N1NeuraminidaseH274Y

N1 Neuraminidase H274Y (pbd ID: 3CKZ) from Alphainfluenzavirus influenza A virus (H5N1) is a mutated protein and is not found in other wild-type influenza viruses and has a singular subunit. Influenza neuraminidase is found on the exterior of the virus and functions by removing sialic acid from cell-surface receptors so that newly formed virus cells can detach from their host cells and propagate throughout a host. The N1 Neuraminidase H274Y (NA) mutated protein has allowed Influenza A to become resistant to Oseltamivir, a popular antiviral medication. Influenza A's resistance to Oseltamivir is owed to an altered hydrophobic pocket in the enzyme's active site that is necessary for Oseltamivir binding. N1 neuraminidase H274Y is currently being studied to determine the effectiveness of Zanamivir, another antiviral medication, to determine if Zanamivir is more effective in neutralizing the mutated virus (1).

N1 Neuraminidase H274Y was crystallized using the sitting drop vapor diffusion method dispensed from an Oryx 8 robot (Douglas Instruments). To do so, the protein mixture (6mg/mL) with 1 mM of Zanamivir was mixed with 100 nL reservoir solution 15% PEG 3350 and 0.1M sodium acetate pH 4.6. Once the crystals formed, they were then transferred to a cryoprotectant reservoir solution augmented with 20% (v/v) PEG 400 and an inhibitor before flash freezing (1). N1 Neuraminidase H274Y has a molecular weight of 42,582.33 Da (2) and an isoelectric point of 5.71 (3). NA has one primary domain that can be crystalized, the catalytic head. The catalytic head is composed of four subunits. Each of the four subunit monomers is a six-bladed propeller structure and all four have antiparallel beta-sheets that are stabilized by disulfide bridges. Each monomer has a functional active site on its surface that is pointed to the side rather than upwards. The total residue count for N1 Neuraminidase H274Y is 385 residues. A discrepancy was derived by the vapour diffusion method used, which introduced a molecule of Ca2+ into the structure of the enzyme, which is not there in-vitro. There were two discrepancies between the modelled data and the enzyme in-vitro. Modeled at residue 252 is Tyr rather than His; and at residue 274 Tyr is modelled rather than His (4). NA has one instance of 3/10 random coils and two instances of alpha helices (5). The three-dimensional structure for the cytoplasmic, stem, and transmembrane domains have not yet been determined because the enzymes used for cleavage of the membrane protein from the virion, and thus are not able to be crystalized. However, it is believed that an alpha-helix exists in the uncrystallized sections of the protein (6).

Unlike other NA enzymes, N1 Neuraminidase H274Y has a substitution of tyrosine at position 274. The substitution alters the function of the active site because the bulkier Tyr in position 274 pushes the carboxyl group of Glu-276 2Å further into the enzyme's binding site. The charged carboxyl group being pushed further into the binding site disrupts the normally hydrophobic pocket that accommodates the pentyloxy substitute of Oseltamivir. Disrupting the hydrophobic pocket results in C9 and C91 being moved 2.5Å from their position in a wild-type protein, which further disallows Oseltamivir to bind to the mutated protein. The NA's active site consists of active binding sites at Arg-118, Asp-151, Arg-152, Arg-224, Glu-276, Arg-292, Arg371, and Tyr406 on the outer-shell of the protein that directly interact with sialic acid; and 10 outer shell residues that do not interact with sialic acid, which are Glu-119, Arg-156, Trp-178, Ser-179, Asp-198, Ile-222, Glu-227, Glu-277, Asn-294, and Glu-425. Arg-118,Arg-292, and Arg-371 interact with the carboxylate of the sialic acid substrate in-vitro, whereas Arg-152 binds to the acetamido group located on the sugar ring. Glu-276 interacts with the 8-hydroxyl and 9-hydroxyl groups on the glycerol sidechain (1,4). Significant hydrogen bonds exist at Tyr-347 and Glu-276, which stabilizes the protein folds. Important ligands for N1 Neuraminidase H274Y include Zanamivir and Ca2+. Zanamivir interacts with the binding pocket located at Glu-276, forming hydrogen bonds and permitting the drug complex to neutralize the virus.Ca2+ bonds to Tyr-347 during the crystallization process and helps in forming the protein crystals (1).

Because neuraminidase proteins are found in many strains of the influenza virus, PSI-BLAST and the Dali server were used to find similar structures so that they may be compared to N1 neuraminidase H274Y. PSI-BLAST searches for proteins with similar sequences in a protein sequence database and compares them based on similarities and gaps in the sequences. An E value is then assigned to the comparison protein: the lower the E-value, the more similar the genetic sequences. The optimal E-value is below 0.05 and an E-value of 0 means that the proteins are identical. Dali server searches for proteins with similar intramolecular distances, which also usually finds proteins with similar folds. It used a sum-of-pairs method to assign Z-scores to comparison proteins: the higher the Z-score, the more similar the proteins. The optimal Z-score is any value above 2.

One of the most similar proteins found in another species of virus was neuraminidase H275Y (pdb ID: 5NWE) from Alphainfluenzavirus influenza A virus (H1N1), which is also a mutant-type protein. The Z-score for neuraminidase H275Y was 74.9 and its E-value was 1.3 x 10^-23 (7,8). The primary, secondary, and tertiary sequences for neuraminidase H275Y are the same as the sequences for N1 Neuraminidase H274Y, as are the binding interactions with Zanamivir and Ca2+, however, the location of the mutated gene is moved up one residue. The result of Tyr being substituted at position 275 rather than at 274 is the polar Tyr side chain pushing E227 into the hydrophobic binding pocket of the enzyme's binding site. E227 encroaching further into the binding pocket led to the terminal carbon (C91) being shifted 2Å from its original position, which differs from C91 and C9 being shifted. Similarly to how the mutation in N1 Neuraminidase H274Y resulted in a decrease in the effectiveness of Oseltamivir, Oseltamivir was far less capable in neutralizing the viral agent in neuraminidase H275Y. Important residues for neuraminidase H275Y include active binding sites at Asp-131 and Tyr-382; substrate bonding sites at Arg-338, Arg-348, Arg-273, Arg-132, and Arg-98; and a binding site at Asn-126 (9).

Viruses containing mutated genes pose a threat to society, which N1 Neuraminidase H274Y and neuraminidase H275Y have shown, because a single substitution in a protein creates a virus that is unable to be effectively neutralized with current medications that are stockpiled. If these strains of viruses were to spread throughout a population, current stockpiles of medications that would be effective in limiting the spread of the virus would be quickly exhausted (1). The importance in studying N1 Neuraminidase H274Y is that with further research, alternative medications can be found that are more effective in neutralizing the viral agent, such as Zanamivir. However, the issue associated with mutated viruses does not end with N1 Neuraminidase H274Y or neuraminidase H275Y because viruses are constantly evolving. Although some mutations do not change the function of a protein to the extent that the virus then becomes resistant to medications used to subdue them, the probability that it will never occur again is improbable. Therefore, it is crucial that further research be applied to both the search for these mutated viruses and the search for medications that can effectively cease the virus's propagation. Further study in the secondary and tertiary sequences may also yield more valuable information into the function of all neuraminidase proteins, which could lead to further understanding of the enzyme and better means of protecting the public against influenza viruses.