Chemical Physics Letters 485 (2010) 191–195
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Computational 3D structures of drug-targeting proteins in the 2009-H1N1 influenza A virus Qi-Shi Du a,b,d,*, Shu-Qing Wang c, Ri-Bo Huang a,b,*, Kuo-Chen Chou d a
Guangxi Academy of Sciences, 98 Daling Road, Nanning, Guangxi 530007, China College of Life Science and Technique, Guangxi University, Nanning, Guangxi 530004, China c School of Pharmaceutical Sciences, Tianjin Medical University, Tianjin 300070, China d Gordon Life Science Institute, 13784 Torrey Del Mar Drive, San Diego, CA 92130, USA b
a r t i c l e
i n f o
Article history: Received 28 September 2009 In final form 14 December 2009 Available online 22 December 2009
a b s t r a c t The neuraminidase (NA) and M2 proton channel of influenza virus are the drug-targeting proteins, based on which several drugs were developed. However these once powerful drugs encountered drug-resistant problem to the H5N1 and H1N1 flu. To address this problem, the computational 3D structures of NA and M2 proteins of 2009-H1N1 influenza virus were built using the molecular modeling technique and computational chemistry method. Based on the models the structure features of NA and M2 proteins were analyzed, the docking structures of drug–protein complexes were computed, and the residue mutations were annotated. The results may help to solve the drug-resistant problem and stimulate designing more effective drugs against 2009-H1N1 influenza pandemic. Ó 2009 Published by Elsevier B.V.
1. Introduction The pandemic of influenza virus was the most dangerous killer for human being in the history, and the continuing mutation of the influenza virus is a consistent threatening to human health and life. Currently, the outbreak of influenza A (H1N1) virus is a pandemic of a new strain of influenza virus [1] identified in April 2009, commonly referred to as ‘swine flu’. The pandemic has caused fatal infection and many deaths from the first detected country Mexico [2] to almost all countries of the World within merely a few months. The H1N1 influenza virus is quite familiar to us because it had caused the 1918–1919 Spain pandemic that had infected 5% of the word population and resulted in 50 million deaths worldwide [3]. In July 2009 the WHO enhanced the warning to phase 6, which means that the spread of H1N1 influenza virus has become a seriously global pandemic. It was anticipated that an even stronger outbreak will happen in the coming winter. The even worse news is that several cases were reported about the drug-resistant strains of H1N1 influenza A virus to oseltamivir (Tamiflu). Influenza virus has two functional surface glycoproteins according to antigenic properties, namely hemagglutinin (HA) and neuraminidase (NA). Sixteen subtypes have currently been defined for the hemagglutinin protein (H1–H16) and nine for the neuraminidase protein (N1–N9) [4]. NA is responsible for
* Corresponding authors. Address: Guangxi Academy of Sciences, 98 Daling Road, Nanning, Guangxi 530007, China. Fax: +86 771 250 3908 (Q.-S. Du). E-mail address:
[email protected] (Q.-S. Du). 0009-2614/$ - see front matter Ó 2009 Published by Elsevier B.V. doi:10.1016/j.cplett.2009.12.037
cleaving the terminal sialic acid moieties from the receptors to facilitate the elution of the progeny virions from the infected cell. The cleavage will facilitate the virus to release and form sites of infection in the respiratory tract [5]. Because of its essential role in influenza virus replication and its highly conserved active sites, NA has become the main target for drug design against influenza virus. The anti-flu drugs oseltamivir (Tamiflu) [6] and zanamivir (Relenza) [7] were developed based on the structure of NA. However, in recent clinic practices the drug-resistant strains of H5N1 and H1N1 viruses have been reported [3,8]. The influenza A virus M2 protein forms a proton-selective transmembrane ion channel, which is activated at acidic pH [9–11]. The M2 channel plays a role in the uncoating of influenza virions in endosomes [12]. The M2 proton channel is the specific target of the anti-influenza drugs amantadine and rimantadine [13–15] because of its important role in the life cycle of influenza virus. However the once powerful drugs lost their effectivity quickly because of the drug-resistant mutations of the influenza A virus. Recent reports show that the resistance of influenza A virus to the adamantane-based drugs in humans, birds and pigs has reached more than 90% [16,17]. In order to solve the drug-resistance problem of the current available drugs and to develop more powerful new drugs against the 2009-H1N1 swine flu, reliable molecular structures of NA and M2 proteins of H1N1 influenza A virus are indispensable [18]. However, so far no experimental structures of the two drug–targeting proteins of 2009-H1N1 swine influenza virus are available. To overcome such a barrier, we adopted the structural bioinformatics tools to build the three dimensional structures of NA and M2 proteins of 2009-H1N1 influenza A virus, followed by analyzing
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their structure features, and providing the docking structures of drug–protein complexes with molecular modeling technique and computational chemistry method.
to the model NA and M2 proteins. In the docking calculations, total of 25 docking conformations with the lowest binding energies were recorded.
