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NEURAMINIDASE
Yen Lai
CHE 442
Nov 13th, 2014
NAs in Virus Life Cycle
Neuraminidases (NAs) are glycoside hydrolase enzymes cleaving the glycosidic linkages of
neuraminic acids, found in many species in all three domains of life, bacteria, archaea, eukarya.
Hemagglutinin and neuraminidase are two molecules that cover the surface of influenza virus,
playing crucial roles in the infectivity of the virus. As the virus particle approaches a cell
hemagglutinin binds to polysaccharide chains on the cell surface, and then the viral genome is
injected into the cell. On the other hand, neuraminidase clip off the ends of these polysaccharide
chains (break α-ketosidic linkage between the sialic (N-acetyl-neuraminic) acid and an
adjacent sugar residue) to liberate a nascent virion from the cell receptor after the previous viral
particle infected the cell.
In fact, NA executes several functions at different stages of infection. Firstly, it helps the virus
approach the target cells by cleavage of sialic acids from respiratory tract mucins. Secondly, it may
take part in the fusion of viral and cell membranes. Thirdly, it facilitates budding of new virions by
preventing their aggregation, caused by the interaction of the HA of one virus with the sialylated
glycans of another.
http://publications.nigms.nih.gov/findings/mar06/agbandje-mckenna_files/textmostly/slide4.html
http://journals.prous.com/journals/servlet/xmlxsl/pk_journals.xml_summary_pr?p_JournalId=2&p_RefId=923098&p_IsPs=N
Sialic acid had been shown to be the cellular receptor for influenza viruses. Neuraminidase is a family
of the receptor destroying enzymes of influenza viruses acts as a sialidase, releasing sialic acids from
macromolecules.
In humans the brain has the highest sialic acid concentration where they have an important role in
neural transmission and ganglioside structure in synaptogenesis.
Sialic acid-rich glycoproteins (sialoglycoproteins) bind selectin in humans and other organisms.
Metastatic cancer cells often express a high density of sialic acid-rich glycoproteins. This
overexpression of sialic acid on surfaces creates a negative charge on cell membranes. This
creates repulsion between cells (cell opposition) and helps these late-stage cancer cells enter the
bloodstream.
R2 = H; alpha linkage to Gal(3/4/6),
GalNAc(6), GlcNAc(4/6), Sia (8/9),
or 5-O-Neu5Gc; oxygen linked to C-7
in 2,7-anhydro molecule; anomeric
hydroxyl eliminated in Neu2en5Ac
(double bond to C-3).
Wikipedia, access on Nov 8, 2014
Varki, Ajit, et al. "Sialic acids." (2009).
Overall Structure: Quaternary, Tertiary, and
Secondary
NA resembles a homotetramer of a mushroom shape with a head of 80*80*40 Å on a thin
stem, 15 Å wide and from 60 to 100 Å long.
Cytoplasmic and transmembrane portions are undetermined. A tetramer is ~240 kDa in
molecular mass (each monomer weighs ~60 kDa).
β-Sheets predominate the secondary level of protein conformation whereas three or four
tiny α-helices in the structure insignificantly contribute to any of the functionality.
NA folds into up-and-down beta sheets in an antiparallel pattern. Folding motifs form a
propeller-like structure. There are a number of loops connecting the motifs and loops
between every second and third strain of each motif. They contain many amino acid residues
important to catalytic activity. They are the variant of the NA structure in length and even in
secondary structure element.
http://www.rcsb.org/pdb/explore/
remediatedSequence.do?structure
Id=1L7F&bionumber=1
Structure
•
•
•
Mushroom shape
Head: four co-planar and roughly
spherical subunits
Stem, cytoplasmic, transmembrane
“Molecule of the Month”
May 2009
1L7F
Amino Acid Sequence
Influenza virus neuraminidases are distinct from the other isoforms.
The N-terminal of the Asn side chain forms glycosidic bond to carbohydrate chain of the lipid
membrane to anchor the protein on the cell surface.
