# Patent

## Title
Crystal structure of the influenza virus polymerase PAC-PB1N complex and uses thereof

## Abstract

Provided are the expression method of influenza virus polymerase PAc-PB1N complex, the co-crystallization method of the complex and the three-dimendional structure of the crystal of PAc-PB1N complex. Also provided are the compounds binding to the influenza virus polymerase PAc and the expression method of influenza virus polymerase PAN. The three-dimensional structure of the crystal of PAc-PB1N complex can be used for screening and designing the drug for the treatment of influenza.

## Background
TECHNICAL FIELD
The present invention relates to a method for expression of influenza virus PA (SEQ ID NO:1) and PB1 N-terminal polypeptide in bacteria, purification and crystallization. It also relates to the crystal three-dimensional structure of complex of C-terminal of PA and N-terminal polypeptide of PB1 and its application in drug design.
BACKGROUND ART
Influenza viruses has already caused great disasters for humanity (Taubenberger and Morens 2007). Due to the lack of sufficient treatments and frequent mutations of the virus itself, the virus remains a threat to humans. In recent years, frequent and severe avian influenza epidemics as well as transmission of avian influenza to humans has constituted a great threat to health and economics of humans, so investigations directed to this kind of virus is of great value to protect human health. Avian influenza virus belongs to influenza virus A type, which are all me the members of the Orthomyxoviridae family. The virus genome consists of 8 negative sense single-stranded RNA. Through comparison and analysis of genes of influenza virus of avian origin and other influenza A viruses, sporadic mutations in the primary structure have been found, and these mutations result in differential pathogenicity of different influenza viruses. Now, it is established that the influenza virus genome encodes 11 proteins, wherein the replication of influenza virus genome RNA and mRNA transcription are dependent on a viral RNA polymerase which has become a potentially important drug target. Recent research suggested that the high pathogenicity of some influenza viruses is directly correlated with the polymerase mutations (Hulse-Post, Franks et al. 2007; Munster, de Wit et 20 al. 2007), further illustrating the necessity of designing drugs aiming at this complex. Investigation into this complex is of great significance to reveal the molecular mechanism underlying virus replication and to design drugs aiming at this complex. The RNA polymerase is a complex composed of PB1 (SEQ ID NO:2), PB2 and PA (SEQ ID NO:1) subunits, wherein PB1 (SEQ ID NO:2) is a subunit with catalytic activity, PB2 is responsible for acquiring cellular mRNA cap (CAP structure) through a snatching mode as primers of virus mRNA transcription, but PB1 (SEQ ID NO:2) acts as an endonuclease in this process. A temperature sensitive mutant ts53 suggests that PA (SEQ ID NO:1) takes part in the replication process of virus genome, but its specific function is still unclear (Sugiura, Ueda et al. 1975; Kawaguchi, Naito et al. 2005). The polymerase has three kinds of RNA activity which are needed in virus synthesis, i.e. mRNA, cRNA and vRNA synthesis, respectively. The mRNA synthesis starts from a capped oligonucleotide primer and ends 15-17 nucleotides prior to the vRNA terminal, followed by addition of a polyA tail. Polymerase can synthesize a cRNA intermediate of the full-length virus de novo and further synthesize full-length vRNA. Respective subunits of the polymerase can be expressed by an insect cell expression system, thus forming three different complexes, wherein one is a ternary complex containg the three subunits PB1 (SEQ ID NO:2)/PB2/PA (SEQ ID NO:1) of polymerase, and the other two are binary complexes: PB1 (SEQ ID NO:2)/PB2 and PB1 (SEQ ID NO:2)/PA (SEQ ID NO:1) binary complexes respectively, PB2/PA (SEQ ID NO:1) complex is not formed (Honda, Mizumoto et al. 2002). 25 amino acids of PB1 N-terminal are sufficient to interact with PA (SEQ ID NO:1) C-terminal, while the PB1 C-terminal is responsible for the interaction with PB2 N-terminal. A synthetic competitive small peptide of the PB1 N-terminal can significantly inhibit the activity of virus polymerase. RNA synthesis experiments using dinucleotide ApG as primers indicated that PB1 (SEQ ID NO:2)/PA (SEQ ID NO:1) complex can effectively initiate the replication of virus genome RNA, and PB1 (SEQ ID NO:2)/PB2 can synthesize virus mRNA in vitro (Honda, Mizumoto et al. 2002), but further studies in which the recombinant polymerase was expressed and purified using 293 cell revealed that all three subuits are necessary for replication and transcription (Deng, Sharps et al. 2006). The idea that PA (SEQ ID NO:1) principally participates in the replication process of virus RNA is derived from a finding that a tempreture sensitive mutant (L226P) can result in replication disorder of virus genome under non-permissible temperatures without affecting transcription activity (Kawaguchi, Naito et al.2005); whereas PB2 is involved in virus mRNA transcription. Further studies found that PA (SEQ ID NO:1) can extensively take part in processes such as transcription, replication and virus stability (Hara, Schmidt et al. 2006). The PB1 (SEQ ID NO:2)/PA (SEQ ID NO:1) complex can bind the 5′ terminal virus promoter, but PB1 (SEQ ID NO:2) by itself does not bind that promoter. Cross-linking experiments indicate that PA (SEQ ID NO:1) can bind vRNA and cRNA promoters (Fodor, Pritlove et al. 1994; Deng, Sharps et al. 2005; Hara, Schmidt et al. 2006) (Fodor 1994, Gonzalez 1999, Jung 2006, Hara 2006), but the specific binding sites are not clear. PA (SEQ ID NO:1) was found to have similar protease activity as chymotrypsins. Sanz-Ezquerro et al. (1996) found that about 250 amino acids at N-terminal are the active region of that protease—(Sanz-Ezquerro, Zurcher et al. 1996). But subsequently the studies by Hara et al. (2001) showed that the Serine at position 624 of C-terminal was the active site of PA (SEQ ID NO:1) protease, as mutation at that site resulted in loss of protease activity (Hara, Shiota et al. 2001). There is still controversy about the effect of PA (SEQ ID NO:1) protease activity on polymerase function. Hara et al. (2006) reported that purified recombinant PA (SEQ ID NO:1) protein can be degraded into two fragments with molecular weight of  ̃25 kDa and  ̃55 kDa through trypsin hydrolysis (Hara, Schmidt et al. 2006). It is known that understanding the three-dimensional structure of a protein is of great help to perform rational drug design, so identifying the three-dimensional structure of PA (SEQ ID NO:1) is of important value to perform drug design and function studies. In addition, previous studies have not reported the expression and purification of influenza virus protein PA (SEQ ID NO:1) in bacteria, so expression and purification of proteins in bacteria is of important benefit to further explore the function of PA (SEQ ID NO:1) and to perform drug screening, thus saving much time and greatly decreasing job cost and labour intensity.
SUMMARY OF THE INVENTION
The present invention provides a method of dividing the wild type or mutant protein of influenza virus polymerase complex subunit PA (SEQ ID NO:1) into N-terminal and C-terminal parts, and cloning and expressing said parts respectively; and a method of expressing, purifying and crystallizing an N-terminal part of PA (SEQ ID NO:1); and a method of expressing a short N-terminal peptide of wild type or mutant protein of influenza virus polymerase subunit PB1 (SEQ ID NO:2); and a method of co-purification of a PA C-terminal and a short N-terminal peptide of PB1. According to the invention, the method of expressing and co-purifying the complex of the first 256 N-terminal amino acids of PA (SEQ ID NO:1) as well of the C-terminal amino acids 257-716 of PA (SEQ ID NO:1) and the N-terminal 25 amino acids of of PB1, and a method of crystallizing a protein complex of the C-terminal part of PA (SEQ ID NO:1) and the N-terminal peptide of PB1 as well as the three-dimensional crystal structure of the resulting complex.
In one aspect, the present invention provides a method of dividing the wild type or mutant protein of influenza virus polymerase complex subunit PA (SEQ ID NO:1) into an N-terminal part and a C-terminal part as well as cloning and expressing these parts respectively; and a method of expressing, purifying and crystallizing an N-terminal part of PA (SEQ ID NO:1); and a method of expressing an N-terminal polypeptide of a wild type or a mutant protein of influenza virus polymerase subunit PB1 (SEQ ID NO:2) and co-purifying the complex of a C-terminal part of PA (SEQ ID NO:1) and an N-terminal short peptide of PB1 for crystallization. In the present invention, preferably a prokaryotic cell expression system of E.coli is used, but other expression systems are not excluded, for example, expression can also be performed in other bacteria or other eukaryotic cells. To express the above-mentioned polypeptides as GST (glutathione-S-transferase) fusion proteins, bacteria expressing the PA (SEQ ID NO:1) C-terminal part are combined with bacteria expressing the PB1 N-terminal peptide. This protein complex comprising the above-mentioned C-terminal part of PA (SEQ ID NO:1) and short N-terminal peptide of PB1 is purified from bacterial expression system, for use in protein crystallization. According to the invention, an expression method of obtaining the first 256 N-terminal amino acids of influenza virus polymerase subunit PA (SEQ ID NO:1) and the C-terminal 257-716 amino acids of influenza virus polymerase subunit PA (SEQ ID NO:1) by expression in E.coli and a method of obtaining a microbial population of E.coli which expresses the polypeptide within the 25 or 48 N-terminal amino acids of influenza virus protein PB1 are described, wherein the methods comprise expressing GST (glutathione-S-transferase)-fused fusion proteins by cloning corresponding genes into pGEX-6p vector.
In another aspect, the present invention provides a method of purifying the complex of a C-terminal part of PA (SEQ ID NO:1) (PAc) and PB1N peptides, comprising: a microbial population expressing the C-terminal amino acids 257-716 of influenza virus PA (SEQ ID NO:1) and a microbial population expressing a short peptide within the 25 to 48 N-terminal amino acids of the of influenza virus PB1 suspended in medium respectively; combining said peptide-expressing bacteria in in such proportion as to generate a desired molar ratio between the total protein content of GST-PAc and GST-PB1N, thereby obtaining a mixture of these two proteins; purifying the protein mixture by affinity column and cleaving the GST fusion protein by PreScission protease; and obtaining a purified complex of C-terminal PA and N-terminal PB1 peptides by methods such as gel filtration and ion exchange chromatography; wherein the protein purity and concentration can be determined by gel electrophoresis.
In another aspect, the present invention provides a method of crystalizing the complex of PAc and PB1N peptide obtained as described above, comprising: concentrating the complex of PAc and PB1N peptide to a concentration of 5-30 mg/ml; screening crystal growth conditions by gas hanging drop at 4-30° C.; and obtaining a crystal of the protein complex.
In another aspect, the present invention provides the crystal of the complex of a PAc and a PB1N peptide.
In another aspect, the present invention provides the three-dimensional crystal structure of the complex of a Pac peptide and a PB1N peptide, wherein the structure describes the interaction mode between PAc and PB1N and corresponding interaction sites, and further describes the composition of secondary structure, peptide side chain orientation and the three-dimensional molecular structure of polypeptides in the complex, wherein X-ray crystal diffraction was performed with the the crystal of the complex of PAc and PB1N peptide in order to obtain the diffraction data from the protein crystal of the complex of PAc and PB1 N-terminal peptide, and wherein a three-dimensional structure model of the complex of PAc and PB1N peptide is constructed by further performing a structure analysis process with the diffraction data of the protein crystal.
In one embodiment, the present invention provides a three-dimensional crystal structure of a complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, wherein the C-terminal part of the influenza virus polymerase subunit PA-PAc comprises amino acids from about amino acid position 201-301 to about 650-terminus, wherein the N-terminal part of of influenza virus polymerase subunit PB1-PB1N is a short peptide within the 48 N-terminal amino acids of the of influenza virus polymerase subunit PB1-PB1N, wherein atoms of the three-dimensional crystal structure of the complex have at least partially the atomic coordinates listed in table 1, or any structure that has an average root mean square deviation (RMSD) smaller than or equal to 1.7 Angstrom with atomic coordinates of main chain backbone carbon atoms of at least 40% amino acids in the three-dimensional crystal structure of the complex.
Preferably, the crystal of a complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N has a space group of P4(1)2(1)2, and the lattice parameters are about: a=b=122 Angstrom, c=133 Angstrom, α=β=γ=90°.
In one embodiment, the influenza virus is selected from influenza virus A, B and C type, preferably influenza virus A type: A/goose/Guangdong/1/96, A/Brevig Mission/1/1918; influenza virus B type: B/Ann Arbor/1/1966 or influenza virus C type: C/JJ/1950.
In one embodiment, the C-terminal part of influenza virus A polymerase subunit PA-PAc consists of a first portion (i.e. constituting the “mouth” section of the crystal structure, as shown in FIG. 4) and a second portion (i.e. constituting the “head” section of the crystal structure, as shown in FIG. 4), wherein the first portion comprises α helix 4, i.e. a fragment of amino acid positions 406-414, α helix 5, i.e. a fragment of amino acid positions 440-450, α helix 8, i.e. a fragment of amino acid positions 583-603, α helix 9, i.e. a fragment of amino acid positions 608-613, α helix 10, i.e. a fragment of amino acid positions 663-649, α helix 11, i.e. a fragment of amino acid positions 653-673, α helix 12, i.e. a fragment of amino acid positions 683-691, α helix 13, i.e. a fragment of amino acid positions 698-714, β sheet 8, i.e. a fragment of amino acid positions 619-623 and β sheet 9, i.e. a fragment of amino acid positions 628-631; wherein the second portion comprises α helix 1, i.e. a fragment of amino acid positions 303-311, α helix 2, i.e. a fragment of amino acid positions 331-349, α helix 3, i.e. a fragment of amino acid positions 364-369, α helix 6, i.e. a fragment of amino acid positions 454-475 and α helix 7, i.e. a fragment of amino acid positions 572-578, β sheet 1, i.e. amino acids fragment of 290-292 positions, β sheet 2, i.e. a fragment of amino acid positions 317-324, β sheet 3, i.e. a fragment of amino acid positions 480-491, β sheet 4, i.e. a fragment of amino acid positions 496-506, β sheet 5, i.e. a fragment of amino acid positions 517-526, β sheet 6, i.e. a fragment of amino acid positions 541-550 and β sheet 7, i.e. a fragment of amino acid positions 557-571 positions; wherein the β sheets of the second portion of the C-terminal of PA-PAc are surrounded by α helix 1, i.e. a fragment of amino acid positions 303-311, α helix 2, i.e. a fragment of amino acid positions 331-349, α helix 3, i.e. a fragment of amino acid positions 364-369, α helix 6, i.e. a fragment of amino acid positions 454-475 and α helix 7, i.e. a fragment of amino acid positions 572-578, wherein fragments of influenza virus B and C type corresponding to α helixes and β sheets of influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B, respectively.
In one embodiment, the C-terminal part of influenza virus type A polymerase subunit PA-PAc interacts with the N-terminal part of PB1-PB1N mainly through α helix 8, α helix 10, α helix 11 and α helix 13, preferably mediated by at least one amino acid selected from a group consisting of Leu666 of α helix 11, Phe710 of α helix 13, Val636 of α helix 10, Leu640 of α helix 10, Trp706 of α helix 13 and Gln670 of α helix 11, and wherein fragments of influenza virus B and C type corresponding to these α helixes of influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, FIGS. 10B, respectively.
In one embodiment, at least one amino acid selected from the group consisting of Ile621, Gly622, Glu623, Thr618 and Pro620 interacts with the influenza virus polymerase subunit PB1, where the Ile621, Gly622, Glu623, Thr618 and/or Pro620 is in the peptide loop between α helix 9 and α helix 10 of the C-terminal of the influenza virus A polymerase subunit PA-PAc, and wherein the fragments of influenza virus B and C type corresponding to the α helixes of influenza virus A type are shown in FIGS. 1A, 1B, and 1C and FIGS. 10A, and 10B respectively.
In one embodiment, a “pocket” structure comprising at least one amino acid selected from the group consisting of Asn647, Gln408, Cys584, Gln587, Gln591, Lys643, Asn647, Ser659, Lys663, Trp699 and Asn703 of the C-terminal of influenza virus A polymerase subunit PA-Pac is provided, where such “pocket” structure binds the influenza virus polymerase subunit PB1N, and wherein the fragments of influenza virus B and C type corresponding to such influenza virus A type fragments are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In one embodiment, “groove” and “channel” structures comprising at least one amino acid selected from the group consisting of Trp406, Glu410, Lys461, Glu524, Phe525, Ser526, Lys536, Lys539, Tyr540, Leu563, Tyr564, Arg566 and Lys574 of the C-terminal of the influenza virus A polymerase subunit PA-Pac are provided, where such “groove” and “channel” structures bind nucleotides, RNA or other small molecules or proteins, and wherein the fragments of influenza virus B and C type corresponding to this influenza virus A type fragment are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In one embodiment, amino acid residues at positions 370-405 of the C-terminal part of PA-PAc constitutes a large loop, wherein the fragments of influenza virus B and C type corresponding to this influenza virus A type fragment are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In one embodiment, α helix 12 and α helix 13, preferably at least one amino acid selected from the group consisting of Ile690, Glu691, Glu692, Cys693 and Asn696 of the α helix 12 and α helix 13, interacts with other proteins.
In one embodiment, at least one amino acid selected from the group consisting of Lys506, Gly507, Arg508, Ser509, His510, Leu511, Arg512, Asn513 and Asp514 interacts with other proteins, wherein His510 constitutes a portion of the polymerase complex RNAse, and wherein the fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In one embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, which binds to at least one member selected from the group consisting of α helix 8, α helix 10, α helix 11 and α helix 13 of a C-terminal part of influenza virus subunit PA-PAc, and which preferably binds to the member selected from the group consisting of Leu666 in α helix 11, Phe710 in α helix 13, Val636 and Leu640 in α helix 10, Trp706 in α helix 13, Gln670 in α helix 11 of N-terminal of influenza virus subunit PA-PAc, wherein fragments of influenza virus B and C type corresponding to influenza virus A are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In one embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to at least one member selected from the group consisting of Ile621, Gly622, Glu623, Thr618 and Pro620 located at the peptide loop between α helix 9 and α helix 10 of a C-terminal part of influenza virus polymerase subunit PA-PAc, wherein fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In one embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to at least one amino acid selected from the group consisting of Asn647, Gln408, Cys584, Gln587, Gln591, Lys643, Asn647, Ser659, Lys663, Trp699 and Asn703 of a C-terminal part of influenza virus A polymerase subunit PA-PAc, wherein fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In one embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to at least one amino acid selected from the group consisting of Trp406, Glu410, Lys461, Glu524, Phe525, Ser526, Lys536, Lys539, Tyr540, Leu563, Tyr564, Arg566 and Lys574 of a C-terminal part of influenza virus A polymerase subunit PA-PAc, wherein fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In one embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to amino acid positions 370-405 of a C-terminal part of influenza virus A polymerase subunit PA-PAc, wherein fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In one embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to helix 12 and α helix 13 of a C-terminal part of influenza virus A polymerase subunit PA-PAc, preferably to at least one amino acid selected from the group consisting of Ile690, Glu691, Glu692, Cys693 and Asn696 in α helix 12 and α helix 13, wherein corresponding fragments of influenza virus B and C type to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In one embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to at least one amino acid selected from the group consisting of Lys506, Gly507, Arg508, Ser509, His510, Leu511, Arg512, Asn513 and Asp514 located at loop region between sheet β 4 and sheet β 5 in the C-terminal of influenza virus A polymerase subunit PA-PAc, wherein fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In one embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate which competes with influenza virus polymerase subunit PB1 (SEQ ID NO:2) for binding PAc, wherein fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In one embodiment, the present invention provides a PAc protein interaction domain comprising a hydrophobic core constituted by the α helix 8, α helix 11, α helix 13 and α helix 10, particularly the interaction domain comprises Met595 in α helix 8, Leu666 in α helix 11, Trp706 and Phe710 in α helix 13, Val636 and Val640 in α helix 10, wherein fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In one embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate which competes with influenza virus polymerase subunit PB1 (SEQ ID NO:2) for binding PAc, wherein the amino acid sequence of the polypeptide, protein, antibody or immunoconjugate comprises at least three amino acids which are identical to amino acids of corresponding position of a short PTLLFL motif of the short helix domain constituted by the 5th-10th residues Pro5, Thr6, Leu7, Leu8, Phe9 and Leu10 of the N-terminal part of wild influenza virus polymerase subunit PB1-PB1N, when the polypeptide or protein is aligned with the PTLLFL motif.
In one embodiment, the present invention provides a composition comprising the above-mentioned polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate.
In one embodiment, the present invention provides use of the above-mentioned composition in the manufacture of a medicament for the treatment of diseases caused by influenza virus.
In one embodiment, the present invention provides a method of expressing and purifying the complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, comprising: (a) constructing a vector with a gene sequence encoding amino acid from about positions 201-301 to about 650 terminus of a C-terminal part of influenza virus polymerase subunit PA-PAc, where the vector may further comprise a protein tag tag fusion, wherein prokaryotic or eukaryotic cells transformed with said vector comprising the protein tag and the PA-PAc sequence can express PAc as a tagged fusion protein; (b) using a method analogous to the method of expressing PAc to express the PB1N with or without a protein tag; (c) Proportionally mixing the cell expressing influenza virus polymerase subunit PAc obtained from step (a) and the cell expressing amino acids within the 48 amino acids of the N-terminal of influenza virus polymerase subunit PB1 obtained from step (b), wherein the resulting protein is isolated by the specific recognition of the specific tag, the protein tag is removed from the protein by enzymolysis, the complex of PAc and PB1N is isolated, and the concentration of the complex is determined;
The atoms of the three-dimensional crystal structure of a complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N have at least 40% of the atomic coordinates listed in table 1, or atomic coordinates of main chain backbone carbons of at least 40% amino acids in the crystal three-dimensional structure of complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N have an average root mean square deviation smaller than or equal to 1.7 Angstrom with respect to the atomic coordinates listed in table 1.
In one embodiment, the present invention provides a method of expressing and purifying the complex of an C-terminal part of influenza virus polymerase subunit PA-PAc and a N-terminal part of influenza virus polymerase subunit PB1-PB1N, wherein a protein tag is selected from GST, Flag-tag, Myc-tag, MBP-tag, specific antibodies?; the vector comprises a selection marker gene; the proportional mixing in step (c) above means that the molar ratio of protein-tagged PAc and protein-tagged PB1N is 0.1:1-1:0.1, preferably the molar ratio of protein-tagged PAc and protein-tagged PB1N is 0.5:1-1:0.5, more preferably the molar ratio of protein-tagged PAc and protein-tagged PB1N is nearly 1:1; wherein preferably the protein tag is GST, wherein the tag is specifically recognized using an affinity column, wherein the tag is removed by proteinases, wherein the complex of PAc and PB1N is separated by gel filtration or ion-exchange chromatography, and wherein the the protein concentration is determined by gel electrophoresis.
In one embodiment, the present invention provides a method of expressing and purifying the complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, wherein the vector is pGEX-6p plasmid vector, and said selective marker gene is penicillin resistance gene, and the proteinase used in step (c) is PreScission proteinase; the restriction site in primers that is employed in the vector is a restriction site selected from a group consisting of SalI and NotI; the restriction site used to insert a gene fragment is a restriction site selected from a group consisting of SalI and NotI; said gene fragment of a C-terminal part of influenza virus polymerase subunit PA-PAc is obtained from the genome of influenza virus A type: A/goose/Guangdong/1/96 by polymerase chain reaction (PCR); said vector and said gene fragment to be inserted are treated by corresponding DNA restriction enzyme respectively, such as those selected from a group consisting of BamHI and XhoI, and ligating the gene to be inserted and the vector by T4 DNA ligase, before transforming prokaryotic cells such as E. coli to obtain cloned plasimds. The cloned plasmid as described above are transformed into E. coli BL21, the resulting transformed bacteria are cultured and induced by using IPTG, wherein the preferred concentration of IPTG is 0.1 mM to 1 mM, and the cultured bacteria are centrifuged to obtain the microbial population that express said fusion protein.
In one embodiment, the present invention provides a method of co-crystallizing the complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, comprising: concentrating the protein concentration of the purified complex of PAc and PB1N to 5-30 mg/ml; screening crystal growth conditions by gas sitting drop or hanging drop method; and obtaining the crystal of a complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N.
In one embodiment, the present invention provides a method of expressing wild type or mutant protein of an N-terminal part of PA-PAN, wherein PAN comprises amino acids from positions 1-50 to about 200-300, the method comprising: constructing an expression vector with a gene sequence encoding amino acids from positions 1-50 to about 200-300 of an N-terminal part of influenza virus polymerase subunit PA-PAN, wherein the vector comprises a protein tag, and transforming cells with the expression vector in order to express tagged PAN fusion protein, wherein the N-terminal part of PA-PAN has at least 40% sequence identity with the amino acids listed in FIG. 1C.
In one embodiment, the present invention provides a method of expressing wild type or mutant protein of an N-terminal part of PA-PAN, wherein gene sequence of the N-terminal part of polymerase subunit PA-PAN is cloned into plasmid vectors for example, a series of pGEX vectors such as pGEX-6p, pGEX-4 T (Amersham Pharmacia), a series of pET vectors (Novagen) and a series of pMAL-c2 (Invitrogen) to express a fusion protein GST-PAN wherein the N-terminus of PAN is fused to GST; wherein the plasmid vector comprises a penicillin resistance gene and a restriction site employed when cloning the vector with the gene of an N-terminal polypeptide of PA-PAN, wherein the restriction site is selected from a group consisting of BamHI and XhoI from multiple cloning sites in pGEX-6p; the restriction site used for cloning a PAN gene fragment is BamHI and XhoI; amplifying a gene fragment of PAN protein from the genome of influenza virus A type: A/goose/Guangdong/1/96 by polymerase chain reaction (PCR) method; treating the vector and the inserted gene fragment with corresponding DNA restriction enzymes respectively, such as those selected from a group consisting of BamHI and XhoI, and ligating gene to be inserted and the vector by T4 DNA ligase, before transforming E. coli to obtain cloned plasmids. The cloned plasmid as described above is transformed into E. coli BL21, the resulting transformed bacteria are cultured and induced by using 0.1 mM to 1 mM IPTG, and the cultured bacteria are centrifuged to obtain the microbial population that express said fusion protein.
In one embodiment, the present invention provides a method of screening candidate compounds which compete with PB1N for binding PAc, the method comprising: (a) attaching PAc to the surface of a fixed support; (b) contacting an excess of tagged PB1N with the attached PAc; (c) eluting thoroughly with an eluent in order to remove un-bound PB1N; (d) contacting a solution with the candidate compound to be detected with the attached PAc with bound PB1N; (e) eluting thoroughly with an eluent in order to obtain a solution to be detected; (f) measuring the concentration of free tagged PB1N in the solution to be detected; (g) calculating the binding capability of the candidate compound to be detected with PAc according to the concentration of free tagged PB1N in the solution to be detected.
In one embodiment, the present invention provides a method of screening candidate compounds which compete with PB1N for binding PAc, wherein attaching PAc to surface of the fixed support in step (a) is achieved by covalently cross-linking or binding PAc with an affinity tag, and determining binding of PAc to the corresponding affinity group attached to the surface of the fixed support.
In one embodiment, the present invention provides a method of screening candidate compounds which compete with PB1N for binding PAc, utilizing an affinity tag such as GST, Flag-tag, Myc-tag, MPB-tag and specific antibody, and wherein the corresponding affinity group is attached to the surface of the fixed support.
In one embodiment, the present invention provides a method of screening candidate compounds which compete with PB1N for binding PAc, wherein the tagged PB1N polypeptide is selected from a protein tagged with an isotope or a protein tagged with other molecules, preferably wherein the other molecular tag is green fluorescent protein or various fusion polypeptides, e.g. binding peroxidase, phosphohydrolase, protein kinase, various group transferase.
In one embodiment, the present invention provides a method of screening candidate compounds which compete with PB1N for binding PAc, wherein the fixed surface is an affinity chromatography column.
In one embodiment, the present invention provides the use of the three-dimensional crystal structure of a complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and N-terminal of influenza virus polymerase subunit PB1-PB1N in designing and screening a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate used in the treatment of diseases caused by the influenza virus infection.
Drug screening can be performed based on the above method of expressing and purifying PAc protein; the above method of expressing and purifying the complex of PAc and PB1N polypeptide and the above method of obtaining protein crystal, and drug design can be performed based on the three-dimensional structure of PAc and PB1N.
In one embodiment, the present invention provides use of the three-dimensional crystal structure of a complex of C-terminal part of influenza virus polymerase subunit PA-PAc and N-terminal part of influenza virus polymerase subunit PB1-PB1N in designing and screening a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate used in the treatment of diseases caused by the influenza virus infection, comprising:
designing a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to a specific portion of the polymerase subunit by computer simulation according to the coordinates of three-dimensional protein structure;
screening for a potential polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to specific portion of the polymerase subunit by computer simulation according to coordinates of three-dimensional protein structure;
analyzing binding characteristics of the designed or searched polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate according to the atomic coordinates of the three-dimensional structure of the protein to bind to any subtype of influenza virus polymerase protein which have at least 50% sequence identity with the PAc and the PB1N sequence;
the designed or searched polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate according to coordinates of three-dimensional protein structure bind to any subtype of influenza virus polymerase protein which has at least 50% sequence identity with the PAc and the PB1N sequence, and then crystallization is preformed, the binding characteristics of polypeptide or compound molecule to protein is analyzed through crystal diffraction method;
wherein a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate which binds to any subtype of influenza virus polymerase protein which has at least 50% sequence identity with the PAc and the PB1N sequence is a candidate compound.
To reveal the role of PA (SEQ ID NO:1) in polymerase and its fine three-dimensional structure, the inventors analyzed the crystal structure with a resolution of 2.9 Angstrom of the complex of a C-terminal fragment of residues 257-716 of PA (PAC) and the N-terminal 25 residues of PB1 (PB1N). This structure clearly indicates the interaction mode between the C-terminal part of PA (SEQ ID NO:1) and the N-terminal part of PB1 as well as the composition of amino acid residues that participate in the interaction and their relative spatial positions. The inventors also determined the three-dimensional structure of the complex of the C-terminal part of PA (SEQ ID NO:1) and the N-terminal part of PB1, the composition of secondary structure of the protein, the binding sites of nucleic acid in the PA (SEQ ID NO:1) protein, a small-molecule channel in the PA (SEQ ID NO:1) protein and the charge distribution on the surface of the PA (SEQ ID NO:1) protein, providing a structural basis for investigating the role of PA (SEQ ID NO:1) in the complex of virus RNA polymerase, and providing a protein crystallization? platform and a three-dimensional platform for designing drugs inhibiting interactions between PA (SEQ ID NO:1) and PB1, as well as interactions between PA (SEQ ID NO:1) and RNA or other proteins, to inhibit the activity of the influenza virus polymerase.
It should be noted that the amino acid sequences of the corresponding fragments in influenza virus B and C types relative to α helixes and β sheets of influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively, which is not listed in detail.



