Created by: Ananya Suram

 

Infection by Zika virus (ZIKV), a flavivirus transmitted by Aedes species mosquitoes, has become an emerging health threat for its role in development of congenital disease during pregnancy. Neutralizing antibodies help protect against a multitude of flaviviruses. The envelope (E) protein of ZIKV has been identified as an antigenic target on account of the ability of antibodies to bind epitopes in all three of its structural domains. Since antibodies that target the DII domain of ZIKV-E can be cross-reactive and less effective, focus has been shifted to neutralizing the DIII domain (1). Zika specific antibody, ZV-48, bound to ZIKA envelope DIII (PDB ID: 5KVE) is a monoclonal antibody generated in Mus musculus that neutralizes ZIKV H/PF/2013 infection (1, 2). 

After immunizing with live ZIKV and boosting with purified DIII, ZV-48 from Mus musculus was expressed by IPTG-induction in Escherichia coli (E. Coli) BL21 for purification and further structural analysis. The protein complexes were crystallized by hanging drop vapor diffusion, under the conditions of 14 mg/mL in 0.2 ammonium sulfate and 15% (w/v) PEG 4000. The crystals were cryoprotected using a solution containing 20% ethylene-glycol and cooled in liquid nitrogen. The ligands on ZV-48, sulfate, 1,2-ethanediol, acetate, and sodium, were used to enhance crystallization (1). The structure of ZV-48 was obtained through X-ray diffraction (2).

ZV-48 has a molecular weight of 38308.28 Da and an isoelectric point of 8.37 (3). It exists as a pentameric single-chain variable fragment composed of 245 residues (1, 2). The variable heavy (VH) and light (VL) domains of the protein are connected by three linker peptides (H1, H3, L3). ZV-48 forms a complex with ZIKV-E via binding of V_H and V_L to the poorly exposed C-C' loop on the DIII epitope (1). The protein is not fully crystallized. Residues 116 through 129 and residue 245 of ZV-48 are unmodeled. Additionally, residues 1 through 5 and 107 through 110 of the DIII domain of the ZIKV-E are not resolved (2).

Antibodies share a basic structure consisting of heavy and light chains made up of beta-sheets that organize into immunoglobulin folds and attach to the stem of the protein by flexible peptides (4). ZV-48 follows this pattern with heavy and light-chain regions of antiparallel beta-sheets connected by linker peptides consisting of random coils and 3/10 helices. The linker peptides are rich in Gly, Ser, and Thr, conferring flexibility and solubility to the protein (4). V_H and V_L contain a mixture of hydrophobic and hydrophilic regions. Nonpolar portions of beta-sheets are interspersed among long spans of polar residues and short but frequent charged regions that exist primarily at the N-terminal and C-terminal regions of beta-strands. Hydrophilic residues, most frequently Tyr, Glu, and Asn, make up the light-chain complementarity-determining region (CDR) that contacts the DIII domain of ZIKV-E to facilitate the formation of an antigen-antibody complex. These residues are highly conserved between ZV-48 and Zika specific antibody, ZV-64, bound to ZIKA envelope DIII (PDB ID: 5KVF), another monoclonal antibody generated in Mus musculus that neutralizes ZIKV H/PF/2013 infection (1, 2). Ten of twelve light chain CDR contact residues are common to ZV-48 and ZV-64, as opposed to two of eleven heavy-chain CDR contact residues (1). The beta-strands of each ZV-48 variable domain pack together to form compressed antiparallel beta-barrels, known as immunoglobulin folds. Folds are stabilized by hydrogen bonding between beta-strands, hydrophobic bonding of nonpolar regions in the interior, and disulfide bonds between beta-sheets (4). V_H and V_L are connected to each other by linker peptides H1, H3, and L3. As ZV-48 is a single-chain variable fragment, it lacks the constant F_c region found in complete antibodies and thus does not share the characteristic Y shape of an antibody (1, 4).

