Quaternary structure of coronavirus spikes in complex with carcinoembryonic antigen-related cell adhesion molecule cellular receptors - PubMed (original) (raw)
Quaternary structure of coronavirus spikes in complex with carcinoembryonic antigen-related cell adhesion molecule cellular receptors
Daniel N Lewicki et al. J Biol Chem. 2002.
Abstract
Oligomeric spike (S) glycoproteins extend from coronavirus membranes. These integral membrane proteins assemble within the endoplasmic reticulum of infected cells and are subsequently endoproteolyzed in the Golgi, generating noncovalently associated S1 and S2 fragments. Once on the surface of infected cells and virions, peripheral S1 fragments bind carcinoembryonic antigen-related cell adhesion molecule (CEACAM) receptors, and this triggers membrane fusion reactions mediated by integral membrane S2 fragments. We focused on the quaternary structure of S and its interaction with CEACAMs. We discovered that soluble S1 fragments were dimers and that CEACAM binding was entirely dependent on this quaternary structure. However, two differentially tagged CEACAMs could not co-precipitate with the S dimers, suggesting that binding sites were closely juxtaposed in the dimer (steric hindrance) or that a single CEACAM generated global conformational changes that precluded additional interactions (negative cooperativity). CEACAM binding did indeed alter S1 conformations, generating alternative disulfide linkages that were revealed on SDS gels. CEACAM binding also induced separation of S1 and S2. Differentially tagged S2 fragments that were free of S1 dimers were not co-precipitated, suggesting that S1 harbored the primary oligomerization determinants. We discuss the distinctions between the S.CEACAM interaction and other virus-receptor complexes involved in receptor-triggered entry.
Figures
Figure 1
Biochemical analysis of S1 quaternary structure.A, recombinant 35S-labeled S1 (strain JHM) was sedimented on a linear 5–20% sucrose gradient, and the 35S-labeled S1 in gradient fractions was visualized after immunoprecipitation onto Sepharose G:N-CEACAMFcbeads, SDS-PAGE, and fluorography. The positions of standards horseradish peroxidase (HRPO 4S), human IgG1 (IgG 7S), and β-galactosidase (β-Gal 16 S) are indicated above the electropherogram. B, recombinant 35S-labeled S1 was incubated at room temperature for 30 min with 0 (lane 1), 0.08 m
m
(lane 2), or 0.25 m
m
DSP (lane 3) and then immunoprecipitated onto Sepharose G:N-CEACAMFc beads and visualized via fluorography following SDS-PAGE on a 4–20% acrylamide gradient gel under non-reducing (NR) and reducing (R) conditions.
Figure 2
Specific capture of assembled S oligomers by N-CEACAMFc. HeLa cells synthesizing S proteins were pulse-labeled with Tran35S-label for 30 min and either lysed immediately (0 hour) or chased for 2 h at 37 °C before lysis (2 hour). Lysates were sedimented on sucrose gradients, and S proteins in each fraction were immunoprecipitated with polyclonal antiserum (anti-S) or with N-CEACAMFc (N-CEACAM Fc) before SDS-PAGE and visualization by fluorography. The sedimentation markers horseradish peroxidase (HRPO 4S), immunoglobulin G (IgG 7S), and β-galactosidase (B-Gal 16S) were identified in fractions from a parallel gradient by enzyme or immunodetection assays, and their positions are indicated above the electropherograms.
Figure 3
Influence of S1 quaternary structure on its ability to bind CEACAM. 35S-Labeled SECTOin sucrose gradient fractions was precipitated with trichloroacetic acid (top panel) or with N-CEACAMFc(bottom panel) and then detected by fluorography following SDS-PAGE. The sedimentation marker immunoglobulin G (IgG 7S) was identified in fractions from a parallel gradient by immunodetection assays, and its position is indicated above the electropherogram. The positions of S1 and S2ECTO are also indicated.
Figure 4
Co-immunoprecipitation of amino-terminal fragments S1330 and S1769. Recombinant S1 fragments of 330 or 769 residues were synthesized alone or together in HeLa cells in the presence of Tran35S-label. S fragments in the media (top panel) and cell lysates (bottom panel) were immunoprecipitated with N-CEACAMFc or with anti-spike mAb J.2.6. The J.2.6 epitope is between S1 residues 510 and 540. 35S-Labeled proteins were visualized by fluorography following SDS-PAGE.
