SARS-CoV-2 spike protein fusion transformation

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Revision as of 18:07, 7 August 2020

The spike protein of SARS-CoV-2 plays a central role in coronavirus attachment to the ACE2 receptor on host cells, and in getting the RNA genome of the virus into the host cell via fusion of the virus and host cell membranes, initiating infection.

Introduction

SARS-CoV-2 spike protein undergoes a dramatic conformational rearrangement () that plays a central role in fusing the coronavirus membrane with the host cell membrane^[1]. Similar conformational transformations have been observed for the spike protein of SARS-CoV^[2] and mouse hepatitis virus^[3], among others. These rearrangements also have much in common with the membrane fusion mechansism of influenza hemagglutinin^[4]. The molecular scenes in this article are based on the cryo-EM pre- and post-fusion structures of SARS-CoV-2 spike protein reported July, 2020, by Cai, Zhang and coworkers with the group of Bing Chen^[1].

Pre-Fusion Structure

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The is a homo-trimer, each chain having a mature length of 1,261 amino acids. This pre-fusion cryo-EM structure 6xr8 is complete except for 110 residues of the C-terminus (narrow end), comprising 48 residues of the stem (heptad repeat 2 domain), a 23-residue trans-membrane domain, and a 39 residue cytoplasmic domain.

Domains

as in Cai, Zhang et al. ^[1] (lengths):

S1 receptor binding region:
- 14-305 (292): N-terminal domain.
- 306-330 (25): Linker.
- 331-527 (197): Receptor binding domain, binds to angiotensin converting enzyme 2 (ACE2).
- 528-590 (63): CTD1 (C terminal region of S1 fragment after furin cleavage).
- 591-687 (97): CTD2 (C terminus of S1 fragment after furin cleavage).
677-688 (12): Pink balls mark the furin cleavage site^[5] (missing in this model due to disorder).
S2 fusion region:
- 686-815 (130): Linker
- 817-825 (9): Fusion peptide (FP).
- 826-833 (8): Fusion peptide proximal region (FPPR).
- 834-909 (76): Linker
- 910-984 (75): Heptad repeat 1 (HR1)
- 985-1034 (50): Central helix (CH)
- 1035-1067 (33): Connector domain (CD)
- 1068-1162 (95): Linker
- 1163-1273 (111): Missing in this model due to disorder. Stem (heptad repeat 2, HR2), transmembrane domain, cytoplasmic domain.

It will be easier to see the domains if you look at only one of the three chains:

Activation

A protease, typically furin, cuts the chains at the position marked with Pink balls^[5], but the assembly, now six chains, remains intact. The cut may facilitate extending one receptor binding domain to engage the ACE2 receptor. This "priming" cut divides each chain of protein S into an N-terminal S1 receptor-binding fragment, and a C-terminal S2 fusion fragment^[1]. , will later separate.

Binding to ACE2 and a second proteolytic cut are believed to trigger release of S1^[1], after which through the stem and transmembrane domains (absent in this model) that extend from the red balls.

Limitations of Models

Next, a dramatic conformational transformation occurs that triggers membrane fusion. We will compare the pre-fusion cryo-EM model (6xr8) with the post-fusion model (6xra). However, these two models have only two protein segments in common, sequence ranges 703-770 and 912-1162 (319 residues or 54% of the 588-residue S2 fusion fragment, 686-1273). The parts of the pre-fusion model . Thus, the pre- to post-fusion comparison will involve .

Fusion

Fusion of the virus and host cell membranes seems to happen when the spike protein brings the virus membrane (near the red balls) very close to the host membrane, after the host membrane is attached to the fusion peptide (near the blue balls).

(1) Aquisition of the host membrane appears to happen when with one long helix, having the fusion peptide at its end (near the blue balls). (The fusion peptide is absent in this model.)

(2) The virus membrane is attached near the red balls. The (near the blue balls), initiating membrane fusion. The virus membrane is brought closer than shown here: these models lack the stem. The red balls would actually end up much closer to the blue balls than these models show.

Use the toggle animation button to restart animation as needed.
Remember that a morph is intended to help you compare two structures. This morph does NOT portray a realistic transition pathway.

(3) The .

