PDBsum entry 3ckz

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Viral protein, hydrolase PDB id
Protein chain
385 a.a. *
Waters ×187
* Residue conservation analysis
PDB id:
Name: Viral protein, hydrolase
Title: N1 neuraminidase h274y + zanamivir
Structure: Neuraminidase. Chain: a. Mutation: yes
Source: Influenza a virus
1.90Å     R-factor:   0.202     R-free:   0.228
Authors: P.Colllins,L.F.Haire,Y.P.Lin,J.Liu,R.J.Russell,P.A.Walker, J.J.Skehel,S.R.Martin,A.J.Hay,S.J.Gamblin
Key ref:
P.J.Collins et al. (2008). Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants. Nature, 453, 1258-1261. PubMed id: 18480754 DOI: 10.1038/nature06956
18-Mar-08     Release date:   20-May-08    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
Q6DPL2  (Q6DPL2_9INFA) -  Neuraminidase
449 a.a.
385 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 
  GO annot!
  Cellular component     membrane   3 terms 
  Biological process     carbohydrate metabolic process   1 term 
  Biochemical function     exo-alpha-sialidase activity     1 term  


DOI no: 10.1038/nature06956 Nature 453:1258-1261 (2008)
PubMed id: 18480754  
Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants.
P.J.Collins, L.F.Haire, Y.P.Lin, J.Liu, R.J.Russell, P.A.Walker, J.J.Skehel, S.R.Martin, A.J.Hay, S.J.Gamblin.
The potential impact of pandemic influenza makes effective measures to limit the spread and morbidity of virus infection a public health priority. Antiviral drugs are seen as essential requirements for control of initial influenza outbreaks caused by a new virus, and in pre-pandemic plans there is a heavy reliance on drug stockpiles. The principal target for these drugs is a virus surface glycoprotein, neuraminidase, which facilitates the release of nascent virus and thus the spread of infection. Oseltamivir (Tamiflu) and zanamivir (Relenza) are two currently used neuraminidase inhibitors that were developed using knowledge of the enzyme structure. It has been proposed that the closer such inhibitors resemble the natural substrate, the less likely they are to select drug-resistant mutant viruses that retain viability. However, there have been reports of drug-resistant mutant selection in vitro and from infected humans. We report here the enzymatic properties and crystal structures of neuraminidase mutants from H5N1-infected patients that explain the molecular basis of resistance. Our results show that these mutants are resistant to oseltamivir but still strongly inhibited by zanamivir owing to an altered hydrophobic pocket in the active site of the enzyme required for oseltamivir binding. Together with recent reports of the viability and pathogenesis of H5N1 (ref. 7) and H1N1 (ref. 8) viruses with neuraminidases carrying these mutations, our results indicate that it would be prudent for pandemic stockpiles of oseltamivir to be augmented by additional antiviral drugs, including zanamivir.
  Selected figure(s)  
Figure 1.
Figure 1: Neuraminidase activity monitored using a fluorescent assay. NA activity for wild type (yellow) and His274Tyr (green) mutant proteins in the absence (labelled 0) and presence (labelled I) of 85 nM inhibitor at 50 M substrate. a, b, Effect of oseltamivir (a) and zanamivir (b). For the His274Tyr mutant the approach to equilibrium occurs at a similar rate for the two inhibitors even though oseltamivir is the much poorer inhibitor. At the oseltamivir concentration used the reduced contribution of the on-rate constant to the observed rate is almost exactly compensated for by the increased contribution from the off-rate constant. Although oseltamivir has sometimes been referred to as a slow-binding inhibitor^21, ^30, the association rate constants that we determined are within the range frequently observed for the interaction of small molecules with proteins. It is the use both here and elsewhere of very low inhibitor concentrations that result in a slow approach to equilibrium, not the kinetics of binding.
Figure 2.
Figure 2: Structure of N1 neuraminidase complexes. a, Sialic acid (coloured blue) docked into the active site of wild-type N1 NA (ribbons coloured yellow) from superposition of the sialic acid complex of N2 (ref. 24) (Protein Data Bank code 2BAT). The positions of some key binding residues are shown with carbons coloured yellow, nitrogens blue and oxygens in red. The side chains of the three mutants examined in this work are shown with their carbons coloured in green (shown as the wild-type residues). b, The structures of sialic acid (carbons coloured blue), zanamivir (carbons coloured grey) and oseltamivir (carbons coloured yellow) are shown in similar orientations with selected carbon atoms numbered. c–e, The overlaid structures of the active sites of wild-type (yellow) and mutant N1 NAs (green) are shown with bound inhibitors coloured similarly; relevant portions of electron density maps are also shown. c, His274Tyr in complex with oseltamivir. d, His274Tyr in complex with zanamivir. e, Asn294Ser in complex with oseltamivir. Dashed lines indicate selected hydrogen bonds. Electron density maps were calculated from 2F[o ]- F[c], [calc] coefficients and are contoured at 1.2 . f, The conformation of oseltamivir and Glu 276 from three complexes is shown after superposition using protein atoms only; the carbon atoms of the inhibitor from the wild-type complex are coloured yellow, the His274Tyr in dark green and the Asn294Ser in light green. The affinities (K[I]) of oseltamivir for the three NAs are given in parentheses.