Figure 5
Sucrose gradient sedimentation analysis of unbound S1 and S1·N-CEACAMFc complexes. A, two possibilities for the CEACAM-binding site architecture on a S1 dimer are illustrated. If only one CEACAM binds each S1 dimer, then divalent N-CEACAMFc would bind one or two S1 dimers. Alternatively, two CEACAM-binding sites could complex S1 and N-CEACAMFc into higher order oligomers. B, unbound 35S-labeled S1 and 35S-labeled S1·N-CEACAMFc complexes were sedimented on linear sucrose gradients and detected in gradient fractions after immunoprecipitation, SDS-PAGE, and fluorography. The sedimentation markers immunoglobulin G (IgG 7S) and β-galactosidase (B-gal 16S) were identified in fractions from a parallel gradient by enzyme or immunodetection assays, and their positions are indicated above the electropherograms. An ∼16 S S1·N-CEACAMFc complex was identified. There was no evidence of larger complexes in pellet fractions (not shown).
Figure 6
Immunoprecipitation of S1· CEACAM complexes.Upper, constant amounts of recombinant S1 were incubated with increasing quantities of 35S-labeled CEACAMECTO for 12 h at 4 °C, and proteins in each aliquot were then immunoprecipitated with immobilized N-CEACAMFc (A and B) or immobilized anti-spike mAb J.2.6 (C). (Lower). Constant amounts of recombinant S1 (35S-labeled as indicated) were incubated with increasing amounts of CEACAMECTO(35S-labeled as indicated) for 12 h at 4 °C, and proteins were then affinity-purified with N-CEACAM6×His(A and B) or immunoprecipitated with mAb J.2.6 (C). The dots on the right of each panel represent the positions of the 113- and 75-kDa molecular mass markers.
Figure 7
Increased capture of mutant S proteins with low affinity CEACAM-binding sites by heteromerization with S1330 fragments. Recombinant mutant S proteins with three point mutations within the CEACAM-binding site were synthesized alone or with increasing amounts of “wild-type” S1330. S1330 synthesis was adjusted by the input multiplicity of infection (MOI) of the vTM3-S1330 vector.35S-labeled spikes were immunoprecipitated with N-CEACAMFc and visualized by fluorography following SDS-PAGE. The amount of 35S associated with the boxed bands was quantitated with a Molecular Dynamics Typhoon 8600 PhosphorImager. The left box contained 2141 cpm and the right box contained 4329 cpm.
Figure 8
**CEACAM-induced conformational changes in S1.**35S-Labeled S1ΔDPR1 was either incubated with (+) or without (−) 10 m
m
NEM prior to incubation for 4 h at 4 °C and then for 1 h at 37 °C with 10 μg of N-CEACAMFc (A) or anti-S1330 mAb number 2 (B) (25). Samples not previously treated with NEM were then incubated with 10 m
m
NEM (+). 35S-Labeled proteins were detected by fluorography using a Molecular Dynamics Typhoon 8600 PhosphorImager following SDS-PAGE under reducing (+ B-ME) and non-reducing (− B-ME) conditions. N-CEACAMFc-induced disulfide-linked high molecular weight spikes are indicated by the *.
Figure 9
Analysis of the oligomeric organization of S2 after separation from S1.A, cDNAs encoding S or EGFP-tagged S (S EGFP) were transfected alone or together into vTF7.3-infected HeLa cells. Following metabolic labeling with Tran35S-label, cytoplasmic extracts were prepared, and EGFP-associated proteins were immunoprecipitated with polyclonal anti-GFP serum. Immunoprecipitates were then electrophoresed and immunoblotted for S2 fragments. Co indicates co-synthesis of S and SEGFP. Mix indicates that equal volumes of independently produced S and SEGFP lysates were mixed before immunoprecipitation. B, autoradiographic image of the immunoblot in A. C, cell lysates were depleted of S1 by sequential immunoprecipitations with N-CEACAMFc at pH 8.5 and 37 °C. Supernatants from the final depletions were collected, and EGFP-associated proteins were immunoprecipitated with anti-GFP serum. Immunoprecipitates were electrophoresed and immunoblotted for S2 fragments. D, autoradiographic image of the immunoblot in C. Arrows indicate the positions of uncleaved SEGFP(S unc-EGFP), S2EGFP, untagged S2, and S1.
Figure 10
Models depicting the quaternary structure of the MHV spike and its interaction with CEACAM during virus entry. To explain a single CEACAM-binding site on S1 dimers, two models are illustrated. Model A appeals to steric hindrance, and model B suggests that a single CEACAM induces global structural rearrangements (illustrated as the change of S1 from an oval to a rectangle). These conformational changes preclude additional CEACAM binding. In both cases, CEACAM binding is hypothesized to displace S1 from S2 (23) and to permit the insertion of an internal fusion peptide (green triangle) into the target cell membrane (71).
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