EbolaGlycoproteinComplex

Ebola virus glycoprotein in complex with EBOV-520 Fab is a glycoprotein/antibody complex (1). The protein (PDB ID: 6OZ9) is classified as a viral protein and is from Homo sapiens and Ebola virus (2). The Ebola virus is an enveloped, negative-strand RNA virus that initiates attachment and fusion of viral and host membranes through interaction with its single surface protein known as glycoprotein (3). The glycoprotein is a trimer that is made up of three non-covalently attached monomers (3). EBOV-520 Fab, which is a therapeutic human monoclonal antibody (mAb), can be used for prevention or treatment of viral infectious diseases, which in this case is homologous Ebola Virus (4). EBOV-520 exhibits multifunctional properties such as epitope recognition, direct virus neutralization and in vivo protection (5). Ebola virus glycoprotein in complex with EBOV-520 Fab demonstrates mAb-antigen (EBOV-520 with Ebola virus glycoprotein) interactions that mediate protection against the virus (4). The glycoprotein/EBOV-520 Fab protein can further aid in developing a design of therapeutic mAb cocktails against other life-threatening viral pathogens such as Sudan virus as well (4).

The co-crystallization of Ebola virus glycoprotein with EBOV-520 Fab was expressed in Drosophila S2 cells using a single pMT-puro plasmid (4). The purification of the protein was performed using StrepTrap HP affinity chromatography column followed by EKMax (4). Glycoprotein with EBOV-520 was crystallized through vapor diffusion and sitting drop methods, under the conditions of 0.1 M HEPES pH 7.0 and 1.4 M Ammonium sulfate at 295 K (2). No ligands were used to induce crystallization (4). The crystal structure of the protein was obtained using X-Ray diffraction, and the structural data of the protein was obtained using single particle negative stain and cryogenic electron microscopy (4).

According to the Expasy protein database, Ebola virus glycoprotein in complex with EBOV-520 Fab has a molecular weight of 77228.98 Da and an isoelectric point of 8.14 (6). The glycoprotein with EBOV-520 has 713 residues, 271 of which belong to Ebola virus glycoprotein and 442 belong to EBOV-520 Fab (2). There are a total of four chains present in glycoprotein with EBOV-520 that are named Chain A, B, L and H (2). Chain A and B are from Ebola virus glycoprotein and make A and B Subunit (2). Chain L and H are from EBOV-520 Fab and make C and D Subunit (2). Chain A is known as small secreted glycoprotein sGP; Chain B as envelope glycoprotein; Chain L as EBOV-520 Fab light chain and Chain H as EBOV-520 Fab heavy chain (2). Small secreted glycoprotein subunit (glycoprotein 1) and Envelop glycoprotein subunit (glycoprotein 2) bind to residues on rEBOV-520 antibody chains (4). The EBOV-520 Fab heavy chain subunit is a base-region specific antibody that binds to epitope on glycoprotein 1 (GP 1) and glycoprotein 2 (GP 2) (4, 5). The EBOV-520 light chain subunit does not participate in the interface with any glycoprotein residues of glycoprotein 1 or 2 (4, 5). The important crystallized domains are the ones present in GP 1 known as 3₁₀ pocket domain and β17 - β18 loop domain and in GP 2 known as internal fusion loop domain (4). The removal of β17 - β18 loop allows the antibody to successfully neutralize the virus by linking to specific residues on 3₁₀ pocket domain and internal fusion loop domain. The domains not present in the crystalized structure include mucin-like domain and transmembrane domain (4).

The secondary structure of Ebola virus glycoprotein in complex with EBOV-520 Fab includes beta sheets, alpha helices and random coils (2, 4). A, B and C Subunits involve all of the secondary structures mentioned above, and D Subunit involves only beta sheets and random coils (2, 4). A and B Subunits are mostly hydrophobic while C and D Subunits appear to be more polar with some hydrophobic sections in the beta sheets (2). Upon folding, the hydrophobic sections of A and B Subunits are predicted to undergo hydrophobic collapse and be located in the middle of the protein while the polar and charged residues are predicted to be mainly located at the surface of the protein. This can be observed when the glycoprotein/EBOV-520 Fab protein forms a trimer that has more hydrophobic A and B Subunits located in the middle of the crystal structure of the protein (2). The glycoprotein/EBOV-520 Fab protein has both intramolecular and intermolecular disulfide bridges (3). Two intramolecular disulfide bonds present in GP 1 are Cys-108 - Cys-135 and Cys-121 - Cys-147 (3). These bonds help in the stabilization of GP 1 subunit (3). Additionally, there are two sets of Beta sheets that link the internal fusion loop and a helix of GP2 through hydrophobic interactions (3). The glycoprotein trimer contains three non-covalently attached monomers and each monomer contains intermolecular disulfide bridges that link GP 1 to GP 2 (3). The residues involved in this intermolecular disulfide bridge are Cys-53 of GP 1 subunit and Cys-609 of GP 2 subunit (3).

