SARS-CoV-2 ORF8 Protein
Leeann Stearns, Ryan Peterson, Allison Robinson, Ryan Vazquetelles, Elizabeth Wiles, Dr. Karen Guzman

This Jmol Exploration was created using the Jmol Exploration Webpage Creator from the MSOE Center for BioMolecular Modeling.

version 2.0
Exploration Content

ORF8 Dimer


ORF8 is a SARS-CoV-2 accessory protein that forms a homodimer, i.e. it is composed of two identical polypeptide subunits. The subunits of the dimer are designated as chain A and chain B. Chain A is shown in the darker tan (Burlywood). Chain B is shown in the lighter tan (Blanched Almond).

Although ORF8 is a symmetrical homodimer, there are slight differences seen when comparing chain A to chain B in the crystal structure. There are residues that do not display in the crystal structure. The missing residues will flash the following colors as we fill them in:

** Chain A: missing 2 residues between Glu64 & Ser67, flashing magenta
** Chain B: missing 3 residues between Ala65 & Ser69, flashing lime green

Polypeptides of the Homodimer

N-termini & C-termini

N-termini are located at Gln18 on Chain A and Glu19 on Chain B. The differing ends are due to differences in the crystal structure. They will blink in blue.

C-termini are represented by ile121. They will blink in red.

N-termini and C-termini

Covalent Interface - Dimerization Domain

There are two interfaces where the ORF8 monomer can interact with another ORF8 monomer: the Covalent Interface and the Noncovalent Interface. Here, we will take a closer look at the Covalent Interface, where the ORF8 homodimer is connected by a disulfide bond between a cysteine residue from each subunit.

In addition to the unique disulfide bond, there are other interactions involved in the Covalent Interface of the ORF8 dimer. These include multiple hydrogen bonds, a salt bridge, and a hydrophobic interaction.

The first hydrogen bond we will explore in the dimer interface is between phenylalanine 120 and alanine 51. Note that, although these are hydrophobic amino acids, the interaction is not between the sidechains, but between the polar groups of the peptide bond on the backbone.

Let's also look at the hydrogen bond that forms between Phenylalanine 120 and arginine 52. This hydrogen bond is between the backbone of Phe120 and the sidechain of Arg52.

Hydrogen bonds are shown in light pink.

To highlight the backbone atoms that form these hydrogen bonds, the atom from Phe120 will first oscillate between pink and cpk, then the atom of Ala51 will do the same. Each hydrogen bond will be displayed in this same manner.

Hydrogen bonds: Phe120-Ala51 and Phe120-Arg52

Arginine 52 has an additional hydrogen bond to isoleucine 121.

Another hydrogen bond present is between lysine 53 and serine 24.

Again the hydrogen bonds are shown in light pink.

Hydrogen bonds: Arg52-Ile 212 and Lys53-Ser24

In addition to the hydrogen bonds, the dimer interface is stabilized by a salt bridge interaction between asparagine 119 and arginine 115. That will be shown in light purple.

There is also a hydrophobic interaction between a valine from each subunit: both Val-117. While you will not see a new bond form, the residues involved in the interaction will be shown in dark orange.

Salt bridge and Hydrophobic Interaction

Lastly, there is a disulfide bond between cysteine residues on each subunit: both Cys-20. This interaction is unique to SARS-CoV-2 ORF8. This will be shown alternating from light yellow to dark yellow.

Disulfide Bond

Now let us put it all together!

Recall hydrogen bonds are light pink, the salt bridge is light purple, the hydrophobic interaction is shown in orange, and finally the covalent disulfide bond is shown in yellow.

Interactions of the Covalent Interface

ORF8 Oligomer

Assembly from Homodimers

As indicated above, the ORF8 protein is a homodimer, composed of two identical polypeptide chains that we designate chain A and chain B. In the crystal structure, these dimers interact to form an oligomer that may be unique to SARS-CoV-2 and potentially play a role in pathogenicity.

Now we will look at how the oligomer is assembled from homodimers by showing you 6 dimers, 12 polypeptides in total, assembled in a chain.

Can you see where the A-chains and B-chains are in the oligomer? Let's look at all of the A-type chains flashing in the darker tan (Burlywood) and the B-type chains flashing in the lighter tan (Blanched Almond).

Oligomer Assembly

Covalent and Noncovalent Interfaces

Next, let's zoom in on one of the dimers and see where the Covalent and Noncovalent Interfaces are located.

Since beta sheets are involved in these two interfaces, we will distinguish these sheets, as well as other features of the oligomer, by coloring:
** Noncovalent Interfaces between separate dimers in dark teal
** Covalent Interfaces within dimer pairs in light blue
** Backbone for better contrast in light grey
** As a point of orientation, the N-terminus is blue and the C terminus as red

After the animation zooms in and spins the dimer, we will draw attention to the Covalent Interface by showing in yellow the covalent disulfide bond between cysteine residues. Then, we will zoom out and add back the other subunits of the oligomer to demonstrate the location of the covalent interfaces along the oligomer, showing the yellow disulfide bonds in the Covalent Interfaces.

Covalent Interfaces of Dimer and Oligomer

Now, we will examine the dark teal Noncovalent Interface within the dimer, as it blinks. Next, we will zoom out and add back the other subunits of the oligomer to display the location of the Noncovalent Interfaces along the oligomer, by showing the dark teal beta sheets of the Noncovalent Interfaces.

Noncovalent Interfaces of Dimer and Oligomer

Let's look at some of the interactions in the Covalent and Noncovalent Interfaces of the oligomer, starting with hydrogen bonds.

In the Covalent Interface, in addition to the disulfide bond shown previously, there is also several hydrogen bonds between the monomers of the dimer, which stabilize the ends of the interface. Those will be shown in light pink.

When looking at the Noncovalent Interface, we will show hydrogen bonds that occur between dimer pairs. The beta sheets of the interface are shown in dark teal, and the hydrogen bonds within them are depicted in magenta.

Hydrogen Bonds in the Covalent and Noncovalent Interfaces

As we look a bit closer, we will see there are more than just hydrogen bonds in the Noncovalent Interface.

Residues that are critical for stabilizing the Noncovalent Interface include the sequence YIDI, which is the single letter abbreviation for the four amino acids that make it up. This sequence motif is unique to SARS-CoV-2.

The sidechains of the beta sheet in the Noncovalent Interface will grow from the backbone as dark teal, then change to cpk coloring to show the primarily hydrophobic nature of the sidechains.

A key amino acid in the YIDI sequence is tyrosine, which forms hydrophobic interactions with several other hydrophobic sidechains. Near the end of the animation, tyrosine flashes in salmon and the other hydrophobic amino acids flash sky blue.

Hydrophobic Interactions of the Noncovalent Interface

Citation & Acknowledgement


Crystal Structure: 7jtl
Article: Flower TG, Buffalo CZ, Hooy RM, Allaire M, Ren X, Hurley JH (2021). Structure of SARS-CoV-2 ORF8, a rapidly evolving immune evasion protein. Proc Natl Acad Sci U S A., Jan 12; 118(2): e2021785118. doi: 10.1073/pnas.2021785118


Special thanks to Cosmo Buffalo, one of the primary authors in the citation above, who helped make this project possible by supplying us with additional information and modified structure files of ORF8.