Now that you understand the various movements taking place, you will better appreciate .

The membrane fusion hypothesis may be clarified by this schematic. It is for influenza virus but the principles are the same.

Lipid Bilayers. Receptor (ACE2 for SARS-CoV-2). Receptor binding domain. Fusion Peptide.
Adapted from Fig. 3 in a 2012 review by Hamilton, Whittaker and Daniel under a CC Attribution License 3.0, in turn adapted from a 2006 original by David Goodsell. Permission given by the authors^[6]

Glycosylation

Both the , and the have extensive N-linked glycosylation (red and gray, exaggerated in size because the cryo-EM can likely resolve only the protein proximal portions). No glycans were observed on the receptor-binding surface. Glycosylation may protect the spike protein from antibodies and other damage from the virion environment^[1].

Unexpected Findings

Spontaneous Fusion Transformation

Fig. 5A (cropped) from Cai, Zhang and coworkers^[1] reproduced in accord with the Creative Commons Attribution 4.0 International license specified in Science. Permission also given by Bing Chen, August 7, 2020.

The fusion transformation, including loss of S1, occurred in a substantial portion of the full-length spike protein purified in mild detergent, in the absence of host cells or ACE2 receptors. Cai, Zhang and coworkers^[1] note that post-fusion S2 conformations have been observed multiple times in electron micrographs by other researchers. They suggest that virions normally contain a mixture of pre-fusion full-length, and post-fusion S2 fragment conformations, and that the post-fusion S2 spike proteins may help to protect the pre-fusion forms, and possibly may induce non-neutralizing antibody responses.

More Compact Structure

Previous studies of soluble extracellular portions of the spike protein have utilized a double proline mutation (K986P, V987P) that stabilizes the pre-fusion conformation^[1]^[7]. K986P appears to eliminate a salt bridge, causing an unsatisfied internal charge^[1]. The wild type salt bridge may contribute to the more compact and better resolved structure observed by Cai, Zhang and coworkers^[1] using the full-length wild type protein.

Methods

The pre-fusion structure 6xr8 was morphed to the post-fusion structure 6xra by linear interpolation, requesting 14 intermediate frames (16 total), using the server provided by Karsten Theis after another method^[8] gave unsatisfactory results. To avoid artifactual movement in the morph, prior to morphing, two changes were required in 6xra: (i) the names of chains B and C needed to be swapped (done with SwissPDBViewer), and (ii) the structure needed to be rotated +120° around the Z axis (Jmol "rotateselected" command). The morph was an 11 MB file, which took 25 sec to load into JSmol. Each script took a minimum of 8 sec to complete. To reduce both the bulk of this file and the processing times for JSmol, the alpha carbons were extracted (along with the MODEL and ENDMDL records) by deleting all other lines in the PDB file^[9]. The resulting 16 model morph PDB file is Image:Morf-6xr8-6xra-theis-cao.pdb.