2. Materials and methods
3. Results and discussion
The amino acid sequences of NA and M2 proteins of 2009-H1N1 influenza A virus are taken from the website NCBI (http:// www.ncbi.nlm.nih.gov/). Up to 11-Sep-2009 at least 3464 NA sequences and 2763 M2 sequences are deposited in the NCBI database. It is believed that as time going on more and more new influenza NA and M2 sequences of H1N1 virus would be submitted to the database. In the homology model construction the query NA sequence of 2009-H1N1 virus is ACV67206 (protein-id in NCBI website http:// www.ncbi.nlm.nih.gov/), which was isolated from an H1N1 virus strain in Texas and deposited to NCBI in 10-Sep-2009. This sequence has the H274Y mutation that confers resistance to Oseltamivir. The structural template for the NA model construction is 3B7E (PDB code in website http://www.rcsb.org/pdb), which was isolated from the 1918-H1N1 virus [18]. In the M2 model construction of 2009-H1N1 virus the query sequence of M2 protein is GQ385303 (protein-id in NCBI website http://www.ncbi.nlm.nih. gov), which is isolated in July 2009 from an H1N1 virus strain in Toronto. The template for the M2 model construction is the highresolution NMR structure of M2 proton channel [19,20] with the PDB code of 2RLF (http://www.rcsb.org/pdb), which was isolated from the Udorn strain of human influenza virus. In this study sequence alignments, homology modeling, and ligand–receptor docking calculations are performed. First, Amino acid sequence alignments were performed based on the signature sequence of the selectivity filter from template proteins to find the location of the active region of query sequences. The consensus structural conserved regions (SCRs) of the target protein were generated from alignment of query sequences to template proteins. Then, using the structural bioinformatics tools [21], the homology 3D structures of the query sequences (NA ACV67206 and M2 GQ385303) were developed by following the same procedures as elaborated in references [22–25]. The newly built homology models were substantially refined to avoid van der Waals radius overlapping, unfavorable atomic distances, and undesirable torsion angles using molecular mechanics and dynamics features. Subsequently, the AUTO DOCK program [26] with the MMFF94 force field and atomic partial charges [27] was utilized to dock the ligands
The results of sequence alignments, the homology models of NA and M2 proteins of 2009-H1N1 virus, and the docking structures of drug–receptor complexes are shown in this section. The important mutations and the structure features of the protein–drug complexes are analyzed and discussed.
Fig. 1. The structure alignment between the homology model of NA sequence (NCBI code: ACV67206) of 2009-H1N1 virus and the template NA (PDB code: 3B7E) of 1918-H1N1 virus. The backbone of query NA (ACV67206) is in green and the backbone of template NA (3B7E) is in red. The backbones of two NA structures overlap very nicely except for some nonstructural loop regions. The insertions and deletions of amino acids in the sequence alignment cause larger deviation in the loop regions. Only 44 different (mutated) residues between the two NA proteins are shown: green for the residues of ACV67206 and red for the residues of 3B7E. The region around the ligand oseltamivir in 6 Å distance is the active pocket. In the active region no mutated residues are found, and all 44 mutated residues are on the surface of NA protein. (For interpretation of the references in color in this figure legend, the reader is referred to the web version of this article.)
Table 1 The sequence alignment of the query NA sequence (ACV67206) of 2009-H1N1 virus and the template NA (3B7E) of 1918-H1N1 virus.a,b,c
a The amino acids are colored according to their properties: acidic (brown), basic (blue), neutral hydrophilic (pink), aliphatic (dark green), aromatic (light green), thiol containing (yellow), and imino (orange). b Only the different residues between the two NA proteins are shown. c The mutation H274Y indicated by grey frame is the drug-resistant mutation confirmed in Ref. [27]. So far this mutation was only reported in the NA sequence ACV67206 of 2009-H1N1 virus.
