ViralNeuraminidaseN5

Viral Neuraminidase N5

Created by Alice Dallstream

   Viral neuraminidase N5 is a protein located on Influenza A viral strains(1,2). Strains of Influenza A using N5 typically are avian(1,2,3). N5 is part of a family of nine neuraminidases(2). Neuraminidase (NA) is involved in the release of new virons from infected cells and prevents virus particles from aggregating. Specifically, NA cleaves sialic acid from glycoproteins containing hemagglutinin(2,3,4). Therefore, NA has been long established as a drug target in guarding against the flu, and several vaccines are in use. Neuraminidases are targeted by the drugs zanamivir and oseltamivir. Recently, this has become increasingly important with the pandemic of H1N1 swine flu in 2009. Studying N5 and other neuraminidases is of vital importance in global medicine for the treatment of all strains of influenza.

   When the sequence of N5 (3SAL) is subjected to a protein BLAST against “Protein Data Bank proteins,” there are many homologs. The closest match is N8 Neuroaminidase with an E value of 0.0 and query coverage of 99%(5). This result is not surprising since N8 and N5 are both contained in Group 1 of the NA family(2-4). The NAs can be separated into two groups based on their active sites, where group 1 is N1, N4, N5, and N8 and group 2 is N2, N3, N6, N7, and N9 (2,3,4). In fact, it has been determined that N8 and N5 are the closest to each other evolutionarily(1,3,4). Another homolog of N5 that is slightly less related, but of medical importance is N1 NA of the 2009 H1N1 strain of swine flu (PDB ID: 3NSS)(5). Their match has an E value of 1 *10^-164 and a query coverage of 98.

   N5 is 395 residues long1. It has eight different disulfide bonds between cysteines in its primary structure. These are between Cys92-Cys417, Cys124-Cys129, Cys183-Cys230, Cys232-Cys237, Cys278-Cys291, Cys280-Cys289, Cys318-Cys337, and Cys421-Cys447. The secondary structure is composed of 3% helices and 45% beta sheets. Overall, N5 is a box-shaped tetramer with four identical subunits(2). Each of these subunits is composed of six four-stranded, anti-parallel beta sheets that form a propeller like arrangement. NAs are surface glycoprotiens(2,3,4), and as such N5 has two N-linked glycolysation sites in the globular head region at Asn 93 and Asn146(2).

   The predominant area of interest in N5 is its active site, mostly because it is a drug target(2). N5 is like other group 1 NAs in that it has a 150-loop and cavity. This allows N5 to have an open conformation and change to a closed conformation when ligands, substrates, or inhibitors bind. The two most important residues involved in binding sialic acid or drugs are Asp151 and Tyr 347. Asp151 will form a hydrogen bond to the drug zanamivir at its 4-guandino region. This causes Asp151 to move 2.04Ᾰ toward the binding site. Meanwhile, Tyr347 will form a hydrogen bond with zanamivir and possibly oseltamivir, another drug, at their carboxylate group. Additionally, Tyr347 will form a bond with calcium as Asp293, Gly297, Asp324, and two water molecules do the same. Only one calcium ion is found in each active site of N5. Tyr347 is highly conserved in avian strains, but is usually Glu, Asp, His or Asn in human strains of influenza. It is hypothesized that this conservation in avian strains is due to the structure of surface sialic acid in birds.

   N8 is 390 amino acids long with a PDB ID of 2HT5(1,5). N8 has an almost identical 150-loop region to N1 and N43. On the other hand, N5 differs from N8 in that it has extended 150 loop region due to Asn147(2). This results in a more open loop conformation. Additionally, N5 has only one calcium site per monomer, while all other NAs, including N8, have two. Even with these differences and the fact that N8 is shorter than N5, N8 and N5 are incredibly similar. Most group 1 NAs have two calcium ions, and some three. The cavity extension seems to be due to a change of Gly147 to Asn147 resulting in several conformational changes. In N5, the Asn146-Asn147 peptide is rotated in relation to other group 1 NAs. The peptide nitrogen of this group will then hydrogen bond with the peptide carbonyl of Ile437-Trp438, the peptide carbonyl of Asn146-Asn147, and the peptide nitrogen of Asn147-Thr148. Additionally, Asn147 will hydrogen bond with Thr439. In other group 1 NAs, the Asn146-Gly147 peptide carbonyl and the Gly147-Thr148 peptide nitrogen form a hydrogen bond to stabilize the loop conformation. The N5 loop is more open than other group 1 NAs. One last difference between N5 and the rest of group 1 is that it only has two glycolysation sites rather than three. Most group 1 NAs have three N-linked glycans at Asn88, Asn146, and Asn234.

   While N5 and the other group 1 NAs have 150-loop cavities, the group 2 NAs do not have such a loop(2,3,4). Most group 1 NAs have a cavity that is about 10Ᾰ long, 5Ᾰ wide and 5Ᾰ deep(3). One could even characterize the group 1 NAs as being open and the group 2 NAs as being closed(4). The group 2 NAs have conserved active sites that hydrogen bond to the carboxylate group of the substrate sialic acid(3).  Additionally, group 2 NAs have Ile149, while group 1 NAs have Val149. The Val149 is 7Ᾰ more open than the Ile149 of group 2.

   In a special case, H1N1 somewhat blurs the distinction between groups 1 and 2(4). H1N1 lacks a 150-loop cavity even though it is a member of group 1. This is due to a change of Val149 to Ile149. This causes a more rigid conformation in the closed position, similar to group 2 NAs. However, this loop likely is much more easily opened than group 2 NAs(2).

   Viral NA N5 is a protein that is integral to the treatment of influenza; its active site is a drug target, because it has the function of cleaving sialic acid, facilitating viral reproduction(2,3,4). N5 is only one of nine serotypes of NA. These serotypes are highly conserved(2,3,4,5) to the point where all E values in BLAST comparing NAs to N5 are in the magnitude less than -60(5), and most differences occur at the active site(2,3,4). The structures of all serotypes of NA are therefore thoroughly studied for continued treatment against the influenza virus.