N9 Neuraminidase-NC41 FAB Complex (PDB ID: 1NCA) from Anous minutus
Created by: William Ball
The
three known types of Influenza viruses are A, B, and C. Influenza A and B are similar in both
structure and function, whereas Influenza C differs in receptor-destroying
enzymes. The influenza virus has two
primary surface antigens, neuraminidase (NA) and hemagglutinin (HA) (1,
2). The neuraminidase portion represents
only 5 to 10% of the virus protein. It
is shaped like the head of a mushroom, protruding from the surface of the virus.
NA is further differentiated in to 2 groups of serotypes, group 1 being N1, N4,
N5, N8 and group 2 being N2, N3, N6, N7, N9. These subtypes are separately
classified based on differing structural characteristics and because they were
found to have no antigenic cross-reactivity (2). The Influenza Virus N9 Neuraminidase and its
antigen-binding fragment (FAB) complex with NC41 antibody (PDB: 1NCA) is one of
the 9 serotypes that will be further analyzed.
N9 Neuraminidase is an antigen protein found on the surface of Influenza A virus,
an avian flu virus isolated from Tern (Anous minutus) (3). The NA on the Influenza A virus is classified
as a hydrolase, meaning it tends to catalyze the removal of terminal sialic
acid from viruses and other glycoconjugates.
In doing so, it helps to facilitate the release of the virus' genetic
material, spreading the virus (1). The
total weight of the N9 Neuraminidase and the NC41 complex is 96,418 daltons,
with the neuraminidase subunit being 52,469 daltons and the immunoglobulin (Ig)
gamma-2A chain subunit being 43,949 daltons.
The isoelectric point of the N9 Neuraminidase is 6.38 (4).
In
general, neuraminidase is a classified as a homotetramer, meaning it has 4
identical subunits arranged circularly.
The subunits are usually spherical and are connected by a 'stalk' made
of amino acids in the center of the 4 subunits.
This stalk attaches the NA homotetramer to the surface of the Influenza
virus. The stalk of the mushroom-shaped complex is comprised of a hydrophobic
region located at the N-terminus; it is followed by 6 polar amino acids, each
which, among the 9 serotypes of NA, exhibit a high degree of conservation (1).
The four identical subunits of the NA contains a total of six, tetra-stranded
anti-parallel beta-sheets; these six centralized beta-strands make up the an
active site in the protein. The
structure of the beta sheets is generally twisted, where both the first and
central strands are parallel with respect to each other. They are also both perpendicular in regards to
the outer beta-strand. Loops, containing
functionally vital binding sites, connect the outer beta-sheets to the central
sheet. After transcription of a
neuraminidase, the methionine residue that initiates it is retained, there is
no signal peptide splitting, no C-terminal processing, and no subsequent
cleavage (1).
The monomeric N9 Neuraminidase-NC41
complex is comprised of 3 subunits: N9 Neuraminidase, IGG2A light
chain, and IGG2A heavy chain. The N9 Neuraminidase portion of the complex is 3% helical, made up of 4 alpha helices,
composed of 12 residues. It is 43% beta sheet, with a total of 29 strands
containing 169 residues. It also
contains random coils (5). This subunit
functions as a sialidase, cleaving terminal alpha sialic acid groups from
polysaccharides, glycoproteins, and glycolipids (3). The IGG2A light chain of
the NC41 complex is 5% helical with 4 helices made up of 12 residues, and 52%
beta sheets with 23 strands made up of 115 residues. The IGG2A heavy chain of the NC41 complex is
5% helical, composed of 3 alpha helices containing 12 residues, and 50% beta
sheets, composed of 21 strands containing 108 residues (5). The Ig Gamma 2A chains on the NC41 antibody,
found in Mus musculus (house mouse), interact selectively and
non-covalently with an antigen in order to induce a specific immune response
(3).
The
N9 Neuraminidase subunit is made up of 389 residues. Arg-327 and Ser-367 are both found on random
coils of the NA structure, yet are important residues in the protein
itself. Both of these residues form
hydrogen bonds that help to stabilize the FAB complex formed with the NC41
antibody. Asn-400 is another important
residue which hydrogen bonds to form a portion of the FAB complex; however, it
is found on one of the protein's beta sheets. Lys-432, found on a turn in the protein's structure, forms a buried salt
link in the center of the NA with the calcium ion (3).
There
are two different types of hydrogen bonds that are involved in the
Neuraminidase and the NC41 FAB complex: intra-molecular and intermolecular. There are between 10 and 15 important
hydrogen bonds found in the Neuraminidase-NC41 FAB complex. Two intra-molecular H-bond in the
neuraminidase portion of the complex are between Ser-370 and Ser-367, and Arg-327 and Ile-368. These H-bonds allow
the NA to maintain its shape, as to allow it to bind with the IGG2A heavy and
light chains in the NC41 antibody.
Intermolecular hydrogen bonds between the NA and NC41 form between
Tyr-49 and Arg-327, and Glu-96 to Ser-367.
These H-bonds, in addition to the van der waal's forces that are
present, allow the NA on the antigen-binding protein to affix to the NC41
antibody (3). By forming these hydrogen
bonds and allowing the complex to form, signals can be transmitted across the
complex to incite a specific immune response.
When the complex forms in an infected mouse, the antibody is either able
to recognize that the virus is foreign and attack it, or it can’t recognize the
NA as coming from a foreign virus and can detach from it, leaving it unscathed. This depends upon the immune system of the
mouse. In addition to the N9
Neuraminidase forming a FAB complex with NC41 antibody, there are also between
zero and three buried salt links found deep in the NA-NC41 complex, binding the
neuraminidase to a calcium ion with a +2 charge (3, 5). The calcium ion is able to bind to the active
sites: Asp-293, Gly-297, Asp-324, and Asn-347 found on NA (5). The presence of
the calcium ion in the center of the NA is necessary for thermostability of the
Influenza virus A.