  The above figures are reprinted by permission from Macmillan Publishers Ltd: Nature (2008, 453, 1258-1261) copyright 2008.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
21489803 A.Albohy, S.Mohan, R.B.Zheng, B.M.Pinto, and C.W.Cairo (2011).
Inhibitor selectivity of a new class of oseltamivir analogs against viral neuraminidase over human neuraminidase enzymes.
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21172786 D.W.Brookes, S.Miah, A.Lackenby, L.Hartgroves, and W.S.Barclay (2011).
Pandemic H1N1 2009 influenza virus with the H275Y oseltamivir resistance neuraminidase mutation shows a small compromise in enzyme activity and viral fitness.
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19844936 E.De Clercq (2011).
The next ten stories on antiviral drug discovery (part E): advents, advances, and adventures.
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21504394 J.L.Myers, and S.E.Hensley (2011).
Oseltamivir-resistant influenza viruses get by with a little help from permissive mutations.
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21326879 P.M.Schmidt, R.M.Attwood, P.G.Mohr, S.A.Barrett, and J.L.McKimm-Breschkin (2011).
A generic system for the expression and purification of soluble and stable influenza neuraminidase.
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21251008 T.Han, and W.A.Marasco (2011).
Structural basis of influenza virus neutralization.
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20631138 A.C.Hurt, S.S.Nor'e, J.M.McCaw, H.R.Fryer, J.Mosse, A.R.McLean, and I.G.Barr (2010).
Assessing the viral fitness of oseltamivir-resistant influenza viruses in ferrets, using a competitive-mixtures model.
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20695427 A.J.Oakley, S.Barrett, T.S.Peat, J.Newman, V.A.Streltsov, L.Waddington, T.Saito, M.Tashiro, and J.L.McKimm-Breschkin (2010).
Structural and functional basis of resistance to neuraminidase inhibitors of influenza B viruses.
  J Med Chem, 53, 6421-6431.
PDB codes: 3k36 3k37 3k38 3k39 3k3a
20221396 C.Pan, B.Cheung, S.Tan, C.Li, L.Li, S.Liu, and S.Jiang (2010).
Genomic signature and mutation trend analysis of pandemic (H1N1) 2009 influenza A virus.
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20519385 E.A.Govorkova, N.A.Ilyushina, B.M.Marathe, J.L.McClaren, and R.G.Webster (2010).
Competitive fitness of oseltamivir-sensitive and -resistant highly pathogenic H5N1 influenza viruses in a ferret model.
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20594934 E.Fadda, and R.J.Woods (2010).
Molecular simulations of carbohydrates and protein-carbohydrate interactions: motivation, issues and prospects.
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20859964 H.Ishikawa, T.Suzuki, H.Orita, T.Uchimaru, and Y.Hayashi (2010).
High-yielding synthesis of the anti-influenza neuraminidase inhibitor (-)-oseltamivir by two "one-pot" sequences.
  Chemistry, 16, 12616-12626.  
20155919 J.C.Sung, A.W.Van Wynsberghe, R.E.Amaro, W.W.Li, and J.A.McCammon (2010).
Role of secondary sialic acid binding sites in influenza N1 neuraminidase.
  J Am Chem Soc, 132, 2883-2885.  
20522774 J.D.Bloom, L.I.Gong, and D.Baltimore (2010).
Permissive secondary mutations enable the evolution of influenza oseltamivir resistance.
  Science, 328, 1272-1275.  
21209916 J.Wu, F.Zhang, M.Wang, C.Xu, J.Song, J.Zhou, X.Lin, Y.Zhang, X.Wu, W.Tan, J.Lu, H.Zhao, J.Gao, P.Zhao, J.Lu, and Y.Wang (2010).
Characterization of neuraminidases from the highly pathogenic avian H5N1 and 2009 pandemic H1N1 influenza A viruses.
  PLoS One, 5, e15825.  
20383144 K.Das, J.M.Aramini, L.C.Ma, R.M.Krug, and E.Arnold (2010).
Structures of influenza A proteins and insights into antiviral drug targets.