BRIEF DESCRIPTION OF THE DRAWINGS
This patent application file contains at least one drawing executed in color. Copies of this patent application publication with color drawings will be provided by the Office upon request and with payment of the necessary fee.
FIGS. 1A-a through 1A-d, 1B, and 1C-a through 1C-c: Sequence of the C-terminal of PA and the protein sequence of PB1N from three types of influenza virus. FIGS. 1A-a through 1A-d. Sequence alignment of PA protein from three types of influenza virus, wherein A_OURS is the sequence of the C-terminal of the PA protein from influenza virus A type: A/goose/Guangdong/1/96 (SEQ ID NO:1); A—1918 is the sequence of the PA protein from the influenza virus A type: A/Brevig Mission/1/1918 (SEQ ID NO:3), which is the cause of a widespread influenza outbreak and a great death event in Europe in 1918; B—1966 is the sequence of the PA protein from the influenza virus B type: B/Ann Arbor/1/1966 (SEQ ID NO:4); C—1950 is the sequence of the PA protein from the influenza virus C type: C/JJ/1950 (SEQ ID NO:5); this result indicates that the protein of influenza virus polymerase subunit PA has highly conservative amino acid residues. FIG. 1B. Sequence alignment of the PB1N protein from four type of influenza virus, wherein A_OURS (SEQ ID NO:2), A—1918 (SEQ ID NO:6), B—1966 (SEQ ID NO:7), C—1950 (SEQ ID NO:8) are as described above. In these figures, “. . . ” is used to indicate amino acid deletion in corresponding fragments. Specific amino acid positions in the specification and claims are illustrated by the example of A_OURS (SEQ IDS NO:1 and 2).
FIGS. 1C-a through 1C-d: Sequence of the N-terminal of the PA protein from three types of influenza viruses, wherein A_OURS (SEQ ID NO:1), A—1918 (SEQ ID NO:3), B—1966 (SEQ ID NO:4), C—1950 (SEQ ID NO:5) are as described above. In these figures, “. . . ” is used to indicate amino acid deletion in the corresponding fragments. Specific amino acid positions in the specification and claims are illustrated by the example of A_OURS (SEQ ID NO:1).
FIG. 2: Purification of a polypeptide fragment of the former 256 amino acid in PA N-terminal of influenza virus A type: A/goose/Guangdong/1/96, wherein A shows the peak of purified protein during gel filtration chromatography Superdex-200 (Amersham Pharmacia Inc.). B represents electrophoresis result from PA N-terminal of corresponding peak. C is the finally purified protein of PA N-terminal.
FIG. 3: Protein crystallization and diffraction obtained from purified protein of PA N-terminal (1-256 residues), wherein A and B are crystals obtained under different crystallization conditions; and C and D show X-ray diffraction pattern of two protein crystals from PA N-terminal.
FIG. 4: PAc and GST-PB1N protein expressed and purified in E.coli, and interaction experiment between PAc and PB1 N-terminal peptide, and ribbon image of structure of the complex between purified PAc and PB1N polypeptide. A. purified PAc protein, and in vitro interaction experiment between purified PAc and PB1 N-terminal peptide. B. Diagram of three-dimensional structure of the complex between PAc and PB1N polypeptide, which shows a overall structure of chewing wolf's head.
FIG. 5: Overall structure of the complex of C-terminal of PA and N-terminal of PB1, wherein: A. side view of overall structure of the complex of PAc and PB1N, to clearly show the direction of protein chain; B. Image in FIG. 5(A) is rotated by 180°, wherein secondary structure is marked, and the two ends of PB1 N-terminal peptide and PAc is indicated by arrows.
FIG. 6: Interaction between PAc and PB1N polypeptide, wherein: A. surface image of PA (SEQ ID NO:1) protein molecule; N-terminal polypeptide of PB1 is illustrated by ribbon image, whereas PA (SEQ ID NO:1) molecule is illustrated by surface charge distribution. B. zoomed to observe that interaction between PA (SEQ ID NO:1) and PB1 is mainly through hydrophobic interaction.
FIG. 7: Surface charge pattern of PA (SEQ ID NO:1) protein molecule, wherein A and B are consistent with the direction of A and B in FIG. 5 respectively.
FIG. 8: Schematic diagrams of big groove and channel present in molecular structure of PAc/PB1N complex. A illustrates the big groove, and some alkaline amino acids residues are marked. B and C are drawings of channel observed from the two sides respectively (rotate 180° about X axis). Some reserved amino acids in or around the channel are marked.
FIG. 9: Illustration of interaction between PA-C and PB1-N: A. ribbon image of the three-dimensional structure of the complex between PA-C and PB1-N polypeptide; B. surface image of the PA (SEQ ID NO:1) protein molecule. The N-terminal polypeptide of PB1 is illustrated by a ribbon image, whereas the PA (SEQ ID NO:1) molecule is illustrated by a surface charge distribution. C. Expanded view of a part of the complex to observe that helices 8, 10, 11 and 13 of the PA (SEQ ID NO:1) molecule interact with the PB1 polypeptide. D. Expanded view of the interaction between PA (SEQ ID NO:1) and PB1. N-terminal polypeptide of PB1 is illustrated by ribbon image, whereas PA (SEQ ID NO:1) molecule is illustrated by surface charge distribution. The two polypeptides bind mainly through hydrophobic interaction.
FIGS. 10-a through 10-e: As described in FIGS. 1A-a through 1A-d and 1B: Sequence of the C-terminal of PA and the protein sequence of PB1N from three types of influenza virus. FIGS. 10-a through 10-d. Sequence alignment of PA protein from three types of influenza virus, wherein A_OURS is the sequence of the C-terminal of the PA protein from influenza virus A type: A/goose/Guangdong/1/96 (SEQ ID NO:1); A—1918 is the sequence of the PA protein from the influenza virus A type: A/Brevig Mission/1/1918 (SEQ ID NO:3), which is the cause of a widespread influenza outbreak and a great death event in Europe in 1918; B—1966 is the sequence of the PA protein from the influenza virus B type: B/Ann Arbor/1/1966 (SEQ ID NO:4); C—1950 is the sequence of the PA protein from the influenza virus C type: C/JJ/1950 (SEQ ID NO:5); this result indicates that the protein of influenza virus polymerase subunit PA has highly conservative amino acid residues. FIG. 10-e. Sequence alignment of the PB1N protein from four type of influenza virus, wherein A_OURS (SEQ ID NO:2), A—1918 (SEQ ID NO:6), B—1966 (SEQ ID NO:7), C—1950 (SEQ ID NO:8) are as described above. In these figures, “. . . ” is used to indicate amino acid deletion in corresponding fragments. Specific amino acid positions in the specification and claims are illustrated by the example of A_OURS (SEQ ID NO:2). The box noted with “Round Loop” indicates a big-ring region in the structure, and the other box (unmarked) is a possible nucleic acid binding region. Arrows indicate amino acid residues in PA that participate in binding with the PB1 short peptide. Specific amino acid positions in the specification and claims are illustrated by the example of A_OURS (SEQ IDS NO:1 and 2).