There
are three types of ligands found in the N9 Neuraminidase-NC41 FAB complex:
N-Acetyl-D-Glucosamine (NAG), Beta-D-Mannose (BMA), and Alpha-D-Mannose
(MAN). N-Acetyl-D-Glucosamine, Beta-D
Mannose, and Alpha-D-Mannose are a carbohydrate glycans that are generally used
in cell signaling and are believed to prevent the enzymatic cleavage and
glycosylation of the certain proteins found in the stalk of the neuraminidase
(4). There are four separate NAG molecules that are present in the NA. NAG N 475 binds to the NA at the active sites
Asn-86, with the residues Asp-83, Asn-234, Val-308 and His-233 forming the
binding pocket. The NAG N 476 binds to
the NA at Asn-146 with Trp-437 helping to form the binding pocket. NAG N 469 binds to the NA at the active sites Asn-199, Asn-200, and Arg-220. These residues form the ligand binding pocket
for the following chain of ligands: NAG N 470 binds to the NAG N 469, which
then binds to a single molecule of BMA N 471 and then to 3 molecules of MAN:
MAN N 472, MAN N 473, MAN N 474 (5).
Programs
such as PSI-BLAST (n-BLAST for nucleic acids) and the Dali Server are used to
help identify different proteins to compare with a specific protein, such as
the N9 Neuraminidase. PSI-BLAST and
n-BLAST are used to find proteins and nucleic acids, respectively, which have
similar primary structures to the protein or nucleic acid that was input. The differences in primary structure are
noted with E values. An E value of less
than 0.5 is ideal, meaning there are only very small differences in the
proteins' primary structures (6). The
Dali Server, on the other hand, is a process that is used on proteins to find
other proteins that have similar tertiary structures to a given protein. It determines similarities by using a sum-of-pairs
method that compares intra-molecular distances, giving a Z-score. 2 or above is a valid Z-score (7).
After
analyzing N9 NA-NC41 FAB complex with both PSI-BLAST and Dali Server the comparison protein, Plasmodium falciparum reticulocyte-binding protein
homologue 5 (PfRH5), was obtained. PfRH5
(PDB ID: 4U1G), unlike N9 NA which was isolated from Australian tern, was
isolated from Oryctolagus cuniculus, or more commonly known as a
rabbit. The E value obtained when
running PSI-BLAST on N9 NA vs PfRH5 was 7E-150, falling under the ideal value
of 0.5 (6). The Z-score obtained with
Dali Server between the two proteins was 24.5, which is considerably larger
than 2 (7). After analyzing these values and the two proteins, it can be
determined that both proteins have similar primary structures and tertiary structures. However, when comparing the
structures of N9 Neuraminidase with Plasmodium falciparum
reticulocyte-binding protein homologue 5, it can be seen that N9 Neuraminidase
is much larger in size, with unit cell lengths of 167.0 angstroms by 167.0
angstroms by 124.0 angstroms, whereas PfRH5 is only 65.12 angstroms by 137.3
angstroms by 228.58 angstroms. However,
PfRH5 is much heavier than N9 Neuraminidase at 234,412.40 daltons, while N9
Neuraminidase is only 96,418 daltons (8, 9).
It also appears that PfRH5 is made up of more alpha helices, while N9
Neuraminidase contains more beta sheets.
N9 NA also has ligands that allow the protein to antibodies and illicit
specific immune responses, whereas PfRH5 doesn't require ligands.
P. falciparum reticulocyte-binding protein-like homolog is a group of proteins
that are classified as parasitic antigens.
These proteins play a vital role in the invasion of red blood cells
which causes immunity to malaria. After
detailed genetic testing, it was determined that PfRH5 is the only protein in
the group of antigen binding proteins that is not able to be genetically deleted. Because of this, scientists believe that it
plays a significant role in the specific immunity to malaria (10). Though it involves viruses instead of
bacteria, N9 Neuraminidase is fairly similar in function to PfRH5. N9 NA functions as a binding protein found on
an antigen on the surface of Influenza A virus.
It is able to bind with antibodies, such as NC41, and cleaves terminal
alpha sialic acid groups of polysaccharides, glycoproteins, and
glycolipids. By doing this, it complexes
with the antibody, and elicits specific immune responses from whatever the
virus is invading (11).
Because
subtype N9 Neuraminidase is found on the surface of Influenza A virus, it has
become the focus of scientific research, in order to prevent viral
transmission. Scientists have developed
a class of drugs known as neuraminidase-inhibitors. These drugs, such as oseltamivir (Tamiflu)
and zanamivir (Relenza), work by
binding to the neuraminidase and forming a complex which inhibits the virus
from transmitting and replicating.
Oseltamivir complexes with Group 2 Neuraminidases, such as N9, around
the ‘150-loop’, comprised of residues 147–152, and the ‘150-cavity’, which is
adjoining the active site. In the group 2 conformations of the NA, the C alpha
position of the 150-loop is approximately 7A? from the isoluecine 149
residue. In addition, there is a
hydrophobic side chain located at residue 149 that points toward the active
site. In this active site, the Arg-292
residue on the NA binds to the carboxylate group of the oseltamivir. That being
said, however, there are resistances in the N9 NA that are forming to
oseltamivir and other NA Inhibiting drugs.
In the case of oseltamivir, a Lysine is substituted for Arginine at
residue 292. This substitution interacts with Glu-276 on the NA, thus hindering its ability to move. This process prevents the hydrophobic
substituent from attaching to C6 of the oseltamivir. Though there are resistances forming in the
NA Inhibiting drugs, scientists are still searching for new ways to prevent the
deadly spread of Influenza viruses (12).