Hemagglutinin

Created by: Sherry He

Hemagluttinin (PDB ID: 4HLZ) is an antigenic glycoprotein on the surface of influenza viruses that is responsible for binding the virus to the targeted cell. Influenza causes respiratory infection in humans that result in severe diseases and millions and high mortality rates during pandemics (1). Hemagluttinin (HA) is the primary target for neutralizing antibodies (Abs) and vaccine design, making it an important protein to study. The mouse antibody C179 was found to target an epitope on the HA stem and inhibit the low-pH conformational change of the HA. C179 can recognize an epitope on the conserved membrane-proximal stem region of the HA by neutralizing the virus inhibiting the fusion process. It can inhibit the low-pH conformational change of the HA by utilizing both its heavy and light chains (1). Typically, the low pH of endosomes, membrane-bound vesicles, trigger conformational changes in HA that mediate fusion of the viral and endosomal membranes, but the C179 antibody uses both its heavy and light chains to inhibit the conformational change (2). The light chain contributes little to no direct contacts with the antigen, but modulates the neutralizing spectrum by affecting the local conformation of the heavy chain (3). Furthermore, antibody C179 was the first anti-HA to cross-neutralize multiple influenza virus subtypes, including H1, H2, H5, H6, and H9 viruses (1).

Hemagglutinin from Mus musculus was crystallized to 2.9 angstrom resolution using the vapor diffusion sitting drop method at a pH of 8.5 and 293K. The crystal structure was formed in the presence of 2M ammonium sulfate and 0.1M Tris-HCl with a pH of 8.5 (2). HCl, NaCl, and ammonium sulfate ligands were present in the crystal structure to induce crystallization. The structure of hemagglutinin was obtained through X-ray diffraction (1, 2).

The hemagglutinin structure has a molecular weight of 319,540 Da (1, 3). The isoelectric point is at a pH of 5.02 (4). There are four unique subunits in hemagglutinin: hemagglutinin HA1, hemagglutinin HA2, Fab C179 heavy chain, and Fab C179 light chain (2, 5). Hemagglutinin HA1 consists of three identical chains making up the globular head region. Hemagglutinin HA2 is a long helical chain with 28 amino acids anchored in the membrane followed by a 10-residue cytosolic tail (2). The Fab C179 heavy chain consists of three identical chains located at the end of the HA2 chain in three groups. The Fab C179 light chain consists of three identical chains located adjacent to the Fab C179 heavy chains (2, 5). Important domains include the membrane-distal receptor binding domain and the C179 VH domain. The membrane-distal receptor binding domain (HA1, "head") is located at the top of a largely alpha-helical membrane fusion region (HA2, "stem") which is important for antibody recognition (1). The C179 VH domain binds using heavy-chain to form a linear cluster of hydrophobic residues that is complementary to the hydrophobic groove just below the HA head to interact with surrounding polar residues (1). Hemagglutinin has a total of 2,832 residues (2). Parts of the protein were not represented in the whole structure. This includes unobserved sequences at position 325-327 of 4HLZ.A, 4HLZ.C, and 4HLZ.E; position 62-65 and 173-174 of 4HLZ.B; position 62-63 and 173-174 of 4HLZ.D; position 173-174 of 4HLZ.F; position 224-229 of 4HLZ.G; and position 1 and 223-229 of 4HLZ.I (2).

Hemagglutinin HA1 chain contains five alpha helices, two 3/10 helices, and 32 beta strands in its secondary structure. Hemagglutinin HA2 chain contains five alpha helices, zero 3/10 helices, and eight beta sheets in its secondary structure (1, 2). The Fab C179 heavy chain consists of zero alpha helices, four 3/10 helices, and 17 beta strands in the secondary structure. The Fab C179 light chain consists of two alpha helices, one 3/10 helices, and 18 beta strands in the secondary structure (2). Fab C179 heavy and light chains bind to HA. The C179 heavy chain contributes 72% of the Fab buried surface area (1). All the subunits have hydrophobic regions in the middle while the polar, basic, and acidic regions are facing out. This allows the protein to recognize other molecules through the interactions, and mediate the entry of viruses (1). The overall structure of the protein resembles a rocket: the head of the rocket is represented by hemagglutinin HA1, the long body is represented by hemagglutinin HA2, and there are three fins to the rocket represented by the Fab C179 heavy and light chains together.

Hemagglutinin has 3 ligands: NAG, SO4, and EDO. NAG ligand aids in the treatment and prevention of osteoarthritis, either by itself or in combination with chondroitin sulfate (6). SO4 ligand increases large intestinal motility, inhibits large intestine fluid/electrolyte absorption, and is a laxative (7). EDO ligand is an osmotically acting laxative and can relieve constipation (8). Hydrogen bonds are important to the structure and function of the protein. Hemagglutinin has a hydrogen bond between Tyr-98 of Fab C179 light chain and Thr-318 of side chain HA1 (1). The light chain also contributes to the hydrophobic interactions where Trp-34, close to the linear cluster of hydrophobic residues, interact with the aliphatic portions of HA2 Lys-38 and Asp-19 (1). These interactions hold the four different subunits together in the protein. Other important residues include Thr-318, which is part of the HA1 chain that interacts with Phe-45 of the HA2 chain. Phe-99, Tyr-98, and Tyr-100 are part of a large hydrophobic residue at the tip of HCDR3 interacting with a second hydrophobic patch on the HA including Thr-318 and His-38. Furthermore, Tyr-100 functions to insert its aromatic side chain into the hydrophobic groove of HA (1). Trp-21 residue makes favorable interactions with HCDR2 hot spot residues Phe-54 and Phe-55 to modulate cross-group reactivity. Residue Val-52 in HA1 epitope overrides neutralization by C179 (1).

PSI-BLAST is used to find proteins with a similar primary structure to a protein query; an E-value of less than 0.05 is considered to be significant for proteins. The Dali Server is used to find proteins with similar tertiary structures to a query; a Z-score of more than 2 means that the protein has similar folds. The hemagglutinin protein with avian receptor (PDB ID: 2WR3) has an E-value of 0.0 from PSI-BLAST as compared to the query protein (9). This shows that the primary structure of the query and comparison protein is very similar. The Z-score between the two proteins is 35.8, meaning that the tertiary structure has many similar folds (10). Although the primary structure of the two were identical, the way the protein is folded could differ which is why the Z-score is not higher than it is. One difference of the comparison protein is that it only has one subunit with three identical chains, whereas the query has 4 unique subunits each with 3 identical chains (2, 5, 11). Furthermore, the chain in the comparison protein has five alpha helices, four 3/10 helices, and 27 beta strands (5, 11). The comparison protein is overall a smaller protein with a molecular weight of 172,730 Da (11). The hemagglutinin protein with avian receptor has a very similar structure to the query protein, however, its functions differ. The comparison protein has many more hydrogen bond networks through bound water molecules to mediate binding to human receptor. The human receptor has a very similar conformation in both human and avian hemagglutinin-receptor complexes that avian viruses can bind to both avian and human receptors (11). The functions of both proteins are relatively similar in that they both mediate the entry of influenza viruses.

Overall, the hemagglutinin protein is an important protein that is responsible for how viruses are spread. It is the key protein that binds viruses to cells, infecting humans and animals until it leads to more severe diseases and deaths. Every year, scientists develop an updated flu vaccine because the influenza virus is rapidly evolving to avoid recognition by the human immune system. Therefore, by studying the hemagglutinin protein, it could lead scientists to produce more broadly applicable vaccines for the future.