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3.1. The sequence alignment of two NA proteins The sequence alignment of the query NA sequence (ACV67206) of 2009-H1N1 virus and the template NA (3B7E) of 1918-H1N1 virus [18] are summarized in Table 1. The amino acids are colored according to their properties: acidic (brown), basic (blue), neutral hydrophilic (pink), aliphatic (dark green), aromatic (light green), thiol containing (yellow), and imino (orange). The residue identity between ACV67206 and 3B7E is 72.9%, implying that after 100 years the mutations in the NA of 2009-H1N1 virus reached 27.1%. In Table 1 only the mutated residues between the two NAs (ACV67206 and 3B7E) are shown. Total 43 residues among the 386 amino acids are different between the two NA proteins. In the 43 mutations 16 residue pairs have different physicochemical properties (shown in different colors). The 16 mutations are on the positions 84, 86, 105, 126, 188, 255, 263, 274, 285, 286, 288, 306, 331, 357, 368, 385, and 430 (in 3B7E numbering). Among them the mutation on position 274 (H274Y) is oseltamivir-resistant, which was confirmed by Collins et al. [8]. However, the NAs
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of 2009-H1N1 viruses, so far deposited in the NCBI website, have no this mutation. 3.2. Homology 3D structure of 2009-H1N1 NA The homology NA structure (ACV67206) of 2009-H1N1 virus is shown in Fig. 1. For facilitating comparison the backbone of ACV67206 is aligned with its template NA (3B7E) of 1918-H1N1 virus. As expected, except for some loop regions the backbones of the two NA structures overlap very nicely owing to their high homology to each other. The insertions and deletions of amino acids in the sequence alignment cause larger deviation in the loop regions, as discussed in [24,25]. Total 5 insertions and deletions are found in the alignment between the two NA proteins. Only 44 different (mutated) residues between the two NA proteins are shown in Fig. 1: green for the residues of ACV67206 and red for the residues of 3B7E. All 44 mutated residues are on the surface of NA protein. After receptor–ligand docking calculation between NA (ACV67206) of 2009-H1N1 and the ligand oseltamivir (Tamiflu), the binding position of oseltamivir is determined and shown in Fig. 2, which is rendered in ball-stick drawing. The region around the oseltamivir in 6 Å distance is the active pocket. In Fig. 2 the residues in active region are rendered in stick drawing. All 20 residues in the active region are conserved, implying that although 44 mutations happened in past 100 years, the active pocket of NA of 2009-H1N1 virus is highly conserved. The confirmed drug-resistant mutation H274Y is indicated in Fig. 2, which is outside the active region. The mutation H274Y blocks the bioactivity of oseltamivir by a complex allosteric inhibition mechanism, as described in Ref. [8]. This may be the reason why the 2009-H1N1 virus was resistant to Tamiflu in some clinical cases. 3.3. The sequence alignment of M2 proteins
Fig. 2. The docking structure of complex between NA (ACV67206) of 2009-H1N1 virus and the ligand oseltamivir (Tamiflu). The oseltamivir is rendered in the space filling drawing. The region around the oseltamivir in 6 Å distance is the active pocket. The residues in active region are shown in stick drawing. In the active region no mutated residues are found. The position of the confirmed drug-resistant mutation H274Y (in 3B7E numbering) is indicated, which is outside of the active region.
The sequence alignment between the query M2 protein (GQ385303) and the template (2RLF) [19,20] is shown in Table 2. The M2 proton channel (GQ385303) of 2009-H1N1 virus is a complete sequence consisting of 97 residues, however, the structure of template M2 protein 2RLF is a segment containing 43 residues from position 18 to 60. In Table 2 the residues highlighted in red indicate the three functional residues (pH sensor His37, channel gate Trp41, and channel lock Asp44) [19,20,28,29] of the M2 channel, which are highly conserved in the M2 proton channel. Those residues with the light-blue frames are the possible binding sites (Thr43, Asp44, and Arg45) for the inhibitors (adamantane-based drugs); while those with the green frames are different between the two sequences. In the sequence alignment no residue insertions and deletions of amino acids are found.