References and Notes

↑ ^1.00 ^1.01 ^1.02 ^1.03 ^1.04 ^1.05 ^1.06 ^1.07 ^1.08 ^1.09 ^1.10 Cai Y, Zhang J, Xiao T, Peng H, Sterling SM, Walsh RM Jr, Rawson S, Rits-Volloch S, Chen B. Distinct conformational states of SARS-CoV-2 spike protein. Science. 2020 Jul 21. pii: science.abd4251. doi: 10.1126/science.abd4251. PMID:32694201 doi:http://dx.doi.org/10.1126/science.abd4251
↑ Fan X, Cao D, Kong L, Zhang X. Cryo-EM analysis of the post-fusion structure of the SARS-CoV spike glycoprotein. Nat Commun. 2020 Jul 17;11(1):3618. doi: 10.1038/s41467-020-17371-6. PMID:32681106 doi:http://dx.doi.org/10.1038/s41467-020-17371-6
↑ Walls AC, Tortorici MA, Snijder J, Xiong X, Bosch BJ, Rey FA, Veesler D. Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion. Proc Natl Acad Sci U S A. 2017 Oct 17;114(42):11157-11162. doi:, 10.1073/pnas.1708727114. Epub 2017 Oct 3. PMID:29073020 doi:http://dx.doi.org/10.1073/pnas.1708727114
↑ Pabis A, Rawle RJ, Kasson PM. Influenza hemagglutinin drives viral entry via two sequential intramembrane mechanisms. Proc Natl Acad Sci U S A. 2020 Mar 31;117(13):7200-7207. doi:, 10.1073/pnas.1914188117. Epub 2020 Mar 18. PMID:32188780 doi:http://dx.doi.org/10.1073/pnas.1914188117
↑ ^5.0 ^5.1 According to Cai, Zhang et al. (their Fig. 4), the initial cut by furin occurs between Arg685 and Ser686 at * in the sequence PRRAR*SVASQ. Thus, S1 is 13-685 (length 673, excluding a 12 residue signal sequence), or 53% of the original chain, leaving S2 as 686-1273, length 588, 47%.
↑ Permission obtained from Gary R. Whittaker August 6, 2020. Permission obtained from David Goodsell August 5, 2020.
↑ Wrobel AG, Benton DJ, Xu P, Roustan C, Martin SR, Rosenthal PB, Skehel JJ, Gamblin SJ. SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects. Nat Struct Mol Biol. 2020 Jul 9. pii: 10.1038/s41594-020-0468-7. doi:, 10.1038/s41594-020-0468-7. PMID:32647346 doi:http://dx.doi.org/10.1038/s41594-020-0468-7
↑ Proteopedia's PyMOL morph server was used in both RigiMOL and linear modes, all atoms or only alpha carbon atoms. Rendering these as backbones or traces by Jmol gave broken lines. The reason for backbone breaking was not investigated further.
↑ Selecting *.ca in the Jmol Java application and saving a PDB file produced a PDB file with numerous errors. The desired result was obtained with this command in macOS Terminal: sed -e /REMARK/d -e /HETATM/d -e /^ATOM\ \ [\ 0-9][0-9][0-9][0-9][0-9]\ \ [CONS][\ B-Z].*$/d <original.pdb >product.pdb.

Proteopedia Page Contributors and Editors (what is this?)

Eric Martz, Karsten Theis

Retrieved from "http://52.214.119.220/wiki/index.php/SARS-CoV-2_spike_protein_fusion_transformation"

@@ Line 99: / Line 99: @@
 Remember that a [[morph]] is intended to help you compare two structures. This morph does NOT portray a realistic transition pathway.</i></center>
-:(3) The <scene name='85/857791/Morf_6xr8_6xra_theis_lin_cao/7'>middle portion of the spike protein remains relatively stable</scene>. (If you view all 3 chains, two swap places. This is an artifact.)
+:(3) The <scene name='85/857791/Morf_6xr8_6xra_theis_lin_cao/7'>middle portion of the spike protein remains relatively stable</scene>.
 Now that you understand the various movements taking place, you will better appreciate <scene name='85/857791/Morf_6xr8_6xra_theis_lin_cao/4'>watching them all at once</scene>.
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 [[Image:Cai-zhang-fig-5a.png|350px]]
 </td></tr><tr><td>
-Fig. 5A (cropped) from Cai, Zhang and coworkers<ref name="cai-zhang" /> reproduced in accord with the [https://creativecommons.org/licenses/by/4.0/ Creative Commons Attribution 4.0 International] license specified in ''Science''.
+Fig. 5A (cropped) from Cai, Zhang and coworkers<ref name="cai-zhang" /> reproduced in accord with the [https://creativecommons.org/licenses/by/4.0/ Creative Commons Attribution 4.0 International] license specified in ''Science''. Permission also given by Bing Chen, August 7, 2020.
 </td></tr></table>
 The <b>fusion transformation</b>, including loss of S1, occurred in a substantial portion of the full-length spike protein purified in mild detergent, in the <b>absence of host cells or ACE2 receptors</b>. Cai, Zhang and coworkers<ref name="cai-zhang" /> note that post-fusion S2 conformations have been observed multiple times in electron micrographs by other researchers. They suggest that virions normally contain a mixture of pre-fusion full-length, and post-fusion S2 fragment conformations, and that the post-fusion S2 spike proteins may help to protect the pre-fusion forms, and possibly may induce non-neutralizing antibody responses.