EMBODIMENTS
The present invention provides a method of expressing fragments of wild type or mutant protein of influenza virus polymerase subunit PA (SEQ ID NO:1), wherein an N-terminal part and a C-terminal part are expressed and purified in E.coli respectively, comprising: a method of only using an N-terminal part of PA for a crystallization experiment; a method of expressing and purifying a fragment comprising residues 257-716 in the C-terminal part of PA as well as the wild type and mutant protein of the N-terminal part of influenza virus subunit PB1 in E. coli respectively. Also provided is a method of crystallizating the complex of a C-terminal part of PA and an N-terminal peptide of PB1, and the crystal structure of complex of PAc/PB1N short peptide obtained therefrom, and a method of drug screening based on these crystallization methods as well as a method of drug design based on these crystal structures.
In one embodiment, the present invention provides a three-dimensional crystal structure of a complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, wherein the C-terminal part of the influenza virus polymerase subunit PA-PAc comprises amino acids from about amino acid position 201-301 to about 650 terminus, wherein the N-terminal part of of influenza virus polymerase subunit PB1-PB1N is a short peptide within the 48 N-terminal amino acids of the of influenza virus polymerase subunit PB1-PB1N, wherein the atoms of the three-dimensional crystal structure have at least 40% of the atomic coordinates listed in table 1, or the atomic coordinates of main chain backbone carbons of at least 40% of amino acids in the three-dimensional crystal structure of a complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N have an average root mean square deviation smaller than or equal to 1.7 Angstrom with respect to the atomic coordinates listed in table 1.
In one embodiment, the present invention provides crystal three-dimensional structure of complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, wherein the influenza virus is selected from influenza virus A, B and C type, preferably influenza virus A type: A/goose/Guangdong/1/96, A/Brevig Mission/1/1918; influenza virus B type: B/Ann Arbor/1/1966 or influenza virus C type: C/JJ/1950.
In a preferred embodiment, the present invention provides a three-dimensional crystal structure of complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, wherein the crystal of the complex has a P4(1)2(1)2 space group of and the lattice parameters are about: a=b=122 Angstrom, c=133 Angstrom, α=β=γ=90°.
In a preferred embodiment, the present invention provides a three-dimensional crystal structure of a complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, wherein the C-terminal part of influenza virus A polymerase subunit PA-PAc consists of a first portion and a second portion, wherein the first portion comprises α helix 4, i.e. a fragment of amino acid positions 406-414, α helix 5, i.e. a fragment of amino acid positions 440-450, α helix 8, i.e. a fragment of amino acid positions 583-603, α helix 9, i.e. a fragment of amino acid positions 608-613, α helix 10, i.e. a fragment of amino acid positions 633-649, α helix 11, i.e. a fragment of amino acid positions 653-673, α helix 12, i.e. a fragment of amino acid positions 683-691, α helix 13, i.e. a fragment of amino acid positions 698-714, β sheet 8, i.e. a fragment of amino acid positions 619-623 and β sheet 9, i.e. a fragment of amino acid positions 628-631; wherein the second portion comprises α helix 1, i.e. a fragment of amino acid positions 303-311, α helix 2, i.e. a fragment of amino acid positions 331-349, α helix 3, i.e. a fragment of amino acid positions 364-369, α helix 6, i.e. a fragment of amino acid positions 454-475 and α helix 7, i.e. a fragment of amino acid positions 572-578, βsheet 1, i.e. amino acids fragment of 290-292 positions, β sheet 2, i.e. a fragment of amino acid positions 317-324, β sheet 3, i.e. a fragment of amino acid positions 480-491, β sheet 4, i.e. a fragment of amino acid positions 496-506, β sheet 5, i.e. a fragment of amino acid positions 517-526, β sheet 6, i.e. a fragment of amino acid positions 541-550 and β sheet 7, i.e. a fragment of amino acid positions 557-571 positions; wherein the β sheets of the second portion of the C-terminal of PA-PAc is surrounded by α helix 1, α helix 2, α helix 3, α helix 6, and α helix 7, wherein fragments of influenza virus B and C type corresponding to α helixes and β sheets of influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B, respectively.
In a preferred embodiment, the present invention provides a three-dimensional crystal structure of a complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, wherein the C-terminal part of influenza virus A polymerase subunit PA-PAc interacts with the N-terminal part of PB1-PB1N mainly through α helix 8, α helix 10, α helix 11 and α helix 13, preferably through at least one amino acid selected from a group consisting of Leu666 of α helix 11, Phe710 of α helix 13, Val636 of α helix 10, Leu640 of α helix 10, Trp706 of α helix 13 and Gln670 of α helix 11, wherein amino acids of fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In a preferred embodiment, the present invention provides a three-dimensional crystal structure of a complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, wherein at least one amino acid selected from the group consisting of Ile621, Gly622, Glu623, Thr618 and Pro620 interacts with the influenza virus polymerase subunit PB1, Ile621, Gly622, Glu623, Thr618 and Pro620 being in the peptide loop between α helix 9 and α helix 10 of the C-terminal part of influenza virus A polymerase subunit PA-PAc, wherein amino acids of fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In a preferred embodiment, the present invention provides a three-dimensional crystal structure of a complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, wherein at least one amino acid selected from the group consisting of Asn647, Gln408, Cys584, Gln587, Gln591, Lys643, Asn647, Ser659, Lys663, Trp699 and Asn703 of the C-terminal of influenza virus A polymerase subunit PA-PAc constitutes a “pocket” of amino acid residues which bind the influenza virus polymerase subunit PB1N, wherein amino acids of fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In a preferred embodiment, the present invention provides a three-dimensional crystal structure of a complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, wherein at least one amino acid selected from the group consisting of Trp406, Glu410, Lys461, Glu524, Phe525, Ser526, Lys536, Lys539, Tyr540, Leu563, Tyr564, Arg566 and Lys574 of the C-terminal of influenza virus A polymerase subunit PA-PAc constitutes a large “groove” and a “channel” structure which bind nucleotide, RNA or other small molecules or proteins, wherein amino acids of fragments of influenza virus B and C type corresponding to the influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In a preferred embodiment, the present invention provides a three-dimensional crystal structure of a complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, wherein amino acid residues of positions 370405 of the C-terminal part of influenza virus A polymerase subunit PA-PAc constitutes a large loop, wherein amino acids of fragments of influenza virus B and C type corresponding to the influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In a preferred embodiment, the present invention provides a three-dimensional crystal structure of a complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, wherein α helix 12 and α helix 13 of C-terminal of influenza virus polymerase subunit PA-PAc interact with other proteins, preferably wherein at least one amino acid selected from the group consisting of Ile690, Glu691, Glu692, Cys693 and Asn696 of the α helix 12 and α helix 13 interacts with other proteins, and amino acids of fragments of influenza virus B and C type corresponding to the influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In a preferred embodiment, the present invention provides a three-dimensional crystal structure of complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, wherein at least one amino acid of the C-terminal part of influenza virus polymerase subunit PA-PAc that is selected from the group consisting of Lys506, Gly507, Arg508, Ser509, His510, Leu511, Arg512, Asn513 and Asp514 interacts with other proteins, wherein His510 constitutes a portion of the polymerase complex RNAse, and amino acids of fragments of influenza virus B and C type corresponding to the influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In another embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound binding to at least one member selected from the group consisting of α helix 8, α helix 10, α helix 11 and α helix 13 of C-terminal of influenza virus polymerase subunit PA-PAc, wherein the influenza virus is selected from influenza virus A, B and C type, preferably influenza virus A type: A/goose/Guangdong/1/96, A/Brevig Mission/1/1918; influenza virus B type: B/Ann Arbor/1/1966 or influenza virus C type: C/JJ/1950; wherein the polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate preferably binds to a member selected from the group consisting of Leu666 in α helix 11, Phe710 in α helix 13, Val636 and Leu640 in α helix 10, Trp706 in α helix 13, Gln670 in α helix 11 of the C-terminal part of influenza virus polymerase subunit PA-PAc, wherein amino acids of fragments of influenza virus B and C type corresponding to the influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In another embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to at least one member selected from the group consisting of Ile621, Gly622, Glu623, Thr618 and Pro620 located at the peptide loop between α helix 9 and α helix 10 of the C-terminal part of influenza virus polymerase subunit PA-PAc, wherein amino acids of fragments of influenza virus B and C type corresponding to the influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In another embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to at least one amino acid selected from the group consisting of Asn647, Gln408, Cys584, Gln587, Gln591, Lys643, Asn647, Ser659, Lys663, Trp699 and Asn703 of the C-terminal part of influenza virus A polymerase subunit PA-PAc, wherein amino acids of fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In another embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to at least one amino acid selected from the group consisting of Trp406, Glu410, Lys461, Glu524, Phe525, Ser526, Lys536, Lys539, Tyr540, Leu563, Tyr564, Arg566 and Lys574 of the C-terminal part of influenza virus A polymerase subunit PA-PAc, wherein amino acids of fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In another embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to amino acids position 370-405 of the C-terminal part of influenza virus A polymerase subunit PA-PAc, wherein amino acids of fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In another embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to helix 12 and α helix 13 of the C-terminal part of influenza virus A polymerase subunit PA-Pac, preferably to at least one amino acid selected from the group consisting of Ile690, Glu691, Glu692, Cys693 and Asn696 in α helix 12 and α helix 13, wherein amino acids of fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In another embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to at least one amino acid selected from the group consisting of Lys506, Gly507, Arg508, Ser509, His510, Leu511, Arg512, Asn513 and Asp514 located at loop region between β sheet 4 and β sheet 5 in the C-terminal part of influenza virus A polymerase subunit PA-PAc, wherein amino acids of fragments of influenza virus B and C type corresponding to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In another embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate which competes with influenza virus polymerase subunit PB1 (SEQ ID NO:2) for binding PAc.
In a preferred embodiment, the present invention provides the polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate which competes with influenza virus polymerase subunit PB1 (SEQ ID NO:2) for binding PAc predominantly by an interaction with PAc through the hydrophobic core constituted by the α helix 8, α helix 11, α helix 13 and α helix 10, preferably interaction with PAc through Met595 in α helix 8, Leu666 in α helix 11, Trp706 and Phe710 in α helix 13, Val636 and Val640 in α helix 10, wherein amino acids of corresponding fragments of influenza virus B and C type to influenza virus A type are shown in FIGS. 1A, 1B, 1C and FIGS. 10A, 10B respectively.
In a preferred embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate which competes with influenza virus polymerase subunit PB1 (SEQ ID NO:2) for binding PAc, wherein the amino acid sequence of the polypeptide or protein comprises at least three amino acids which are identical to amino acids of corresponding position of a short PTLLFL motif of the short helix domain constituted by the 5th-10th residues Pro5, Thr6, Leu7, Leu8, Phe9 and Leu10 of the N-terminal part of wild influenza virus polymerase subunit PB1-PB1N, when the polypeptide or protein is aligned with the PTLLFL motif.
In another embodiment, the present invention provides a composition comprising above-mentioned polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate, and optionally comprising a carrier and an excipient.
In another embodiment, the present invention provides use of the composition in the manufacture of a medicament used in the treatment of diseases caused by influenza virus.
In another embodiment, the present invention provides a method of expressing and purifying the complex of an C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, comprising:
(a) constructing a vector with a gene sequence encoding amino acid from about positions 201-301 to about 650 terminus of a C-terminal part of influenza virus polymerase subunit PA-PAc, where the vector can further comprise a protein tag, wherein prokaryotic cells or eukaryotic cells are transformed with said vector in order to express a tagged PAc fusion protein;
(b) using a method analogous to the method of expressing PAc to express the PB1N with or without a protein tag;
(c) Proportionally mixing the cells expressing influenza virus polymerase subunit PAc obtained from step (a) and the cells expressing amino acids within the 48 amino acids of the N-terminal of influenza virus polymerase subunit PB1 obtained from step (b), wherein the resulting protein is isolated by the specific recognition of the specific tag, the protein tag is removed from the protein by enzymolysis, the complex of PAc and PB1N is isolate, and the concentration of the complex is determined;
wherein the atoms of the three-dimensional crystal structure of the complex of the C-terminal part of influenza virus polymerase subunit PA-PAc and the N-terminal part of influenza virus polymerase subunit PB1-PB1N have at least 40% of the atomic coordinates listed in table 1, or atomic coordinates of main chain backbone carbons of at least 40% amino acids in the three-dimensional crystal structure of the complex of the C-terminal part of influenza virus polymerase subunit PA-PAc and the N-terminal part of influenza virus polymerase subunit PB1-PB1N have an average root mean square deviation smaller than or equal to 1.7 Angstrom with respect to the atomic coordinates listed in table 1.
In a preferred embodiment, wherein the protein tag is selected from GST, Flag-tag, Myc-tag, MBP-tag, specific antibodies; the vector comprises a selection marker gene; the proportional mixing in step (c) above means that the molar ratio of protein-tagged PAc and protein-tagged PB1N is 0.1:1-1:0.1, preferably the molar ratio of protein-tagged PAc and protein-tagged PB1N is 0.5:1-1:0.5, more preferably the molar ratio of protein-tagged PAc and protein-tagged PB1N is nearly 1:1; wherein preferably the protein tag is GST, wherein the tag is specifically recognized using an affinity column, wherein the tag is removed by a proteinase, wherein the complex of PAc and PB1N is separated by gel filtration or ion-exchange chromatography, and wherein the the protein purity is determined by gel electrophoresis.
In a more preferable embodiment, the procaryotic cell is E coli. 
In another embodiment, the present invention provides a method of co-crystallizing the complex of an C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N, comprising:
adjusting the protein concentration of the purified complex of PAc and PB1N to 5-30 mg/ml;
screening crystal growth conditions by gas sitting drop and hanging drop;
obtaining a crystal of the complex of the C-terminal part of influenza virus polymerase subunit PA-PAc and the N-terminal part of influenza virus polymerase subunit PB1-PB1N.
In another embodiment, the present invention provides a method of expressing wild type or mutant protein of an N-terminal part of PA-PAN, wherein PAN comprises amino acids from about position 1-50 to about position 200-300, the method comprising: constructing an expression vector with a gene sequence encoding amino acid from about position 1-50 to about 200-300 of the N-terminal part of influenza virus polymerase subunit PA-PAN, the vector comprising a gene for protein tag fusion or no protein tag fusion, wherein eukaryotic cells or prokaryotic cells are transformed with said vector in order to express PAN with or without the protein tag, wherein the amino acid sequence of the N-terminal part of PA-PAN exhibits at least 40% sequence identity with the amino acids sequence listed in FIG. 1C.
In a preferred embodiment, the procaryotic cell is E coli. 
In another embodiment, the present invention provides a method of screening candidate compounds which compete with PB1N for binding PAc, the method comprising:
(a) attaching PAc to the surface of a fixed support;
(b) contact the excess tagged PB1N with the attached PAc;
(c) eluting thoroughly with an eluent in order to remove un-bound PB1N;
(d) contacting a solution with the candidate compound to be detected with the attached PAc with bound PB1N;
(e) eluting thoroughly with an eluent in order to obtain a solution to be detected;
(f) measuring the concentration of free tagged PB1N in the solution to be detected;
(g) calculating the binding capability of the candidate compound to be detected with PAc according to the concentration of free tagged PB1N in the solution to be detected.
In a preferred embodiment, PAc is attached to the surface of the fixed support in step (a) by covalently cross-linking or binding PAc with an affinity tag, and attaching PAc to the fixed support by affinity binding.
Preferably, the affinity tag is selected from GST, Flag-tag, Myc-tag, MBP-tag and specific antibody and there is corresponding binding group of the affinity tag on the surface of the fixed support.
Preferably, the PB1N polypeptide is tagged with an isotope or another chemical molecule; preferably, the other chemical molecular tag is green fluorescent protein or various fusion polypeptides.
Preferably, the fixed surface can be affinity chromatography columns.
In one embodiment, the present invention provides use of the three-dimensional crystal structure of complex of a C-terminal part of influenza virus polymerase subunit PA-PAc and an N-terminal part of influenza virus polymerase subunit PB1-PB1N in designing and screening a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate used in the treatment of diseases caused by the influenza virus infection, comprising:
designing polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate binding to a specific portion of the polymerase by computer simulation technology according to the coordinates of the three-dimensional protein structure;
screening for a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate potentially binding to a specific portion of the polymerase by computer simulation technology according to the coordinates of the three-dimensional protein structure;
analyzing the binding characteristics of the designed or screened polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate according to the coordinates of three-dimensional protein structure in binding to any type of influenza virus polymerase protein which has at least 50% sequence identity with the PAc and the PB1N sequence;
crystallizing the designed or screened polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate according to the coordinates of three-dimensional protein structure in binding to any type of influenza virus polymerase protein which has at least 50% sequence identity with the PAc and the PB1N sequence, and
analyzing the binding characteristics of the polypeptide or compound molecule to the protein by crystal diffraction;
wherein the polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate is a candidate compound in that it binds to any type of influenza virus polymerase protein which has at least 50% sequence identity with the PAc and the PB1N sequence.
In one embodiment, the present invention provides a structure of the three subunits i.e. PA, PB1, PB2 of any subtype of influenza virus polymerase or the complex of PA, PB1 and PB2, wherein a protein contained in it or a fragment thereof has 40% identical sequence with the PAc protein.
In one embodiment, the present invention provides a three-dimensional stereochemical structure of the three subunits i.e. PA, PB1, PB2 of any subtype of influenza virus polymerase or that of the complex of PA, PB1 and PB2, wherein the coordinates of the three-dimensional structure in main chain of a protein contained in it or a fragment thereof has an average root mean square deviation smaller than or equal to 1.7 Angstrom with respect to the three-dimensional atomic coordinates of main chain backbone carbons having at least 40% amino acid residues of the PAc protein sequence.
In one embodiment, the present invention provides structure of subunit PA, PB1, PB2 or that of the complex of subunit PA, PB1 and PB2 from any subtype of influenza virus, wherein a protein fragment contained in it has 20% sequence homology with the fragment of amino acids 1-11 of the PB1N polypeptide, preferably 40% sequence homology.
In one embodiment, the present invention provides a polypeptide or a small molecule, characterized in that it interacts with any amino acid of the influenza virus subunit PA.
In one embodiment, the present invention provides use of the crystal three-dimensional structure in drug screening and drug design.
In one embodiment, the present invention provides a method of screening a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate which binds to the protein based on a three-dimensional crystal structure of PAc and PB1N protein, comprising: obtaining a PAc-containing crystal by protein crystallization, or obtaining the coordinates of a three-dimensional crystal structure of the protein complex containing PAc and PB1N; wherein the three-dimensional structure comprises any structure that has an average root mean square deviation smaller than or equal to 1.7 Angstrom with respect to the three-dimensional coordinates of a main chain backbone carbons having at least 40% amino acid residues of the atomic coordinates.
In one embodiment, the present invention provides a method of expressing and purifying the influenza virus subunit PA through expressing PA fragments in bacteria and eukaryotic cell expression systems.
In one embodiment, the present invention provides a polypeptide, protein, inorganic compound or organic compound, antibody or immunoconjugate that interacts with amino-acid residues on a protein, wherein the protein has at least 40% identical amino acids with any fragment of α helix 8, 10, 11 and 13 in complex of C-terminal of influenza virus polymerase subunit PA-PAc and N-terminal of influenza virus polymerase subunit PB1-PB1N.
It should be noted that the fragments in influenza virus B and C type corresponding to α helix and β sheet of influenza virus A are shown in FIGS. 1A, 1B, 1C and FIG. 10A, FIG. 10B respectively, which will not be listed in detail. The alignment methods of protein or polypeptide sequence that can be used is for example: CLUSTALW (http://www.ebi.ac.uk/Tools/clustalw2/index.html).
Expression and Purification Methods of Influenza Virus PA (SEQ ID NO:1) and PB1 (SEQ ID NO:2) Protein
The protein sequences encoded by virus gene derived from Avian influenza virus A/goose/Guangdong/1/96 are respectively as follows:
(1) Protein Sequence of PA:







MEDFVRQCFNPMIVELAEKAMKEYGEDPKIETNKFAAICTHLEVCFMYS


 


DNIFIDERGESTIIESGDPNALLKHRFEIIEGRDRTMAWTVVNSICNTT


 


GVEKPKFLPDLYDYKENRFIEIGVTRREVHTYYLEKANKIKSEKTHIHI


 


FSFTGEEMATKADYTLDEESRARIKTRLFTIRQEMASRGLWDSFRQSER


 


GEETIEERFEITGTMCRLADQSLPPNFSSLEKFRAYVDGEEPNGCIEGK


 


LSQMSKEVNARIEPFLKTTPRPLRLPDGPPCSQRSKFLLMDALKLSIED


 


PSHEGEGIPLYDAIKCMKTFFGWKEPNIVKPHEKGINPNYLLAWKQVLA


 


ELQDIENEEKIPKTKNMRKTSQLKWALGENMAPEKVDFEDCKDVSDLRQ


 


YDSDEPKPRSLASWIQSEFNKACELTDSSWIELDEIGEDVAPIEHIASM


 


RRNYFTAEVSHCRATEYIMKGVYINTALLNASCAAMDDFQLIPMISKCR


 


TKEGRRKTNLYGFIIKGRSHLRNDTDVVNFVSNIEFSLTDPRLEPHKWE


 


KYCVLEIGDMLLRTAIGQVSRPMFLYVRTNGTSKIKMKWGMEMRRCLLQ


 


SLQQIESMIEAESSVKEKDMTKEFFENKSETWPIGESPKGMEEGSIGKV


 


CRTLLAKSVFNSLYASPQLEGFSAESRKLLLIVQALRDNLEPGTFDLGG


 


LYEAIEECLINDPWVLLNASWFNSFLTHALK; i.e.,


 


( SEQ ID NO: 1 )


Met Glu Asp Phe Val Arg Gln Cys Phe Asn Pro Met 


 


Ile Val Glu Leu Ala Glu Lys Ala Met Lys Glu Tyr 


 


Gly Glu Asp Pro Lys Ile Glu Thr Asn Lys Phe Ala 


 


Ala Ile Cys Thr His Leu Glu Val Cys Phe Met Tyr 


 


Ser Asp Phe His Phe Ile Asp Glu Arg Gly Glu Ser 


 


Thr Ile Ile Glu Ser Gly Asp Pro Asn Ala Leu Leu 


 


Lys His Arg Phe Glu Ile Ile Glu Gly Arg Asp Arg 


 


Thr Met Ala Trp Thr Val Val Asn Ser Ile Cys Asn 


 


Thr Thr Gly Val Glu Lys Pro Lys Phe Leu Pro Asp 


 


Leu Tyr Asp Tyr Lys Glu Asn Arg Phe Ile Glu Ile 


 


Gly Val Thr Arg Arg Glu Val His Thr Tyr Tyr Leu 


 


Glu Lys Ala Asn Lys Ile Lys Ser Glu Lys Thr His 


 


Ile His Ile Phe Ser Phe Thr Gly Glu Glu Met Ala 


 


Thr Lys Ala Asp Tyr Thr Leu Asp Glu Glu Ser Arg 


 


Ala Arg Ile Lys Thr Arg Leu Phe Thr Ile Arg Gln 


 


Glu Met Ala Ser Arg Gly Leu Trp Asp Ser Phe Arg 


 


Gln Ser Glu Arg Gly Glu Glu Thr Ile Glu Glu Arg 


 


Phe Glu Ile Thr Gly Thr Met Cys Arg Leu Ala Asp 


 


Gln Ser Leu Pro Pro Asn Phe Ser Ser Leu Glu Lys 


 


Phe Arg Ala Tyr Val Asp Gly Phe Glu Pro Asn Gly 


 


Cys Ile Glu Gly Lys Leu Ser Gln Met Ser Lys Glu


 


Val Asn Ala Arg Ile Glu Pro Phe Leu Lys Thr Thr


 


Pro Arg Pro Leu Arg Leu Pro Asp Gly Pro Pro Cys 


 


Ser Gln Arg Ser Lys Phe Leu Leu Met Asp Ala Leu 


 


Lys Leu Ser Ile Glu Asp Pro Ser His Glu Gly Glu 


 


Gly Ile Pro Leu Tyr Asp Ala Ile Lys Cys Met Lys


 


Thr Phe Phe Gly Trp Lys Glu Pro Asn Ile Val Lys 


 


Pro His Glu Lys Gly Ile Asn Pro Asn Tyr Leu Leu 


 


Ala Trp Lys Gln Val Leu Ala Glu Leu Gln Asp Ile 


 


Glu Asn Glu Glu Lys Ile Pro Lys Thr Lys Asn Met 


 


Arg Lys Thr Ser Gln Leu Lys Trp Ala Leu Gly Glu 


 


Asn Met Ala Pro Glu Lys Val Asp Phe Glu Asp Cys 


 


Lys Asp Val Ser Asp Leu Arg Gln Tyr Asp Ser Asp 


 


Glu Pro Lys Pro Arg Ser Leu Ala Ser Trp Ile Gln 


 


Ser Glu Phe Asn Lys Ala Cys Glu Leu Thr Asp Ser 


 


Ser Trp Ile Glu Leu Asp Glu Ile Gly Glu Asp Val 


 


Ala Pro Ile Glu His Ile Ala Ser Met Arg Arg Asn 


 


Tyr Phe Thr Ala Glu Val Ser His Cys Arg Ala Thr 


 


Glu Tyr Ile Met Lys Gly Val Tyr Ile Asn Thr Ala 


 


Leu Leu Asn Ala Ser Cys Ala Ala Met Asp Asp Phe 


 


Gln Leu Ile Pro Met Ile Ser Lys Cys Arg Thr Lys 


 


Glu Gly Arg Arg Lys Thr Asn Leu Tyr Gly Phe Ile 


 


Ile Lys Gly Arg Ser His Leu Arg Asn Asp Thr Asp 


 


Val Val Asn Phe Val Ser Met Glu Phe Ser Leu Thr


 


Asp Pro Arg Leu Glu Pro His Lys Trp Glu Lys Tyr 


 


Cys Val Leu Glu Ile Gly Asp Met Leu Leu Arg Thr 


 


Ala Ile Gly Gln Val Ser Arg Pro Met Phe Leu Tyr 


 


Val Arg Thr Asn Gly Thr Ser Lys Ile Lys Met Lys 


 


Trp Gly Met Glu Met Arg Arg Cys Leu Leu Gln Ser 


 


Leu Gln Gln Ile Glu Ser Met Ile Glu Ala Glu Ser 


 


Ser Val Lys Glu Lys Asp Met Thr Lys Glu Phe Phe 


 


Glu Asn Lys Ser Glu Thr Trp Pro Ile Gly Glu Ser 


 


Pro Lys Gly Met Glu Glu Gly Ser Ile Gly Lys Val 


 


Cys Arg Thr Leu Leu Ala Lys Ser Val Phe Asn Ser 


 


Leu Tyr Ala Ser Pro Gln Leu Glu Gly Phe Ser Ala 


 


Glu Ser Arg Lys Leu Leu Leu Ile Val Gln Ala Leu 


 


Arg Asp Asn Leu Glu Pro Gly Thr Phe Asp Leu Gly 


 


Gly Leu Tyr Glu Ala Ile Glu Glu Cys Leu Ile Asn 


 


Asp Pro Trp Val Leu Leu Asn Ala Ser Trp Phe Asn 


 


Ser Phe Leu Thr His Ala Leu Lys.