Table 2 The sequence alignment between the query M2 sequence (GQ385303) of 2009-H1N1 virus and the template M2 proton channel (2RLF).a,b
a
The M2 (GQ385303) is a complete sequence consisting of 97 residues and the template M2 (2RLF) is a segment containing 43 residues from position 18 to 60. The three functional residues (pH sensor His37, channel gate Trp41, and channel lock Asp44) of the M2 channel are highlighted in red. Those residues with the blue frames are the possible binding sites (Thr43, Asp44, and Arg45) of the inhibitors (adamantane-based drugs); while those with the green frame are different residues between the two sequences. b
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Fig. 4. The docking structure of complex between M2 proton channel (GQ385383) of 2009-H1N1 virus and the ligand rimantadine. In the docking complex the channel lock Asp44 holds the channel gate Trp41 through a hydrogen bond, keeping it in the closed conformation in the middle or higher pH (7.5) condition. But in the lower pH environment, the pH sensor His37 and the indole amine of Trp41 are protonated, so as to weaken the hydrogen bond between Trp41 and Asp44, making it easily broken by the repulsive interaction between the positively charged His37 residues of two adjacent helix chains. After rimantadine binds at the Asp44 through two hydrogen bonds between amino group of rimantadine and carboxyl group of Asp44, the pKa value of Asp44 is lowered by the two hydrogen bonds.
Fig. 3. The homology model of 2009-H1N1 M2 proton channel (NCBI code: GQ385383) and its template NMR structure of M2 proton channel (PDB code: 2RLF). The two M2 proteins are highly homologous, no differences are found on the backbones. The three functional residues (pH sensor His37, channel gate Trp41, and channel lock Asp44) of the M2 channel are rendered in ball-and-stick drawing, which are located at the middle of the channel. The mutated residues between two M2 proteins are show in green for GQ385383 and blue for 2RLF, respectively. Most mutated residues are on the top and bottom of the channel, which have little effects on the functional residues. However, the mutation Leu43Thr is found in the active region: the non-polar residue Leu43 of 2RLF is replaced by the polar residue Thr43 in the 2009-H1N1 M2 protein. Ligand rimantadine (in purple) binds at four equivalent sites near the channel lock Asp44 on the lipid-facing side of the channel. (For interpretation of the references in color in this figure legend, the reader is referred to the web version of this article.)
channel [19,20,28]. The channel lock Asp44 holds the channel gate Trp41 through a hydrogen bond, keeping it in the closed conformation in the middle or higher pH (7.5) condition. But in the lower pH environment, the pH sensor His37 and the indole amine of Trp41 are protonated, so as to weaken the hydrogen bond between Trp41 and Asp44, making it easily broken by the repulsive interaction between the positively charged His37 residues of two adjacent helix chains. After rimantadine binds at the Asp44 through two hydrogen bonds between amino group of rimantadine and carboxyl group of Asp44, the pKa value of Asp44 is lowered by the two hydrogen bonds [30,31]. Therefore, in the lower pH environment, it becomes difficult for Asp44 to be protonated. That is why in the acidic condition the ligand rimantadine can help Asp44 to keep the channel in the closed conformation.
3.4. Homology 3D structure of 2009-H1N1 M2 protein 4. Conclusion A superposition of the modeled 2009-H1N1 M2 (GQ385383) structure with its template of M2 protein channel (2RLF) is shown in Fig. 3, from which we can see that their backbone structures are basically the same as expected due to their high sequence homology without any residue insertions and deletions. The three functional residues (His37, Trp41, and Asp44) of the M2 channel are rendered in ball-and-stick drawing. They are located at the middle of the channel. The different residues between the two channel structures are shown in green for 2RLF and blue for GQ385383. Most mutated residues in H1N1-M2 protein are at the top and bottom of the channel, which have little effects on the functional residues (His37, Trp41, and Asp44). However, one mutation, namely L43T, is found in the active region: the non-polar residue Leu43 of 2RLF is replaced by the polar residue Thr43 in the 2009-H1N1 M2 protein. Ligand rimantadine binds at four equivalent sites near the channel lock Asp44 on the lipid-facing side of the channel. Illustrated in Fig. 4 is the docking structure of complex between M2 proton channel (GQ385383) and ligand rimantadine, which revealses the gating and inhibiting mechanism of the M2 proton
The neuraminidase (NA) and M2 proton channel of influenza A viruses are the two drug-targeting proteins for the drug discovery fighting with the current influenza pandemic. The computational 3D structures developed in this study for the NA and M2 protein channel of 2009-H1N1 virus provide a structural basis for this purpose. A comparison of the NA (ACV67206) of 2009-H1N1 virus with the NA (3B7E) of 1918-H1N1 virus indicates that the residues in active region are highly conserved. All 44 mutated residues are located outside the active region. The confirmed drug-resistant mutation H274Y perturbs the bioactivity of oseltamivir through a long region allosteric inhibition mechanism [8]. Among the 44 mutations there may be some other similar drug-resistant mutations. It may be equally important for researchers to focus their attentions on this type drug resistant mechanism as well. In the 3D structure modeled for the M2 proton channel (GQ385383) of 2009-H1N1 virus the mutation L43T is observed; i.e., the non-polar residue Leu43 of 2RLF is replaced by the polar
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residue Thr43 in the 2009-H1N1 M2 protein. This mutation is very close to the functional residue Trp44, the channel lock and the binding site of rimantadine. Therefore, the mutation L43T may also have impact on the bioactivity of rimantadine.
