NEURAMINIDASE

Yen LaiCHE 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 -ketosidic linkageα 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).

-Sheetsβ predominate the secondary level of protein conformation whereas three or four tiny -helices in the structure insignificantly contribute to any of the functionality. α

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?structureId=1L7F&bionumber=1

Structure• Mushroom shape• Head: four co-planar and roughly

spherical subunits• Stem, cytoplasmic, transmembrane

1L7F

“Molecule of the Month”May 2009

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, N-acetyl, 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.

Active site: Arg118, Asp151, Arg152, Arg224, Glu276, Arg292, Arg371, and Tyr406,

[4]

R292

E276E151

R118

H274

Mutant: R292K, E119G, H274T R292K: BCX-1812< oseltamivir, = zanamivir

E119G: BCX-1812< zanamivir

Thank you!

References[1] The Biosphere: Life on Earthhttp://www.ucmp.berkeley.edu/alllife/threedomains.htmlaccessed on 9/20/14[2]

http://www.virology.ws/2013/11/05/the-neuraminidase-of-influenza-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.