(2) Protein Sequence of PB1:








MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQY


 


SEKGKWTTNTETGAPQLNPIDGPLPEDNEPSGYAQTDCVLEAMAFLEKS


 


HPGIFENSCLETMEIVQQTRVDKLTQGRQTYDWTLNRNQPAATALANTI


 


EVFRSNGLTANESGRLIDFLKDVMESMDKGEMEIITHFQRKRRVRDNMT


 


KKMVTQRTIGKKKQRLNKRSYLIRALTLNTMTKDAERGKLKRRAIATPG


 


MQIRGFVYFVETLARSICEKLEQSGLPVGGNEKKAKLANVVRKMMTNSQ


 


DTELSFTITGDNTKWNENQNPRMFLAMITYITRNQPEWFRNVLSIAPIM


 


FSNKMARLGKGYMFESKSMKLRTQIPAEMLASIDLKYFNESTRKKIEKI


 


RPLLIDGTASLSPGNIMMGMFNMLSTVLGVSILNLGQKRYTKTTYWWDG


 


LQSSDDFALIVNAPNHEGIQAGVDRFYRTCKLVGINMSKKKSYINRTGT


 


EEFTSFFYRYGFVANFSMELPSFGVSGINESADMSIGVTVIKNNMINND


 


LGPATAQMALQLFIKDYRYTYRCHRGDTQIQTRRSEELKKLWEQTRSKA


 


GLLVSDGGPNLYNIRNLHIPEVCLKWELMDEDYQGRLCNPLNPFVSHKE


 


IESVNNAVVMPAHGPAKSMEYDAVATTHSWIPKRNRSILNTSQRGILED


 


EQMYQKCCNLFEKFFPSSSYRRPVGISSMVEAMVSRARIDARIDFESGR


 


IKKEEFAEIMKICSTIEELRRQK; i.e.,


 


( SEQ ID NO: 2 )


Met Asp Val Asn Pro Thr Leu Leu Phe Leu Lys Val 


 


Pro Ala Gln Asn Ala Ile Ser Thr Thr Phe Pro Tyr 


 


Thr Gly Asp Pro Pro Tyr Ser His Gly Thr Gly Thr 


 


Gly Tyr Thr Met Asp Thr Val Asn Arg Thr His Gln 


 


Tyr Ser Glu Lys Gly Lys Trp Thr Thr Asn Thr Glu 


 


Thr Gly Ala Pro Gln Leu Asn Pro Ile Asp Gly Pro 


 


Leu Pro Glu Asp Asn Glu Pro Ser Gly Tyr Ala Gln 


 


Thr Asp Cys Val Leu Glu Ala Met Ala Phe Leu Glu 


 


Lys Ser His Pro Gly Ile Phe Glu Asn Ser Cys Leu 


 


Glu Thr Met Glu Ile Val Gln Gln Thr Arg Val Asp 


 


Lys Leu Thr Gln Gly Arg Gln Thr Tyr Asp Trp Thr 


 


Leu Asn Arg Asn Gln Pro Ala Ala Thr Ala Leu Ala 


 


Asn Thr Ile Glu Val Phe Arg Ser Asn Gly Leu Thr 


 


Ala Asn Glu Ser Gly Arg Leu Ile Asp Phe Leu Lys 


 


Asp Val Met Glu Ser Met Asp Lys Gly Glu Met Glu 


 


Ile Ile Thr His Phe Gln Arg Lys Arg Arg Val Arg 


 


Asp Asn Met Thr Lys Lys Met Val Thr Gln Arg Thr 


 


Ile Gly Lys Lys Lys Gln Arg Leu Asn Lys Arg Ser 


 


Tyr Leu Ile Arg Ala Leu Thr Leu Asn Thr Met Thr 


 


Lys Asp Ala Glu Arg Gly Lys Leu Lys Arg Arg Ala 


 


Ile Ala Thr Pro Gly Met Gln Ile Arg Gly Phe Val 


 


Tyr Phe Val Glu Thr Leu Ala Arg Ser Ile Cys Glu 


 


Lys Leu Glu Gln Ser Gly Leu Pro Val Gly Gly Asn 


 


Glu Lys Lys Ala Lys Leu Ala Asn Val Val Arg Lys 


 


Met Met Thr Asn Ser Gln Asp Thr Glu Leu Ser Phe


 


Thr Ile Thr Gly Asp Asn Thr Lys Trp Asn Glu Asn 


 


Gln Asn Pro Arg Met Phe Leu Ala Met Ile Thr Tyr 


 


Ile Thr Arg Asn Gln Pro Glu Trp Phe Arg Asn Val 


 


Leu Ser Ile Ala Pro Ile Met Phe Ser Asn Lys Met 


 


Ala Arg Leu Gly Lys Gly Tyr Met Phe Glu Ser Lys 


 


Ser Met Lys Leu Arg Thr Gln Ile Pro Ala Glu Met 


 


Leu Ala Ser Ile Asp Leu Lys Tyr Phe Asn Glu Ser 


 


Thr Arg Lys Lys Ile Glu Lys Ile Arg Pro Leu Leu 


 


Ile Asp Gly Thr Ala Ser Leu Ser Pro Gly Met Met 


 


Met Gly Met Phe Asn Met Leu Ser Thr Val Leu Gly 


 


Val Ser Ile Leu Asn Leu Gly Gln Lys Arg Tyr Thr 


 


Lys Thr Thr Tyr Trp Trp Asp Gly Leu Gln Ser Ser 


 


Asp Asp Phe Ala Leu Ile Val Asn Ala Pro Asn His 


 


Glu Gly Ile Gln Ala Gly Val Asp Arg Phe Tyr Arg 


 


Thr Cys Lys Leu Val Gly Ile Asn Met Ser Lys Lys 


 


Lys Ser Tyr Ile Asn Arg Thr Gly Thr Phe Glu Phe 


 


Thr Ser Phe Phe Tyr Arg Tyr Gly Phe Val Ala Asn 


 


Phe Ser Met Glu Leu Pro Ser Phe Gly Val Ser Gly 


 


Ile Asn Glu Ser Ala Asp Met Ser Ile Gly Val Thr 


 


Val Ile Lys Asn Asn Met Ile Asn Asn Asp Leu Gly 


 


Pro Ala Thr Ala Gln Met Ala Leu Gln Leu Phe Ile 


 


Lys Asp Tyr Arg Tyr Thr Tyr Arg Cys His Arg Gly 


 


Asp Thr Gln Ile Gln Thr Arg Arg Ser Phe Glu Leu


 


Lys Lys Leu Trp Glu Gln Thr Arg Ser Lys Ala Gly 


 


Leu Leu Val Ser Asp Gly Gly Pro Asn Leu Tyr Asn 


 


Ile Arg Asn Leu His Ile Pro Glu Val Cys Leu Lys 


 


Trp Glu Leu Met Asp Glu Asp Tyr Gln Gly Arg Leu 


 


Cys Asn Pro Leu Asn Pro Phe Val Ser His Lys Glu 


 


Ile Glu Ser Val Asn Asn Ala Val Val Met Pro Ala 


 


His Gly Pro Ala Lys Ser Met Glu Tyr Asp Ala Val 


 


Ala Thr Thr His Ser Trp Ile Pro Lys Arg Asn Arg 


 


Ser Ile Leu Asn Thr Ser Gln Arg Gly Ile Leu Glu 


 


Asp Glu Gln Met Tyr Gln Lys Cys Cys Asn Leu Phe 


 


Glu Lys Phe Phe Pro Ser Ser Ser Tyr Arg Arg Pro 


 


Val Gly Ile Ser Ser Met Val Glu Ala Met Val Ser 


 


Arg Ala Arg Ile Asp Ala Arg Ile Asp Phe Glu Ser 


 


Gly Arg Ile Lys Lys Glu Glu Phe Ala Glu Ile Met 


 


Lys Ile Cys Ser Thr Ile Glu Glu Leu Arg Arg Gln  


 