[7] [8] [9] [10] [11]
Acknowledgements This work is financially supported by the National Basic Research Program (‘973’) of China under the Project 2009CB724703, and by the Chinese National Science Foundation (NSFC) under the Project 30970562. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cplett.2009.12.037. References [1] J.T. Wu, G.M. Leung, M. Lipsitch, B.S. Cooper, S. Riley, PLoS Med. 6 (5) (2009) e1000085. doi:10.1371/journal. pmed.1000085. [2] D.K. Shay, B. Ridenhour, PLoS Med. 6 (6) (2009) e1000103. doi:10.1371/ journal.pmed.1000103. [3] S.H. Hauge, S. Dudman, K. Borgen, A. Lackenby, O. Hungnes, Emerg. Infect. Dis. 15 (2009) 155. [4] World Health Organization (WHO), Bull. WHO 58 (1980) 585. [5] J.L. McKimm-Breschkin, Antiviral Res. 47 (2000) 1. [6] W. Lew, X. Chen, C.U. Kim, Curr. Med. Chem. 7 (2000) 663.
[12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31]
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C.J. Dunn, K.L. Goa, Drugs 58 (1999) 761. P.J. Collins et al., Nature 453 (2008) 1258. L.H. Pinto, L.J. Holsinger, R.A. Lamb, Cell 69 (1992) 517. I.V. Chizhmakov, F.M. Geraghty, D.C. Ogden, A. Hayhurst, M. Antoniou, A.J. Hay, J. Physiol. 494 (1996) 329. J.A. Mould, R.G. Paterson, M. Takeda, Y. Ohigashi, P. Venkataraman, R.A. Lamb, L.H. Pinto, Dev. Cell 5 (2003) 175. K. Martin, A. Helenius, Cell 67 (1991) 117. N. Kolocouris et al., Bioorg. Med. Chem. Lett. 17 (2007) 4358. G. Stamatiou, A. Kolocouris, N. Kolocouris, G. Fytas, G.B. Foscolos, J. Neyts, E. De Clercq, Bioorg. Med. Chem. Lett. 11 (2001) 2137. D. Tataridis et al., Bioorg. Med. Chem. Lett. 17 (2007) 692. R.A. Bright, D.K. Shay, B. Shu, N.J. Cox, A.I. Klimov, J. Am. Med. Assoc. 295 (2006) 891. V.M. Deyde et al., J. Infect. Dis. 196 (2007) 249. X. Xu, X. Zhu, R.A. Dwek, J. Stevens, I.A. Wilson, J. Virol. 82 (2008) 10493. J.R. Schnell, J.J. Chou, Nature 451 (2008) 591. R.M. Pielak, R. Jason, J.R. Schnell, J.J. Chou, Proc. Natl. Acad. Sci. USA 106 (2009) 7379. K.C. Chou, Curr. Med. Chem. 11 (2004) 2105. K.C. Chou, J. Proteom. Res. 4 (2005) 1657. K.C. Chou, K.D. Watenpaugh, R.L. Heinrikson, Biochem. Biophys. Res. Commun. 259 (1999) 420. K.C. Chou, D. Jones, R.L. Heinrikson, FEBS Lett. 419 (1997) 49. K.C. Chou, A.G. Tomasselli, R.L. Heinrikson, FEBS Lett. 470 (2000) 249. G.M. Morris et al., J. Comput. Chem. 19 (1998) 1639. W.D. Cornell et al., J. Am. Chem. Soc. 177 (1995) 5179. R.-B. Huang, Q.-S. Du, C.-H. Wang, K.-C. Chou, Biochem. Biophys. Res. Commun. 377 (2008) 1243. Q.-S. Du, R.-B. Huang, C.-H. Wang, X.-M. Li, K.-C. Chou, J. Theoret. Biol. 259 (2009) 159. H. Li, A.D. Robertson, J.H. Jensen, Proteins 55 (2004) 689. H. Li, A.D. Robertson, J.H. Jensen, Proteins 61 (2005) 704.
