Crossref PubMed Scopus (94) Google Scholar). The 43 NMR-derived models for SCR2 and SCR3 (33Uhrinova S. Lin F. Ball G. Bromek K. Uhrin D. Medof M.E. Barlow P.N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4718-4723Crossref PubMed Scopus (40) Google Scholar) exhibit a large spread of interdomain angles that fan out parallel to the capsid surface. Only three chains showed any significant correlation with the EM density, and none of these models were compatible with the overall packing seen for all four domains in our reconstruction.FIGURE 2Calculation of a quasi-atomic model for EV12·DAF. A, variation in SCR2 orientation for the 14 crystal forms of DAF1234, with each model superimposed onto the capsid-docked SCR3. Only SCR2 is shown for each model. B, variation in SCR2 orientation for the 43 different NMR models. C, the optimal SCR2 position is from chain B of the x-ray structure of Protein Data Bank code 1OK3. D, points of contact on DAF1234 with the symmetry partner across the 2-fold axis. The green surface represents a steric clash between Arg102 and Arg103 and identical residues of the symmetry partner. This is resolved by side chain rearrangement. The blue surface is a van der Waals contact between Pro137 and Pro109 of the symmetry partner. The red and orange surfaces are an overlap between the main chain atoms of residues 174-180 of SCR3 (red) and a surface composed of residues 95-98 and 75-77 of the symmetry partner SCR2 (orange). This clash can be resolved only by a remodeling of loop 174-180. E, electron density of SCR1. The strong density at the center of each lobe is shown as a red mesh, whereas the lower contours are shown as a blue mesh. The major and minor lobes, as well as the position of SCR2, are highlighted. F, superposition of all 14 possible SCR1 orientations from the crystal structures. These orientations are consistent only with the minor lobe density. G, optimal “minor lobe” SCR1 model from the side. Also highlighted is the remodeled loop 174-180 on SCR3. H, complete DAF model based on a hybrid of the original DAF34 fit (with the remodeled loop 174-180 on SCR3) and the two crystal structures that gave optimal SCR1 and SCR2 positions (green). Also shown in magenta is the alternative position for SCR1 proposed to explain the major lobe density. The symmetry partner DAF molecule is shown in red. I, radially depth-cued atomic model of the virus capsid (blue) decorated with 60 copies of the DAF1234 hybrid model (green).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Resolving Steric Collisions across the 2-fold Symmetry Axes—Our previous model for EV12·DAF1234 predicted a minor steric collision between the SCR2-proximal re" @default.
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