Lys






The gene of influenza virus subunit PA (SEQ ID NO:1) was divided into a C-terminal part and an N-terminal part, and then the C-terminal part and the N-terminal part were cloned by molecular cloning techniques, wherein the N-terminal part contains the first 256 amino acids and the C-terminal part contains amino acids 257-716. The two parts were cloned into pGEX-6p vector (from Amersham Pharmacia Inc.) respectively so as to express fusion proteins of N-terminal fused with GST (GST-PAN and GST-PAc). The cloned plasmids were transformed into E. coli BL21 respectively, and E.coli were induced by using 0.1 to 1 mM IPTG (isopropyl-β-thiogalactoside) in BL21 to express these two proteins respectively, thus obtaining respectively expressing bacteria of these two protein, for details see Example 1.
The gene encoding N-terminal 48 amino acids of PB1 (including the peptide of the first 25 amino acids) was also cloned into pGEX-6p vector, in order to express a fusion protein of fused GST-PB1 peptide.
Likewise, the short peptide of GST fused the former 25 amino acids or former 48 amino acids of PB1N was expressed respectively. The vectors were transformed into E. coli BL21 in the same way. E.coli were induced by using 0.1 to 1 mM IPTG in BL21 to express proteins, thus obtaining bacteria expressing the protein.
The bacteria that express GST-PA-N were suspended in buffer, lysed and centrifuged to obtain supernatant. Then affinity chromatographic column was used to purify GST-PA-N fusion protein from the supernatant.
The GST-PAc expressing bacteria and the GST-PB1 expressing bacteria were suspended with a buffer (which contains about 20 mM Tris-HCl (pH 8.0) and 250 mM NaCl) respectively and mixed with a molar ratio between the protein content of GST-PAc and GST-PB1N is 0.1:1-1:0.1, preferably the molar ratio between the protein content of GST-PAc and GST-PB1N is 0.5:1-1:0.5, more preferably the molar ratio between the protein content of GST-PAc and GST-PB1N is near 1:1.
Subsequently, Glutathione-Sepharose affinity column (from Amersham Pharmacia Inc.) was used to purify the GST fusion protein. After enzymolysis with PreScission protease (from Amersham Pharmacia Inc.), the complex of PAc/PB1 short peptide was separated and purified through such methods as gel filtration Superdex-200 and ion exchange chromatography (Q sepharose), wherein complex can be used for further crystallization experiment after determining the protein concentration by SDS-PAGE gel electrophoresis.
Crystallization and Optimization of Protein
The complex that has been expressed and purified through above methods was condensed to a concentration of 5-30 mg/ml, and crystal growth condition is screened with crystallization reagents (from Hampton Research) by gas hanging drop so as to obtain original crystals under conditions of multiple crystallization reagents.
Through further optimization, crystals with good appearance were obtained in solution containing about 1M sodium acetate with different buffer conditions under pH 4-9, wherein larger triangle-cone crystal was obtained in solution containing about 1-1.3M sodium acetate (Sigma) with different buffers under pH 4-9, and the resolution of said crystal is about 4 Angstrom.
When collecting X-ray diffraction data, the crystal required by diffraction was transferred from hanging drops to about 10 μl corresponding crystallization buffer containing 1.4M sodium acetate and 10% glycerol (Sigma). After the fluid drops are left open for air dehydration for more than one hour, parent crystal and selenium-containing crystal with a resolution of about 3 Angstrom as well as corresponding X-ray diffraction data were obtained.
Collection of Crystal Data and Structure Analysis
A set data of parent crystal with a resolution of 2.9 Angstrom from the complex crystal of PA-PB1 N-terminal (this PB1 N-terminal contains 25 amino acids) was first collected by using FR-E X-ray diffractometer (Rigaku) under a wave length 1.5418 Angstrom. Then under wave length of 0.9783 and 0.9785 Angstrom, two sets of data from derivative crystal of selenium atom were collected, i.e. peak and edge, using synchrotron radiometer located at APS, Chicago, USA (station number: SBC 191D; detection screen: ADSC Q315), the resolution of said crystal is about 3.3 Angstrom. The three sets of data were treated by HKL2000 (Otwinowski 1997) and found to have spacegroup of P4(1)2(1)2. Phase was calculated by multi-wavelength anomalous scattering (Hendrickson 1991), and sas file resulted from treatment was searched for selenium atoms by SHELXD (Sheldrick 1998). The protein itself has 14 methionines, and the inventors found 14 selenium atoms in all. Coordinates of selenium atoms and two sets of data (i.e. Peak and Edge) were input into Program autoSHARP (Vonrhein, Blanc et al. 2007) to calculate phase and to modify the electron density map, and several secondary structures (including α helixes and β sheets) can be clearly found from the calculated electron density map. Then phase can be expanded by Program CAD, and the phase was expanded to 2.9 Angstrom by collected parent data so as to construct a structure model, wherein the Programs used to construt model are ARP/wARP (Perrakis, Morris et al. 1999) and Phenix (Adams, Grosse-Kunstleve et al. 2002). Automatic model construction performed by these two programs can amount to 60% of the whole structure, and the rest is manually constructed through Program COOT (Emsley and Cowtan 2004).
Finally, the resulting model was modified by Program CNS (Brunger, Adams et al. 1998) and REFMAC5 (Murshudov, Vagin et al. 1997) to achieve the protein structure analysis, and the final factor R and factor R-free for modify structure are 0.22 and 0.26 respectively.
Atomic coordinates in crystal structure of protein complex of PAc/PB1N short peptide, see Table 1.
EXAMPLE
Method for Expressing Influenza Virus PA and PB1 Polypeptides
In one embodiment of the present invention, PA (SEQ ID NO:1) was divided into two fragments so as to express former 256 amino acid residues fragments and 257-716 amino acid residues fragments of the PA, respectively, and two gene fragments encoding these two protein polypeptides were cloned into an Escherichia coli expression vector, respectively so as to expressing proteins in a bacteria. The PA N-terminal polypeptides were purified from a PA N-terminal (1-256 amino acids) expressing bacteria and used for protein crystallization. C-terminal of PA expressing bacteria was centrifugally collected for later use so as to be co-purified with the N-terminal of PB1 polypeptides.
Polypeptide containing former 25 or no more than 48 amino acids of the N-terminal of PB1 (not containing first-position methionine) was expressed in the form of GST fusion protein in a bacteria. The influenza virus polymerase protein subunit PA was expressed by fragments in a bacteria or other eukaryotic cells such that at least 50% fragments were part of amino acid fragments of positions 257-716 of the PA protein.
Expression and Purification of N-terminal of Influenza Virus PA in Escherichia Coli 
The N-terminal of the influenza virus PA (amino acids 1-256) was cloned into a pGEX-6p vector (from Amersham Pharmacia Inc.) via a molecular cloning technique, the cloning sites thereof being BamHI and XhoI. Expression plasmids with a PA N-terminal gene, obtained by cloning, were transformed into Escherichia coli BL21 for protein expression, such that the bacteria could express the N-terminal (amino terminal) of the PA protein which was connected with the GST fusion protein and has protease cleavage sites cleaved by ProScission protease to further separate a GST protein tag from the target protein-PA polypeptide. IPTG with a final concentration of about 0.1-1 mM was used in the cultured Escherichia coli BL21 cells to induce Escherichia coli in order to obtain the expressing bacteria of said protein. The used vector contained an ampicillin-resistance gene. After the cloning-constructed expression plasmids of the fusion protein were transformed into Escherichia coli such as BL21 (Novagen), bacterium were cultured overnight using bacteria culture media such as LB and so on at 37° C., and after about 12 hours transferred to a mass culture medium in a proportion of about 1:100, and cultured in a shake flask at 37° C. until OD is approximately 1.0, and then added 0.1-1 mM IPTG for inducing expression. After about 3 to 6 hours, the bacterium were collected centrifugally, and the collected precipitated bacterium could be stored at −20° C. to −80° C. in a refrigerator for later use or be used directly for purification of the PA N-terminal protein.
Expression and Purification of Complex of C-terminal of Influenza Virus PA and PB1 Polypeptide
The C-terminal (amino acids 257-716) of the influenza virus PA was cloned into a pGEX-6p vector (from Amersham Pharmacia Inc.) via a molecular cloning technique, the cloning sites thereof being BamHI and NhoI. Expression plasmids with a PA C-terminal gene, obtained by cloning, were transformed into Escherichia coli BL21 for protein expression, such that the bacteria could express the N-terminal (amino terminal) of the protein which was connected with the GST fusion protein and has protease cleavage sites cleaved by ProScission protease to further separate a GST protein tag from the target protein-PA polypeptide. IPTG with a final concentration of about 0.1-1 mM was used in the cultured Escherichia coli BL21 cells to induce Escherichia coli in order to obtain the expressing bacteria of said protein. The used vector contained an ampicillin-resistance gene. After the cloning-constructed expression plasmids of the fusion protein were transformed into Escherichia coli such as BL21 (Novagen), bacterium were cultured overnight using bacteria culture media such as LB and so on at 37° C., and after about 12 hours transferred to a mass culture medium in a proportion of about 1:100, and cultured in a shake flask at 37° C. until OD is approximately 1.0, and then added 0.1-1 mM IPTG for inducing expression after the culture temperature is lowered to 16° C. After about 12 to 24 hours, the bacteria were collected centrifugally, and the collected precipitated bacterium could be stored at −20° C. to −80° C. in a refrigerator for later use or be used directly for purification.
The gene of the N-terminal of PB1 with no more than 48 amino acids (the inventor had expressed the former 48-amino acid polypeptide and the former 25-amino acid polypeptide of the N-terminal of PB1) was likewise cloned into multiple cloning sites of the pGEX-6p vector, wherein the used cloning sites were BamHI and XhoI, such that the bacteria could express a fusion protein containing GST, there is protease cleavage sites cleaved by ProScission protease in the fusion protein in order to further separate a GST protein tag from the target protein of PB1 polypeptide. The fusion protein of a GST-PB1N peptide was expressed in Escherichia coli BL21 in the same way as the PA fusion protein was expressed above. The resistance gene was ampicillin-resistance gene. The protein expression was carried out at 37° C., and the used inducer was IPTG. Finally, the expressing bacterium were collected centrifugally, used directly for protein purification and could be stored temporarily at −20° C. to −80° C. in a refrigerator.
The centrifugally collected expressing bacteria expressing the GST-PA N-terminal polypeptide was suspended using a buffer solution containing 20 mM Tris-HCl (pH 8.0) and 250 mM NaCl or a buffer solution of 1×PBS (pH 7.4) phosphoric acid. An ultrasonic breaker was used to break cells. The insoluble precipitation was centrifugally separated and removed in order to collect soluble supernatant. A Glutathione affinity chromatographic column was used to purify the GST-PA-N-terminal polypeptide, and the ProScission protease was further used to enzymolyze the fusion protein into two fragments of GST (glutathione S-transferase) and PA-N. Ion exchange chromatography and gel exclusion chromatography were then used to purify the PA-N protein polypeptide. The protein was concentrated to 5-30 mg/mL for crystal growth.
The expressing bacteria expressing the GST-PAC C-terminal polypeptide and the expressing bacteria expressing the GST-PB1N short peptide were suspended using a buffer solution containing 20 mM Tris-HCl (pH 8.0) and 250 mM NaCl or a buffer solution of 1×PBS (pH 7.4) phosphoric acid, and then mixed in proportion, such that the molar ratio of the total protein of GST-PA to GST-PB1 was 0.1:1 to 1:0.1, preferably 0.5:1 to 1:0.5, most preferably close to 1:1.
The cell in the mixed bacterial suspension was lysed using ultrasonic wave or other cell lysing methods. An insoluble portion and a soluble portion of the bacterial lysates were centrifugally separated. The supernatant obtained by high speed centrifugation (about 20,000 g) was preliminarily separated using a Glutathione-Sepharose affinity column (from Amersham Pharmacia Inc.) to purify such mixed protein. The protein containing a GST tag could bind to the Glutathione-Sepharose affinity column, while other proteins could not bind to said affinity column. The above mentioned bacterial suspension buffer solution was used to rinse impurity after the protein bound to the affinity column. A suitable amount of ProScission protease (from Amersham Pharmacia Inc.) was used to enzymolyze the mixed GST fusion protein of the affinity column. This process generally needs about 24 hours. Then, the enzymatically cleaved PAC and PB1N proteins were further separated using methods of gel filtration superdex-200 (from Amersham Pharmacia Inc.), Q sepharose ion exchange chromatography (from Amersham Pharmacia Inc.) and so on to purify the PAC/PB1N short peptide complex (the chromatography column comes from Amersham Pharmacia Inc.). The protein purity was determined via SDS-PAGE gel electrophoresis, and the purity generally reached more than 90%. The protein purified by the above steps was concentrated to about 5-30 mg/mL using an evaporating pipe (coming from Millipore Inc.) for a further crystallization experiment.
The person skilled in the art would know that, the N-terminal of influenza virus PA-PAN and the C-terminal of influenza virus PA-PAC as well as the N-terminal of PB1-PBN could be expressed not only in the prokaryotic cell such as Escherichia coli described hereinabove but also in an eukaryotic cell such as insect cells; any other endonuclease, protease cleavage site, and ligase could be used; the target polypeptide to be purified may be fused with other tags such as GST, and the corresponding separating and purifying method was then selected for purification, and finally the tag fused into the target polypeptide was removed. Various change and modification of the present invention as described above fall within the scope of protection of the present invention.
It shall be noted that, the fragments of an α helix and a β sheet of type B or type C influenza corresponding to type A influenza virus are as shown in FIGS. 1A, 1B and 1C as well as FIGS. 10A and 10B, respectively. Herein, the description thereof is omitted.
EXAMPLE 2
Crystallization of PAC/PB1N Short Peptide Complex
The complex of the PA and PB1 polypeptides expressed and purified with the above method was concentrated to about 5 to 30 mg/mL. A crystallization reagent (from reagent kits such as Screen Kit I/II, Index, and so on from Hampton Research incorporation, etc.) was used in gas sitting drop to screen crystal growth conditions. Upon preliminary screening, the inventor could obtain an original crystal with multiple different crystallization reagents.
Upon further optimization, a crystal with a fairly good appearance was obtained using a solution containing about 1M of sodium acetate in cases of using buffer solutions with different PH values (PH 4-9). A relatively larger triangle-cone crystal was obtained in a sodium acetate buffer solution (PH4-9) with a concentration of 1 to 1.3 M (from Sigma Inc.), the resolution being about 4 Angstrom.
When X-ray diffraction was performed to collect data, crystal needed for the diffraction was transferred from suspension drops to a 10 microliter corresponding crystal buffer solution containing 1.4M sodium acetate and 10% glycerol (from Sigma). The liquid drops were placed in the air for dehydration for more than one hour to obtain a protein crystal with a high resolution, and the resolution of parental and selenium-containing (selenium substituted) protein crystal could reach more than 2.9 Angstrom.
It shall be noted that, the fragments of an α helix and a β sheet of type B or type C influenza corresponding to type A influenza virus are as shown in FIGS. 1A, 1B and 1C as well as FIGS. 10A and 10B, respectively. Herein, the description thereof is omitted.
EXAMPLE 3
Crystal Structure of PAC/PB1N Short Peptide Complex
A set data of parent crystal with a resolution of 2.9 Angstrom from the complex crystal of PA-PB1 N-terminal (this PB1 N-terminal contains 25 amino acids) was first collected by using FR-E X-ray diffractometer (Rigaku) under a wave length 1.5418 Angstrom. Then under wave length of 0.9783 and 0.9785 Angstrom, two sets of data from derivative crystal of selenium atom were collected, i.e. peak and edge, using synchrotron radiometer located at APS, Chicago, USA (station number: SBC 191D; detection screen: ADSC Q315), the resolution of said crystal is about 3.3 Angstrom. The three sets of data were treated by HKL2000 (Otwinowski 1997) and found to have spacegroup of P4(1)2(1)2. Phase was calculated by multi-wavelength anomalous scattering (Hendrickson 1991), and sas file resulted from treatment was searched for selenium atoms by SHELXD (Sheldrick 1998). The protein itself has 14 methionines, and the inventors found 14 selenium atoms in all. Coordinates of selenium atoms and two sets of data (i.e. Peak and Edge) were input into Program autoSHARP (Vonrhein, Blanc et al. 2007) to calculate phase and to modify the electron density map, and several secondary structures (including α helixes and β sheets) can be clearly found from the calculated electron density map. Then phase can be expanded by Program CAD, and the phase was expanded to 2.9 Angstrom by collected parent data so as to construct a structure model, wherein the Programs used to construt model are ARP/wARP (Perrakis, Morris et al. 1999) and Phenix (Adams, Grosse-Kunstleve et al. 2002). Automatic model construction performed by these two programs can amount to 60% of the whole structure, and the rest is manually constructed through Program COOT (Emsley and Cowtan 2004).
Finally, the resulting model was modified by Program CNS (Brunger, Adams et al. 1998) and REFMAC5 (Murshudov, Vagin et al. 1997) to achieve the protein structure analysis, and the final factor R and factor R-free for modify structure are 0.23 and 0.26 respectively.
EXAMPLE 4
Crystallization of N-terminal of PA
The PA N-terminal polypeptide complex expressed and purified by the above method was concentrated to about 5 to 30 mg/mL. A crystal agent (kits such as Screen Kit I/II, Index, and so on from Hampton Research Incorporation, etc.) was used in gas sitting drop to screen crystal growth conditions. Upon preliminary screening, the inventor obtained an original crystal with different crystal agents.
For further optimization, a crystal well diffracted by an X-ray was obtained from a solution containing about 20% PEG 8000 or 20% PEG 3350 and 0.1 M magnesium chloride or 0.1 M magnesium acetate in a case of using a MES buffer solution with −PH 6.5 (all the used regents came from Sigma Inc.) (see A and B crystal pictures and C and D diffraction pictures in FIG. 3). The X-ray diffracting resolution of the obtained crystal is about 2 to 4 Angstrom.
EXAMPLE 5
Method for Screening Small Molecules Competing with PB1N for Binding to PAC 
During a process of screening a small molecular medicament capable of disintegrating the PAC/PB1N short peptide complex, the PB1N short peptide gene was fused with a gene expressing GFP (green fluorescent protein). The PB1N short peptide protein fused with GFP was expressed as an indicator molecule during a small molecular compound disintegrating a protein complex. The PB1 short peptide gene fragment was connected to the GFP gene fragment using a molecular cloning method so as to express PB1 small peptide fusion protein of which one N-terminal or C-terminal was connected with GFP.
Method 1: a complex of PA C-terminal fusion protein with the N-terminal fused GST, viz. GST-PAC fusion protein, and GFP-PB1N short peptide fusion protein was expressed with the above method of expressing and purifying the C-terminal of PA and the PB1 short peptide. The complex of the GST-PAC fusion protein and the GFP-PB1N short peptide fusion protein flew through and bound to the Glutathione affinity column. Since said complex contained the GFP protein, the GFP-PB1N fusion protein binding to GST-PAC enabled the affinity column to show a green color after GST-PAC bound to the affinity column. The affinity column binding with the complex protein of GST-PAC and GFP-PB1N was rinsed sufficiently with a buffer solution to thoroughly elute and remove unbound proteins. Then, a mixture of small molecular compounds to be screened flew through the affinity column (said mixture should not contain Glutathione or other eluted compounds for detaching GST from the affinity column). If the mixture contained small molecules substituted for the PB1N polypeptide to bind to the PAC, part of the GFP-PB1N polypeptide fusion protein binding to PAc was replaced and eluted. The eluted solution flowing out showed a green color due to containing said GFP fusion protein when observed under a fixed wavelength fluorescent microscope. The small molecules were further sequentially separated and purified from the mixture, and the components substituted for the PB1N polypeptide were traced by the above green GFP protein tracing method to finally determine a small molecular compound interfering with the binding of PA to the PB1 small peptide. In the above method, besides using GST as an affinity matrix, other polypeptides such as Flag-tag, Myc-tag, MBP (Maltose binding protein)-tag, specific antibody, etc. could be used as combining groups of affinity matrix. Correspondingly, the affinity chromatographic column needs a corresponding affinity matrix, for example, if the Flag-tag was used, an antibody against the Flag-tag (e.g. an anti-flag monoclonal antibody from Sigma Inc.) was used to be fixed to the affinity chromatographic column as a gel medium binding to the Flag. The compound molecules binding to PA and replacing a PB1 small peptide (specific structure) could be determined via methods such as mass spectrometry, etc.
Method 2: PAC was purified separately (fusion protein or non-fusion protein) and covalently crosslinked to a gel medium by chemical crosslinking, but protein was not denatured. GFP-PB1N flew through the covalently binding gel column. GFP-PB1N bound to the PAC protein such that the gel column presents a green fluorescent light of GFP. The solution of the small molecular mixture flew through the gel column, if a compound substituted for GFP-PB1N to bind to PAC was present, the GFP-PB1N fusion protein was eluted. The eluent showed a green color by stimulation of a specific-wavelength light, and the compound molecules substituted for GFP-PB1N bound to the PAC molecules of the gel column. A buffer solution was used to elute the gel column to remove impurities, and urea and the like was then used to denature PAC in order that the small molecules binding thereto were eluted. Methods of mass spectrometry and the like were used to analyze small molecules binding to PA to obtain structural information of the small molecules. The compound may be a small molecular medicament capable of disintegrating the PAC/PB1N short peptide complex.
EXAMPLE 6
Method of Using Crystal Three-Dimensional Structure of PAC/PB1N Complex to Design and Screen Various Polypeptides, Proteins, or Inorganic or Organic Compounds for Treating Diseases Caused by Influenza Virus Infection
A crystal three-dimensional structure of a complex of the C-terminal of influenza virus polymerase subunit PA-PAC and the N-terminal of influenza virus polymerase subunit PB1-PB1N was used to design and screen various polypeptides, proteins, or inorganic or organic compounds for treating diseases caused by influenza virus infection. The specific steps are as follows: polypeptides and compound molecules binding to specific portion were designed through computer stimulation technology according to coordinates of three-dimensional structure of protein; potential polypeptides and compound molecules binding to specific portion were searched for through computer stimulation technology according to coordinates of three-dimensional structure of protein; the polypeptides and compound molecules designed or searched according to the coordinates of three-dimensional structure of protein bound to any type of influenza virus polymerase protein which has at least 50% sequence identity with the PAC and PB1N sequences, and binding information was then analysed; the polypeptides and compound molecules designed or searched according to the coordinates of three-dimensional structure of protein bound to any type of influenza virus polymerase protein which has at least 50% sequence identity with the PAC and the PB1N sequences, and then crystallized; and the binding information of the polypeptides or compound molecules to protein is analyzed through a crystal diffraction method.