Moreover, Ebola virus glycoprotein in complex with EBOV-520 Fab contains several functionally important residues that allow the interaction within GP 1 subunit, between the two glycoprotein subunits, and between the glycoproteins and antibody subunits. The residues that participate in interactions, in the form of disulfide bridges, within GP 1 and between the two glycoproteins, GP 1 and GP 2, are both mentioned in the above paragraph. Additional functionally important residues include Asn-514, which is a conserved residue on GP 2 that is not fully neutralized by mAb rEBOV-520 (4). Asn-101, Val-102, Ala-103, Thr-104, and Val-105 of EBOV-520 Fab heavy chain binds to the 3₁₀ pocket of GP 1 (4). EBOV-520 Fab also binds to 26 of glycoprotein residues, of which 16 are located in GP 1 and 10 are located in GP 2 (4). Some additional important residues located on EBOV-520 Fab heavy chain include Trp-100 and Tyr-108 that bind to GP 2, Tyr-106 that binds to GP1 and Thr-104 that binds to α1 loop of GP 1 (4). Finally, another functionally important residue is Asn-512 because this is a key epitope residue for rEBOV-520 as a mutation at this residue decreases binding of antibody to the glycoprotein (4). Thus, decreasing the direct virus neutralization property of the EBOV-520 Fab antibody. There is only one ligand present in the glycoprotein/EBOV-520 Fab protein, which is known as glycerol, and it functions as an intermediate in carbohydrate and lipid metabolism (2). Glycerol, which is present on GP 1, enhances protein stabilization in aqueous solutions (7).

Furthermore, two online servers: Dali Server and PSI-BLAST, were used to compare Ebola virus glycoprotein in complex with EBOV-520 Fab with proteins that have similar structures. Dali Server is used for finding proteins with tertiary structure similarities to a protein of interest. It uses sum-of-pairs method which produces a measure of similarity by comparing intramolecular distances. It measures similarities by assigning Z-scores to the comparison proteins. A score above 2 is significant. Dali Server only works for proteins because it requires an amino acid backbone as it compares the three-dimensional structures of the protein of interest with comparison proteins. Thus, it cannot work for a nucleic acid. PSI-BLAST is a program that is used to find proteins with similar primary structure to a protein of interest. The proteins with similar primary structure are known as subjects. PSI-BLAST uses sequence homology to assign E-scores to the comparison proteins. An E-score below 0.5 is significant whereas an E-score of 0 means that the comparison protein is the same as the protein of interest. A score of 0 indicates that there is no difference in the sequence of the proteins, thus there are no structural changes in the folded protein.

The results from Dali Server and PSI-BLAST are used to compare the protein, Cleaved Ebola GP in complex with a broadly neutralizing human antibody ADI-15946 (PDB ID: 6MAM), with the protein of interest, Ebola virus glycoprotein in complex with EBOV-520 Fab (8). According to the two servers, the Z-score for the comparison protein is 26 and the E-score is 6 x 10^-156 (9, 10). These scores are significant according to the criteria mentioned above and these scores indicate that there is high sequence homology and similarity in tertiary structure between the comparison protein and the protein of interest. The comparison structure is also an Ebola virus glycoprotein but with a different antibody linked to it, this further explains why the two servers produced significant Z-score and E-score values. The comparison structure does not contain any ligands in the crystal structure, has twelve chains and two oligosaccharides unlike glycoprotein/EBOV-520 Fab protein that has one ligand, four chains and one oligosaccharide (2, 8). The main difference between the functions of the two proteins is that the antibody of the comparison protein has a different angle of approach than the angle of approach of EBOV-520 antibody. This makes the EBOV-520 have a more extended activity against not only Ebola virus but Sudan virus as well (4).

In conclusion, Ebola virus glycoprotein in complex with EBOV-520 Fab is a glycoprotein antibody trimer that can be used for prevention or treatment of viral infectious diseases. EBOV-520 antibody uses its multifunctional properties to neutralize both Ebola virus and Sudan virus. The information gathered from the interactions in Ebola virus glycoprotein in complex with EBOV-520 Fab can be used to develop an antibody cocktail that can further enhance the multifunctional antibody properties and protection against the virus. This is a significant interest to scientists because developing a cocktail for deadly Ebola Virus disease using various mAbs can save individuals infected with this disease. The knowledge from the mAbs-antigen interactions of Ebola virus can be used to further develop therapeutic cocktails against other viral diseases as well.