Flaviviruses are positive-sense RNA viruses. To enter the host cell, ZIKV interacts with target cell receptors to mediate E protein reorganization, low pH-dependent endocytosis, and membrane fusion that permits release of the viral genome into the target cell. DIII of ZIKV-E is an immunoglobulin-like domain that facilitates attachment of ZIKV to the host cell via membrane receptor binding (5). ZV-48 neutralizes the virus by impeding viral attachment and E protein reorganization (6). It also facilitates recognition by B cells and subsequent differentiation that prompts immune response (5).

The light-chain CDR is the primary binding site for DIII, though there is some interaction between the heavy-chain CDR and the antigen as well. The following ZIKV/ZV-48 residue pairs constitute binding sites between the antigen and the antibody. Some light-chain pairs are conserved in ZV-64: Thr-353/Tyr-97 (hydrogen bond), Lys-340/Glu-30 (hydrogen bond), Pro-354/Tyr-92 (Van der Waals contact). Other light-chain pairs are unique to ZV-48: Pro-354/Tyr-31 (Van der Waals), Gln-350/Tyr-31 (Van der Waals), Gln-344/Asn-30 (hydrogen bond). Few binding sites exist between ZIKV/ZV-48 V_H CDR: Thr-351/Leu-30 (Van der Waals), Thr-351/Gly-96 (Van der Waals), Asp-384/Asn-56 (hydrogen bond). None of the heavy-chain binding sites are conserved in ZV-64. Compared to other Zika-specific antibodies that neutralize ZIKV H/PF/2013 infection, ZV-48 exhibits reduced inhibitory activity against other strains of virus on account of its lower binding affinity for recombinant proteins and shorter half-life (1).

The heavy chain of ZV-48 is structurally similar to that of anopheline anti-platelet protein (AAPP) with 8H7 Fab antibody (PDB ID: 4OKV), an anti-coagulant that inhibits blood aggregation without prolonging duration of bleeding (9). The primary structure of ZV-48 was compared to AAPP-8H7 using the Position-Specific-Iterated Basic Local Alignment Search Tool (PSI-BLAST). PSI-BLAST assigns an E-value to proteins based on their sequence homology. An E-value below 0.5 indicates significant similarities in primary structures of proteins. The heavy chain of AAPP-8H7 showed an E-value of 6e-57 (7). The tertiary structure of ZV-48 was compared to that of AAPP-8H7 using the Dali Server. The Dali Server uses a sum-of-pairs method to compare intramolecular distances between proteins and assign a Z-score. A Z-score above 2 indicates significant similarities in tertiary structures of proteins. The heavy chain of AAPP-8H7 showed a Z-score of 23.0 (8).

AAPP-8H7 is an anti-coagulant protein generated in Anopheles stephensi that directly binds type-I and type-III collagen at sites of vascular injury to prevent interaction with platelet receptors, internal calcium release, and subsequent platelet aggregation (9). The protein has a molecular weight of 54594.07 Da and an isoelectric point of 6.06 (3). The AAPP domain consists of two α-helices connected by a turn region. The protein is stabilized by disulfide bonds at the N-terminal and C-terminal regions but is still sensitive to proteolysis. AAPP was complexed with 8H7 monoclonal antibody to stabilize the protein and allow crystallization. The 8H7 domain follows the pattern of basic antibody structure with antiparallel beta-barrels and antigen-binding sites on the heavy and light chain CDRs. The antibody interacts with the turn region of AAPP, leaving the helical regions intact. Nevertheless, AAPP-8H7 is significantly less effective at inhibiting platelet aggregation than free AAPP (9). Residues 96 through 98 of AAPP-8H7 are part of a helical structure absent in ZV-48. Apart from these, the first 110 residues of ZV-48 participate in the same secondary antiparallel beta-sheets as those in AAPP-8H7, though with some substitutions. Notably, substitutions were frequent at transition sites between secondary structures and in the linker peptides, or residues 26 through 33 in ZV-48 (7).