Generally, Asparagine residues are strictly conserved in NAs in order to perform
glycosylation, especially Asn 146. Besides, proline and cysteine residues are crucial for
folding of the polypeptide chains and maintaining the three dimensional structure of the
molecule. There are eight invariant disulfide bonds formed by cysteine/threonine to
stabilize the structure of NA.
Amino acid residues Arg118, Asp151, Arg152, Arg224, Glu276, Arg292, Arg371, and
Tyr406 are essential constituents of the enzyme active site whilst Glu119, Arg156,
Trp178, Ser179, Asp (or Asn in N7 and N9) 198, Ile222, Glu227, Glu277, Asp293, and
Glu425 are structurally significant.
Hydropathy Index
MNPNQ KIITI GSICL VVGLI SLILQ IGNII SIWIS HSIQT GSQNH TGICN QNIIT YKNST
WVKD TTSVI LTGNS SLCPI RGWAI YSKDN SIRIG SKGDV FVIRE PFISC SHLEC RTFFL TQGAL
LNDRH SNGTV KDRSP YRALM SCPVG EAPSP YNSRF ESVAW SASAC HDGMG WLTIG ISGPD
NGAVA VLKYN GIITET IKSWR KKILR TQESE CACVN GSCFT IMTDG PSDGL ASYKI FKIEK GKVT
KSIEL NAPNS HYEEC SCYPD TGKVM CVCRD NWHG SNRPW VSFDQ NLDYQ IGYICS GVFGD
NPRPK DGTGS CGPVY VDGAN GVKGF SYRYG NGVW IGRTK SHSSR HGFEM IWDPN GWTE TDSKF
SVRQD VVAMT DWSG YSGSF VQHPE LTGLD CIRPC FWVEL IRGRP KEKTI WTSAS SISFC GVNSD
TVDWS WPDGA ELPFT IDK
Amino Acid Alignment
Amino Acid Alignment
E119
R156
W178
E425
NA Inhibition and Influenza Drugs
Many of antiviral drug molecules for influenza are NA inhibitors (e.g. zanamivir and
oseltamivir are already used as drug products, whereas BCX-1812 has entered the last
phase of clinical trials), which mimics the transition state of the hydrolysis reaction.
The functional groups common between BCX-1812 and zanamivir, the carboxylate, Nacetyl, and guanidinium groups, all have the same relative positions in the active site. The
carboxylate forms salt bridges with the anidinyl groups of R118, R371, and R292. The
carbonyl oxygen of the N-acetyl group forms a hydrogen bond with a distal nitrogen atom
of the side chain of R151, while the amide nitrogen forms a hydrogen bond with a water
molecule at the base of the active site. The guanidinium group interacts with the acidic
groups E119 and E227.
Despite of the sophistication of drug molecule design, the virus has the ability to develop
some mechanism to defense for itself. R292K, E119G, H274T are some frequently observed
mutants in influenza virus NA leading to drug resistance.
R292
E151
E276
H274
R118
[4]
Mutant: R292K, E119G, H274T
R292K: BCX-1812< oseltamivir, = zanamivir
E119G: BCX-1812< zanamivir
Active site:
Arg118,
Asp151,
Arg152,
Arg224,
Glu276,
Arg292,
Arg371,
and Tyr406,
Thank you!
References
[1] The Biosphere: Life on Earth
http://www.ucmp.berkeley.edu/alllife/threedomains.html
accessed on 9/20/14
[2]http://www.virology.ws/2013/11/05/the-neuraminidase-ofinfluenza-virus/
Accessed on 9/20/14
[3] Shtyrya, Y. A., Mochalova, L. V., & Bovin, N. V. (2009). Influenza
virus neuraminidase: structure and function. Acta naturae, 1,
26.
[4] Smith, B. J., McKimm-Breshkin, J. L., McDonald, M., Fernley, R.
T., Varghese, J. N., & Colman, P. M. (2002). Structural studies of
the resistance of influenza virus neuramindase to
inhibitors. Journal of medicinal chemistry, 45, 2207-2212.