  Nat Struct Mol Biol, 17, 530-538.  
20498070 L.J.Calder, S.Wasilewski, J.A.Berriman, and P.B.Rosenthal (2010).
Structural organization of a filamentous influenza A virus.
  Proc Natl Acad Sci U S A, 107, 10685-10690.  
  20885781 L.Le, E.H.Lee, D.J.Hardy, T.N.Truong, and K.Schulten (2010).
Molecular dynamics simulations suggest that electrostatic funnel directs binding of Tamiflu to influenza N1 neuraminidases.
  PLoS Comput Biol, 6, 0.  
20523902 N.A.Ilyushina, J.P.Seiler, J.E.Rehg, R.G.Webster, and E.A.Govorkova (2010).
Effect of neuraminidase inhibitor-resistant mutations on pathogenicity of clade 2.2 A/Turkey/15/06 (H5N1) influenza virus in ferrets.
  PLoS Pathog, 6, e1000933.  
20161706 R.H.Friesen, W.Koudstaal, M.H.Koldijk, G.J.Weverling, J.P.Brakenhoff, P.J.Lenting, K.J.Stittelaar, A.D.Osterhaus, R.Kompier, and J.Goudsmit (2010).
New class of monoclonal antibodies against severe influenza: prophylactic and therapeutic efficacy in ferrets.
  PLoS One, 5, e9106.  
20538598 S.J.Gamblin, and J.J.Skehel (2010).
Influenza hemagglutinin and neuraminidase membrane glycoproteins.
  J Biol Chem, 285, 28403-28409.  
20037767 T.Rungrotmongkol, M.Malaisree, N.Nunthaboot, P.Sompornpisut, and S.Hannongbua (2010).
Molecular prediction of oseltamivir efficiency against probable influenza A (H1N1-2009) mutants: molecular modeling approach.
  Amino Acids, 39, 393-398.  
20410266 Y.P.Lin, V.Gregory, P.Collins, J.Kloess, S.Wharton, N.Cattle, A.Lackenby, R.Daniels, and A.Hay (2010).
Neuraminidase receptor binding variants of human influenza A(H3N2) viruses resulting from substitution of aspartic acid 151 in the catalytic site: a role in virus attachment?
  J Virol, 84, 6769-6781.  
19651908 A.C.Hurt, J.K.Holien, and I.G.Barr (2009).
In vitro generation of neuraminidase inhibitor resistance in A(H5N1) influenza viruses.
  Antimicrob Agents Chemother, 53, 4433-4440.  
19156651 B.Carbain, P.J.Collins, L.Callum, S.R.Martin, A.J.Hay, J.McCauley, and H.Streicher (2009).
Efficient synthesis of highly active phospha-isosteres of the influenza neuraminidase inhibitor oseltamivir.
  ChemMedChem, 4, 335-337.  
19503932 B.Carbain, S.R.Martin, P.J.Collins, P.B.Hitchcock, and H.Streicher (2009).
Galactose-conjugates of the oseltamivir pharmacophore--new tools for the characterization of influenza virus neuraminidases.
  Org Biomol Chem, 7, 2570-2575.  
19525932 G.Neumann, T.Noda, and Y.Kawaoka (2009).
Emergence and pandemic potential of swine-origin H1N1 influenza virus.
  Nature, 459, 931-939.  
19133796 I.Stephenson, J.Democratis, A.Lackenby, T.McNally, J.Smith, M.Pareek, J.Ellis, A.Bermingham, K.Nicholson, and M.Zambon (2009).
Neuraminidase inhibitor resistance after oseltamivir treatment of acute influenza A and B in children.
  Clin Infect Dis, 48, 389-396.  
19321726 J.M.Laplante, S.A.Marshall, M.Shudt, T.T.Van, E.S.Reisdorf, L.A.Mingle, P.A.Shult, and K.St George (2009).
Influenza antiviral resistance testing in new york and wisconsin, 2006 to 2008: methodology and surveillance data.
  J Clin Microbiol, 47, 1372-1378.  
19440354 J.T.Wu, G.M.Leung, M.Lipsitch, B.S.Cooper, and S.Riley (2009).
Hedging against Antiviral Resistance during the Next Influenza Pandemic Using Small Stockpiles of an Alternative Chemotherapy.
  PLoS Med, 6, e1000085.  
  20029609 L.Le, E.Lee, K.Schulten, and T.N.Truong (2009).
Molecular modeling of swine influenza A/H1N1, Spanish H1N1, and avian H5N1 flu N1 neuraminidases bound to Tamiflu and Relenza.
  PLoS Curr, 1, RRN1015.  
19137067 M.J.Tuvim, S.E.Evans, C.G.Clement, B.F.Dickey, and B.E.Gilbert (2009).
Augmented lung inflammation protects against influenza A pneumonia.
  PLoS ONE, 4, e4176.  
19461872 M.Lawrenz, R.Baron, and J.A.McCammon (2009).
Independent-Trajectories Thermodynamic-Integration Free-Energy Changes for Biomolecular Systems: Determinants of H5N1 Avian Influenza Virus Neuraminidase Inhibition by Peramivir.
  J Chem Theory Comput, 5, 1106-1116.  
  19961611 N.Goni, A.Fajardo, G.Moratorio, R.Colina, and J.Cristina (2009).
Modeling gene sequences over time in 2009 H1N1 Influenza A Virus populations.
  Virol J, 6, 215.  
  19678928 N.Sriwilaijaroen, P.Wilairat, H.Hiramatsu, T.Takahashi, T.Suzuki, M.Ito, Y.Ito, M.Tashiro, and Y.Suzuki (2009).
Mechanisms of the action of povidone-iodine against human and avian influenza A viruses: its effects on hemagglutination and sialidase activities.
  Virol J, 6, 124.  
  19309695 N.X.Wang, and J.J.Zheng (2009).
Computational studies of H5N1 influenza virus resistance to oseltamivir.
  Protein Sci, 18, 707-715.  
19254207 P.M.Colman (2009).
New antivirals and drug resistance.
  Annu Rev Biochem, 78, 95.  
19457254 S.Maurer-Stroh, J.Ma, R.T.Lee, F.L.Sirota, and F.Eisenhaber (2009).
Mapping the sequence mutations of the 2009 H1N1 influenza A virus neuraminidase relative to drug and antibody binding sites.
  Biol Direct, 4, 18; discussion 18.  
19144639 T.Kuzuhara, D.Kise, H.Yoshida, T.Horita, Y.Murazaki, A.Nishimura, N.Echigo, H.Utsunomiya, and H.Tsuge (2009).
Structural Basis of the Influenza A Virus RNA Polymerase PB2 RNA-binding Domain Containing the Pathogenicity-determinant Lysine 627 Residue.
  J Biol Chem, 284, 6855-6860.
PDB code: 3cw4
19927983 T.Naito, A.Kawaguchi, and K.Nagata (2009).
[Function of influenza virus RNA polymerase based on structure]
  Uirusu, 59, 1.  
19909463 T.Sawabuchi, S.Suzuki, K.Iwase, C.Ito, D.Mizuno, H.Togari, I.Watanabe, S.R.Talukder, J.Chida, and H.Kido (2009).
Boost of mucosal secretory immunoglobulin A response by clarithromycin in paediatric influenza.
  Respirology, 14, 1173-1179.  
19580433 V.M.Deyde, and L.V.Gubareva (2009).
Influenza genome analysis using pyrosequencing method: current applications for a moving target.
  Expert Rev Mol Diagn, 9, 493-509.  
19124660 V.M.Deyde, T.Nguyen, R.A.Bright, A.Balish, B.Shu, S.Lindstrom, A.I.Klimov, and L.V.Gubareva (2009).
Detection of molecular markers of antiviral resistance in influenza A (H5N1) viruses using a pyrosequencing method.
  Antimicrob Agents Chemother, 53, 1039-1047.  
19513050 V.Soundararajan, K.Tharakaraman, R.Raman, S.Raguram, Z.Shriver, V.Sasisekharan, and R.Sasisekharan (2009).
Extrapolating from sequence--the 2009 H1N1 'swine' influenza virus.
  Nat Biotechnol, 27, 510-513.  
18978531 A.Lackenby, C.I.Thompson, and J.Democratis (2008).
The potential impact of neuraminidase inhibitor resistant influenza.
  Curr Opin Infect Dis, 21, 626-638.  
18558099 D.J.Vocadlo, and G.J.Davies (2008).
Mechanistic insights into glycosidase chemistry.
  Curr Opin Chem Biol, 12, 539-555.  
19079604 M.Throsby, E.van den Brink, M.Jongeneelen, L.L.Poon, P.Alard, L.Cornelissen, A.Bakker, F.Cox, E.van Deventer, Y.Guan, J.Cinatl, J.ter Meulen, I.Lasters, R.Carsetti, M.Peiris, Kruif, and J.Goudsmit (2008).
Heterosubtypic neutralizing monoclonal antibodies cross-protective against H5N1 and H1N1 recovered from human IgM+ memory B cells.
  PLoS ONE, 3, e3942.  
18706999 M.von Itzstein (2008).
Disease-associated carbohydrate-recognising proteins and structure-based inhibitor design.
  Curr Opin Struct Biol, 18, 558-566.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.