EXAMPLE 7
Designing and Screening Small Peptides Using Crystal Three-Dimensional Structure of PAC/PB1N for Treating Diseases Caused by Influenza Virus Infection
Upon verification by experiment, a short peptide containing M1, D2, V3, N4, P5, T6, L7, L8, F9, L10, and K11 bound to the C-terminal of PA. The inventors cloned gene encoding PB1N polypeptide containing the first-position M1 to the eleventh-position K11 into a pFEX-6p vector, purified said GST-PB1N fusion protein, and used the fusion protein fixed to the affinity chromatographic gel column to bind to PA-C in a solution through an in vitro binding experiment. The inventors found that said fusion protein maintained the PB1N's capability of binding to PA-C.
Using the same experimental method, the inventors found that a fusion protein containing M1, D2, V3, N4, P5, T6, L7, L8, F9, L10, K11, V12 and p13 also maintained the PB1N's capability of binding to PA-C. Thus, these two short peptides had a capacity of binding to PA-C to make themselves potential polypeptide medicaments interfering with influenza virus polymerase activity or models for further medicament design.
Likewise, the selected polypeptide having at least three amino acid sequence alignment identical with the above polypeptide might be a potential polypeptide medicament interfering with influenza virus polymerase activity.
The structure of subunits PA, PB1, PB2 or the complex of PA, PB1 and PB2 of any type of influenza virus polymerase contains one protein or one fragment thereof having at least 40% sequences identical with those of the PAC protein.
In the structure of subunits PA, PB1, PB2 or the complex of PA, PB1 and PB2 of any type of influenza virus polymerase, at least 40% coordinates of main chain carbon backbone of three-dimensional structure of one protein or one fragment thereof has average root mean square deviation smaller than or equal to 1.7 Angstrom with respect to the atomic coordinates of PAC.
In the structure of subunit PA, PB1, PB2 or the complex of PA, PB1 and PB2 of any type of influenza virus polymerase, the protein fragment has 40% sequence identity of 2-12 amino acid fragments of the PB1N polypeptide.
Any polypeptide or small molecule that interacts with key amino acids of the influenza virus subunit PA is included in the invention.
The structure is used in medicament screening and medicament designing.
A method for screening a compound or polypeptide binding to a protein based on the three-dimensional structure of the PAC and PB1N comprises: obtaining a crystal of the complex of PAC and PB1N proteins by protein crystallization, wherein the crystal of the complex protein has a spacegroup of P4(1)2(1)2, and the crystal cell parameters are: a=b32 122 Angstrom, c=133 Angstrom, and α=β=γ=90°; obtaining the coordinate of the three-dimensional structure of the complex of the PAC and PB1N proteins by an X-ray diffracting crystal technique, wherein any structure containing at least 40% amino acid residues of which coordinates of main chain carbon backbone have average root mean square deviation smaller than or equal to 1.7 Angstrom with respect to said coordinate is included.
A method of expressing and purifying influenza virus subunit PA comprises expressing PA in segments in a bacteria or a eukaryotic expressing system, and the method is used to express and purify any protein fragments which has 40% sequence identity with PA.
In a preferred embodiment, the PAC/PB1N complex is used in designing and screening polypeptides, proteins, compounds, or medicaments in the treatment of diseases caused by influenza virus infection.
In a preferred embodiment, polypeptides for treating diseases caused by the influenza virus infection comprises polypeptides interacting with the above complex, at least one of the α helix or β sheet, and at least one amino acid site.
In a preferred embodiment, proteins for treating diseases caused by the influenza virus infection comprises proteins interacting with the above complex, at least one of the α helix or β sheet, and at least one amino acid site.
In a preferred embodiment, compounds for treating diseases caused by the influenza virus infection comprises compounds interacting with the above complex, at least one of the α helix or β sheet, and at least one amino acid site.
In a preferred embodiment, a pharmaceutical composition comprises the above polypeptides, proteins, or compounds.
The pharmaceutical composition of the present invention generally includes one carrier or excipient. An antibody and/or immunoconjugate are dissolved in a pharmaceutically acceptable carrier, wherein an aqueous carrier is preferred. Many types of aqueous carriers can be applied, e.g. buffer saline, etc. These solutions are sterilized and generally free from undesired substances. These components can be disinfected through a conventional, well-known disinfecting technique. These components may include auxiliary substances required by physiological conditions, such as a buffering agent adjusting PH, toxicity regulator, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The fusion proteins of these components vary greatly in concentration mainly depending on the selected administration manner, the liquid amount and viscosity required by a patient, body weight, etc.
Therefore, about 1.2 to 1200 μg of one typical pharmaceutical immunotoxin component in the present invention shall be applied daily for brain administration. One typical component for treating neoplasms of breast, ovary and lung via intravenous administration shall be applied to one patient 0.1 to 10 mg per day. The dosage of 0.1 to 100 mg per day for one person may be allowed, especially in a case that the medicament is administrated to a closed position without entering blood circulation or a lymphatic system, for example, it is administrated to a body cavity or an organ lacuna. The actual procedures for preparing applicable medical components are understood or acquired by the person skilled in the art and are described in detail in some publications, e.g. Remington's PHARMACETUTICAL SCIENCE, 19th ed., Mack Publishing Company, Easton, Pa. (1995).
The components of the present invention can be used for a treatment. In treatment application, the components are applied to a patient suffering from a certain disease (for example, glioblastoma, breast cancer, ovarian cancer, and lung cancer), the dosage of which shall be enough to at least alleviate or partially control said disease and the complications thereof. The dosage enough to complete these tasks is called as “therapeutically effective dosage”. The applied effective dosage depends on illness severity and patient's general health conditions. The effective dosage of the component can achieve subjectively-recognized alleviation of a certain symptom or objective improvement recorded by a clinician or other qualified observer.
Whether to be administrate once only or for several times depends on desired and tolerated dose and frequency by a patient. Nevertheless, an adequate amount of the immunotoxin shall be provided to treat a patient effectively. Preferably, the medical dosage might be administrated only once or administrated periodically until a certain therapy efficacy or an adverse reaction inhibits continuation of the treatment. Generally, these dosages are enough to treat or improve disease conditions without incurring unbearable toxicity for a patient.
The immunoconjugate of the present invention can be prepared into gastrointestinal sustained release formulations (e.g. an implant, an oil injection, or a microparticle system). A protein delivery system can be fully understood by referring to Banga, A. J., THERAPEUTIC PEPTIDES AND PROTEINS: FORMULATION, PROCESSING, AND DELIVERY SYSTEMS, Technomic Publishing Company, Inc., Lancaster, Pa., (1995). The microparticle system includes microspheres, particles, microcapsules, nano-microcapsules, nano-microspheres, and nano-particles. The microcapsule uses therapeutic protein as a core. In globules, therapeutic substances are dispersed in the particles. Particles, microspheres, and microcapsues which are smaller than about 1 μm are generally called as nano-microparticles, nano-spheres, and nano-microcapsules. Capillary vessels are about 5 μm in diameter. Therefore, only nano-particles are intravenously administrated. The microparticles are about 100 μm in diameter and are intravenously and intramuscularly administrated. Examples are Kreuter, J., COLLOIDAL DRUG DELIVERY SYSTEMS, J. Kreuter, ed., Marcel Dekker, Inc., New York, N.Y., pp. 219-342 (1994); and Tice&Tabibi, TREATISE ON CONTROLLED DRUG DELIVERY, A. Kydonieus, ed., Marcel Dekker, Inc. New York, N.Y., pp. 315-339, (1992), both of which are cited herein.
Polymers can be used for ion controlled release of immunoconjugate components in the present invention. Multiple degradable and nondegradable polymers for drug controlled release are well-known in the art (Langer, R., Accounts Chem. Res. 26:537-542 (1993)), for example, a retarding polymer polaxamer 407 is viscous and flowable at low temperature, but is formed as a semisolid gel at body temperature, and is proved to be an effective carrier for forming and delivering continuously recombinant interleukin-2 and urease (Johnston etc., Pharm. Res. 9:425-434 (1992)) and Pec etc., J. Parent. Sci. Tech. 44(2):58-65(1990)). Likewise, hydroxyapatite can be used as a microcarrier for protein controlled release (Ijntema etc., Int. J. Pharm. 112:215-224 (1994)), while liposome is used for controlled release and targeting transport processes of a liplid-coated medicament (Betageri, etc., LIPOSOME DRUG DELIVERY SYSTEMS, Technomic Publishing Co., Inc., Lancaster, Pa. (1993)). Many other therapeutic protein controlled release systems have been known, for example, U.S. Pat. Nos. 5,055,303, 5,188,837, 4,235,871, 4,501,728, 4,837,028, 4,957,735, 5,019,369, 5,055,303, 5,514,670, 5,413,797, 5,268,164, 5,004,697, 4,902,505, 5,506,206, 5,271,961, 5,254,342, and 5,534,496, any of which is cited herein.
Experimental Results
The atomic coordinate of the structure of the complex of PAC and PB1N is shown in the following Table 1.
Polymerase subunit PA protein derived from avian H5N1 influenza virus strains A/goose/Guangdong/1/96 is compared with PA protein sequences of type A influenza virus strains A/BrevigMission/1/1918 that outbreaks on a large scale in Europe, 1998, and two types of type B influenza virus strains B/Ann Arbor/1/1966 and type C influenza virus strains C/JJ/1950, the results of which are as shown in FIG. 1.
PA (SEQ ID NO:1) is divided by the inventor into two parts, and multiple fragments of varying lengths of two parts of a PA gene are cloned and expressed in Escherichia coli, wherein residues 1-256 (see FIG. 1 and FIG. 2) and residues 257-716 (see FIG. 4A) of the N-terminal of PA are well expressed and purified by being fused with GST (Glutathione S-Transferase) and a well-diffracted parental crystal of purified PA amino-terminal (PA_N) is obtained (see FIG. 3).
A preliminary crystallization experiment shows that the C-terminal of PA fails to obtain a crystal despite a lot of efforts has been made. Therefore, the inventor also uses a GST fusion method to express PB1 N-terminal peptides of 25 amino acids and 48 amino acids (see FIG. 4A, which is an electrophoretogram before and after the C-terminal of PA is fused with the GST or the N-terminal of PB1 is fused with the GST, wherein strand 1 PA_C CT is a control sample for the C-terminal of PA, strand 2 GST-PB1+PAC is a sample in which the complex of the C-terminal of PA and the N-terminal of PB1 is fused with GST, strand 3 GST-PB1 is a sample in which the PB1N terminal is fused with GST, strand 4 GST+PA_C is a sample in which the C-terminal of PA is fused with GST, and strand 5 GST CT is a GST control sample; wherein PA_C denotes an electrophoretic band of PA_C, GST-PB1 is an electrophoretic band in which the PB1N terminal is fused with GST, and GST is a control electrophoretic band for GST). The results show that: GST-PB1 can bind to purified PA_C protein.
The in vitro binding experiment shows that the expressing bacterium of the corresponding polypeptides of PA and PB1 are mixed in proportion, and then these two proteins are co-purified through the Glutathione affinity column and gel exclusion chromatograph, and so on. It is determined therefrom that the co-purification of the two can be realized, indicating the purified 460 amino acids of the C-terminal of PA may form a stable complex with the GST-PB1 polypeptide.
Further, the PAC/PB1N polypeptide complex is separated and purified after using protease to cleave the GST fusion peptide and then used in a crystallization experiment to obtain a crystal under multiple conditions, wherein a well-diffracted crystal is obtained under one condition. However, the separately purified PA C-terminal protein fails to obtain a crystal, implying that the addition of PB1 polypeptide helps to stabilize the PA protein.
Three-Dimensional Structure of PAC/PB1N Polypeptide Complex
A method of MAD is used to analyze a crystal structure of a complex of PA C-terminal 460 amino acids and PB1 N-terminal 25 amino acids, wherein the resolution is 2.9 Angstrom, the finally corrected R factor is 23%, and the Rfree factor is 26%. In general, if the structure is indicated by lines, the PA part is, vividly speaking, like a wolf's head viewed from the side, which has an extruding mouth, a skull thereafter and a ringed neck (FIG. 4B). PB1 is like a bone bite by the mouth of PA. The PA part mainly comprises 13 α helixes, one small 310 helix, 7 β sheets and loops connected the preceding structures (FIGS. 1, 4B, and FIG. 5). In terms of structure, the PA protein part can be deemed to have two domains. One domain is an area binding to the PB1 polypeptide (domain I, viz. the first part of the PA C-terminal structure) and is composed of helixes and loops, wherein helixes 4, 5, 8, 9, 10, 11, 12, and 13 constitute the mouth and cheek parts of the wolf's head. Such a domain is relative narrow viewed from the top part of the wolf's head. The other domain (domain II, viz. the second part of the PA C-terminal structure) is a latter half part of the wolf's head composed of the remaining helixes and β sheets, which is relatively large viewed from the top, with a large loop connected below, and resembles the neck part of the wolf' head. The N-terminal of the PB1 polypeptide is located on the side surface of the wolf's head, and the C-terminal is located at the other side surface, which are equivalent to two check sides of the wolf's head. PA of the crystal in the present invention is a polypeptide with the PA N-terminal 256 residues removed. It is reported that the N-terminal is a main active area of PA protease, while the other relative far active site Ser624 is located on the other check side. It is thought that Ser624 is an amino acid of the PA protease active centre which constitutes a protease active area together with the removed part of PA. Therefore, the PA N-terminal removed part is located on the other side surface of the cheek. The second domain of the latter half part of the wolf's head is composed of the remaining helixes and β sheets. Seven differently oriented β sheets of varying lengths constitute a slight twisted plane and located in the center position of the domain, while α helixes are distributed at the periphery of the helix and constitute a Rossmann fold structure. There is a large groove at the junction of two domains to form a large loop about 25 Angstrom in diameter with the lower cervical ring (FIG. 5).
Interaction Between PA and PB1 Polypeptide
Helixes binding to the PB1 polypeptide in domain I are helix 4 (406-415), 8 (582-604aa), 10 (633-650aa), 11 (653-674aa), and 13 (698-714aa), wherein three helixes (8, 11, 13) extend outwards nearly in parallel to form a nearly right-angled triangle viewed from a helix axis direction, the fourth helix (10) extends obliquely, the PB1 polypeptide is interposed therebetween (FIG. 5 and FIG. 6), and helix 4 interacts with the PB1N peptide residue on the side surface. Four helixes forms a hydrophobic core in a region binding to the PB1 polypeptide, and the side chains thereof and the hydrophobic amino acid residues of PB1 are combined together by means of hydrophobic interaction and hydrogen bonds. The second-position aspartic acid to the fifteenth-position glutamine of the PB1 polypeptide can be observed in an electron density map. Such a result coincides with the previous 12 amino acids of the PB1N terminal are key amino acids for PA binding to PB1 reported by Perez, etc. (Perez and Donis 2001). Further, the inventors analyze interaction between these amino acid residues and PA. The conserved LLFL peptide fragment of PB1 reported by the articles of Perez, etc. (Perez and Donis 2001) is present in a short helix and suspended in the middle of the hydrophobic core formed by PA. The amino acid residues of PA involved in forming the hydrophobic core are Leu666 of helix 11, Phe710 of helix 13, and Ile 633, Val636, and Leu640 of helix 10, etc. The amino acids of PA involved in binding to the PB1 polypeptide include Trp706 of helix 13, Gln670 of helix 11, etc., wherein Trp706 interacts with amino acids of Val3, N4, etc. of the PB1 short peptide by means of hydrophobic bonds, van der Waals forces, etc. Gln670 interacts with F9, V12, P13 and A14 of PB1 by means of hydrogen bonds, hydrophobic bonds, van der Waals forces, etc. T639 of helix 10 is involved in the interaction with Val3 (van der Waals forces). Q408 and N412 of helix 4 are involved in the reaction with Val3 and D2 of PB1. In addition, a loose loop interposed between helix 9 and helix 10 is also involved in the interaction with PB1, wherein I621, G622, and E623 interact with D2 and N5, respectively, and T618 and P620 interact with L8 and K11, respectively. Helix 8 is distant from the PB1 polypeptide, mainly due to van der Waals forces. Such a result is substantially consistent with the function of the residues of the PB1 polypeptide being involved in binding reported by the documents. These amino acids involved in binding to PA are conserved across type A, B and C influenza viruses, and the PA residues involved in binding to the PB1 polypeptide are mostly conserved (FIG. 1). The analysis of a fine three-dimensional structure of a mode of binding PA to PB1 provides a powerful three-dimensional information platform for designing corresponding medicaments to inhibit the function of the influenza virus polymerase.
It is found from the results of analyzing the PA structure that a large groove (domain III) is provided at the conjunction of two domains below domain I (FIG. 8A). The surface of the large groove is enriched with basic amino acids, wherein some amino acids, such as K536, K328, R566, and so on, are highly conserved in the three types of influenza viruses. In addition, the 399th-position Lys and 401st-position Arg are also conserved (FIG. 1). Mutants at the sites of K536 and so on prove the importance of these sites, indicating that the large groove part is associated with the binding of nucleotide or RNA. In addition, a large annular chain part is provided below the groove. This fragment of polypeptide is formed by being pulled apart via interaction between the annular polypeptide fragment with another neighboring molecular surface in crystal packing. The electron density of part of amino acids can not be observed clearly. The large groove and the neck ring constitute a large loop about 25 Angstrom in diameter, and such a space is enough to accommodate a double RNA helix. As described above, on the surface of the groove inside the ring, there are mainly distributed conserved basic amino acids. Although the amino acids of the chain fragment are not highly conserved, the three types of influenza viruses are mainly composed of acidic amino acids. The conservativeness and structure specificity of the groove and the annular structure in the PA molecule imply their critical roles in the polymerase to make them become second drug targets besides the area in which PA binds to PB1.
RNA promoter binding capacity. In terms of the structure analyzed by the inventors, the inventors find that the large groove is a binding site for nucleotide or RNA, wherein the sites of K539, E538, and K328 are highly conserved across the three types of influenza viruses. The inventors deem that these amino acids are involved in binding to RNA nucleotide, especially in binding to RNA, indicating that the PA subunit plays an important role in binding to a promoter, RNA, and a process of RNA synthesis.
Between domain I and domain II, the helixes of domain I and the β sheets of domain II form a channel with a diameter of about 8 Angstrom to 15 Angstrom (FIGS. 8B and 8C). In such a structure analyzed by the inventors, one loose fragment of the N-terminal of PA resembles a rope lying in the channel. Since the N-terminal fragment is relatively far from the two ends of the channel, the inventors think that the N-terminal fragment is absent from the channel when the polymerase complex interacts with other host proteins to exhibit the polymerase's function. Thus, a channel is present in the middle of the domain, extending through the channel from the PA groove to the other side of the cheek of the wolf's head. On the surface of the channel, there exist some amino acids which are highly conserved in the three types of influenza viruses, e.g. R566, K539, and K574 located on the surface of the large groove, conserved E410, K460, E524, and K536 in the middle of the channel, and the like. It is found that some residues thereof have an influence on the polymerase activity. The researches of Fodor, etc. indicate that mutation of E524A abolishes the virus's capacity of RNA synthesis to inhibit virus production. Mutation of E410 decreases the polymerase activity (Fodor, Crow et al. 2002). Mutation of K539A is found not to influence mRNA synthesis but significantly influence gene replication of cRNA and vRNA, indicating that such an area is critical for replication of viral genome. It is known that the nucleotide has a size close to that of the channel (e.g. ATP has a length of about 14 Angstrom and a width of about 8 Angstrom, and other three nucleotides UCG have a size similar thereto). The inventors, thus, deem that such a channel allows the passage of nucleotide or other small molecules or interaction with other proteins in some cases. Therefore, the mutation of the surface residues will cause significant change of the polymerase activity. The unique structure and the functional importance of the channel make it become a third drug designing site for binding target.
In a specific embodiment, there is provided in the present invention a polypeptide, a protein, an inorganic compound, or an organic compound competing with the influenza virus polymerase PB1 to bind to PAC, wherein the amino acid sequences of the polypeptide or protein contain a short LLFL motif of the short helix region formed by the PB1N residues 5 to 11 of wild type influenza virus polymerase PB1.
Herein, part of atoms between Met595 and Val12, between Leu666 and Phe9, between Leu640 and Leu8, between Leu636 and Leu8, between Met628 and Leu7, between Phe710 and Thr6, between Trp706 and Thr6, between Trp706 and Pro5, between Phe411 and Pro5, and between Trp706 and Asn4 are involved in interaction within the scope of 4 Angstrom. Therefore, it can be seen that they bind to one another by means of hydrophobic interaction. Thus, polypeptides or compounds involved in hydrophobic interaction with the corresponding amino acids of the C-terminal of PA can be used as medicaments for inhibiting influenza virus.
There are many successful examples for designing a medicament based on a structure of target protein (Schneider, G. and Fechner, U., Nature Reviews Drug Discovery 2005, 4, 649). The LigBuilder program developed by the research group set up by Professor Lu Hua from Peking University has more than 700 worldwide registration users (Wang, R. X.; Gao, Y.; Lai, L. H., LigBuilder: A multi-purpose program for structure-based drug design. J. Mol. Mod. 2000, 6, 498). Many examples has been reported of designing a highly active inhibitor successfully using the LigBuilder program, for example, the Boehringer Ingelheim pharmaceutical company uses LigBuilder 1.2 to access and implement aided design for optimizing a highly active kinase inhibitor (Goldberg D R, Hao, M-H., et al., J. Med. Chem. 2007, 50, 4016).
Novel medicament design and calculation is conducted using the LigBuilder 2.0 program according to the crystal structure of the PAC-PB1N complex of the H5N1 virus RNA polymerase. First, directed at the PAC protein, an analysis of binding sites is made, wherein two binding sites wining highest scores are located in the “mouth” region and the “channel” region of the structure. Novel medicament design and calculation for these two sites are conducted using the LigBuilder 2.0 program to obtain some easily-synthesized compounds with high prediction activity.
Molecules binding to the “channel” region of the PA molecules are exemplified as follows:




R1=—CH3, —CH2CH3 
R2=—CH3, —CH2NH2, —CH(OH)CH3, —CH(CH3)2 
Predicted Kd: 8.64 to 9.60




R1=—NH2 
R2=—COCH3, —CH2COCH3, —CO, —OCH2CH3 
R3=—CH2NH2, —CH2(NH2)CH2CH3, —CH2(NH2)CH2CH2CH3 
Predicted Kd: 8.51 to 9.65
Molecules binding to the “mouth” region of the PA molecule are exemplified as follows:




R1=—OH
R2=—OH, —CO, —CONH2, —CONHCH3, COOH
R3=—COOH, —NH2, —C(NH2)2+
R4=—CH(OH)CH3, CONH2, CH(NH2)CH2OH
Predicted Kd: 8.52 to 8.96




R1=—OH, —NHCO
R2=—CH2(OH)CH3, —NHCH3, —CH2OH, —CH2NH2, —NH2, —C(NH2)2+
R3=—NH2, —C(NH2)2+, —CH2OH, —CO, —CH2CO, —NHCO
R4=—OH, —OCH3 
REFERENCE


    Adams, P. D., R. W. Grosse-Kunstleve, et al. (2002). “PHENIX: building new software for automated crystallographic structure determination.” Acta Crystallogr D Biol Crystallogr 58(Pt 11): 1948-54.
    Brunger, A. T., P. D. Adams, et al. (1998). “Crystallography & NMR system: A new software suite for macromolecular structure determination.” Acta Crystallogr D Biol Crystallogr 54(Pt 5): 905-21.
    Deng, T., J. Sharps, et al. (2005). “In vitro assembly of PB2 with a PB1-PA dimer supports a new model of assembly of influenza A virus polymerase subunits into a functional trimeric complex.” J Virol 79(13): 8669-74.
    Deng, T., J. L. Sharps, et al. (2006). “Role of the influenza virus heterotrimeric RNA polymerase complex in the initiation of replication.” J Gen Virol 87(Pt 11): 3373-7.
    Emsley, P. and K. Cowtan (2004). “Coot: model-building tools for molecular graphics.” Acta Crystallogr D Biol Crystallogr 60(Pt 12 Pt 1): 2126-32.
    Fodor, E., M. Crow, et al. (2002). “A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs.” J Virol 76(18): 8989-9001.
    Fodor, E., D. C. Pritlove, et al. (1994). “The influenza virus panhandle is involved in the initiation of transcription.” J Virol 68(6): 4092-6.


Hara, K., F. I. Schmidt, et al. (2006). “Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding.” J Virol 80(16): 7789-98.

    Hara, K., M. Shiota, et al. (2001). “Influenza virus RNA polymerase PA subunit is a novel serine protease with Ser624 at the active site.” Genes Cells 6(2): 87-97.
    Hendrickson, W. A. (1991). “Determination of macromolecular structures from anomalous diffraction of synchrotron radiation.” Science 254(5028): 51-8.
    Honda, A., K. Mizumoto, et al. (2002). “Minimum molecular architectures for transcription and replication of the influenza virus.” Proc Natl Acad Sci U S A 99(20): 13166-71.
    Hulse-Post, D. J., J. Franks, et al. (2007). “Molecular changes in the polymerase genes (PA and PB1) associated with high pathogenicity of H5N1 influenza virus in mallard ducks.” J Virol 81(16): 8515-24.
    Kawaguchi, A., T. Naito, et al. (2005). “Involvement of influenza virus PA subunit in assembly of functional RNA polymerase complexes.” J Virol 79(2): 732-44.
    Munster, V. J., E. de Wit, et al. (2007). “The molecular basis of the pathogenicity of the Dutch highly pathogenic human influenza A H7N7 viruses.” J Infect Dis 196(2): 258-65.
    Murshudov, G. N., A. A. Vagin, et al. (1997). “Refinement of macromolecular structures by the maximum-likelihood method.” Acta Crystallogr D Biol Crystallogr 53(Pt 3): 240-55.
    Otwinowski, Z. M., Wladek (1997). “Processing of x-ray diffraction data collected in oscillation mode” Methods in Enzymology 276 (Macromolecular Crystallography, Part A): 307-326
    Perez, D. R. and R. O. Donis (2001). “Functional analysis of PA binding by influenza a virus PB1: effects on polymerase activity and viral infectivity.” J Virol 75(17): 8127-36.
    Perrakis, A., R. Morris, et al. (1999). “Automated protein model building combined with iterative structure refinement.” Nat Struct Biol 6(5): 458-63.
    Sanz-Ezquerro, J. J., T. Zurcher, et al. (1996). “The amino-terminal one-third of the influenza virus PA protein is responsible for the induction of proteolysis.” J Virol 70(3): 1905-11.
    Sheldrick, G. M., Ed. (1998). Direct Methods for Solving Macromolecular Structures. Dordrecht, The Netherlands, Kluwer Academic Publishers.
    Sugiura, A., M. Ueda, et al. (1975). “Further isolation and characterization of temperature-sensitive mutants of influenza virus.” Virology 65(2): 363-73.
    Taubenberger, J. K. and D. M. Morens (2007). “The Pathology of Influenza Virus Infections.” Annu Rev Pathol.
    Vonrhein, C., E. Blanc, et al. (2007). “Automated structure solution with autoSHARP.” Methods Mol Biol 364:215-30.

## Claims

1. A crystal complex of the influenza A virus polymerase PA-PAc subunit C-terminus and the influenza A virus polymerase PB1-PB1N subunit, wherein the PA-PAc subunit C-terminus consists of amino acids 257-716 of SEQ ID NO:1, and the influenza A virus polymerase PB1-PB1N subunit consists of amino acids 2-26 of of SEQ ID NO:2, and wherein said crystal complex is in space group P41212 having unit cell dimension of a=b=122 Angstrom, c=133 Angstrom, α=β=γ=90°.

2. The crystal complex of claim 1, wherein said crystal diffracts X-ray, and the X-ray diffraction pattern is solved to produce the three dimensional structure of the complex defined by the atomic coordinates listed in Table 1, or atomic coordinates having an average root mean square deviation smaller than or equal to 1.7 Angstrom with respect to the atomic coordinates listed in Table 1.

3. The crystal complex of claim 2, wherein the PA-PAc subunit C-terminus interacts with the PB1-PB1N N-terminus through at least one amino acid of the PA-PAc subunit selected from group consisting of Leu666 , Phe710 , Val636 , Leu640 , Trp706 and Gln670 of SEQ ID NO:1.

4. The crystal complex of claim 2, wherein at least one amino acid selected from the group consisting of Ile621, Gly622, Glu623, Thr618 and Pro620 of the PA-PAc subunit of SEQ ID NO:1 interacts with the PB1 subunit.

5. The crystal complex of claim 2, wherein the PA-PAc subunit comprises a pocket structure which interacts with the PB1N subunit, said pocket structure comprising at least one amino acid selected from the group consisting of Asn647, Gln408, Cys584, Gln587, Gln591, Lys643, Asn647, Ser659, Lys663, Trp699 and Asn703 of SEQ ID NO:1.

6. The crystal complex of claim 2, wherein the PA-PAc subunit comprises groove and channel structures which bind to nucleotides, RNA, or other small molecules or proteins, said groove and channel structures comprising at least one amino acid selected from the group consisting of Trp406, Glu410, Lys461, Glu524, Phe525, Ser526, Lys536, Lys539, Tyr540, Leu563, Tyr564, Arg566 and Lys574 of SEQ ID NO:1.

7. A method of expression and purification of a complex of the influenza A virus polymerase PA-PAc subunit C-terminus, said C-terminus consisting of amino acids 257-716 of SEQ ID NO:1, and the influenza A virus polymerase PB1-PB1N subunit N-terminus polymerase consisting of residues 2-26 of of SEQ ID NO:2 according to claim 3, said method comprising:
(a) Expressing said PA-PAc subunit C-terminus consisting of residues 257-716 of SEQ ID NO:1 and said PB1-PB1N subunit N-terminus consisting of residues 2-26 of SEQ ID NO:2 in a suitable expression system, where either the PA-PAc subunit C-terminus or the PB1-PB1N subunit N-terminus is expressed as a fusion protein comprising an affinity tag;
(b) Combining said expressed PA-PAc subunit C-terminus and said PB1-PB1N subunit N-terminus proteins for a time sufficient for the subunits to complex; and
(c) Isolating and purifying said complex.

8. The method of claim 7, wherein the affinity tag is selected from the group consisting of GST, Flag-tag, Myc-tag, and MBP-tag.

9. The method of claim 8, wherein the expression system is an Escherichia coli expression system.

10. A method to obtain a crystal complex of the influenza A virus polymerase PA-PAc subunit C-terminus, said C-terminus consists of amino acids 257-716 of SEQ ID NO:1, and the influenza A virus polymerase PB1-PB1N subunit consisting of residues 2-26 of SEQ ID NO:2 according to claim 1, said method comprising:
(a) Concentrating a purified complex of the PA-PAc subunit C-terminus and the PB1-PB1N subunit N-terminus to 5-30 mg/ml; and
(b) Obtaining the crystal from 1-1.3 M sodium acteate, pH 4.9, using the sitting or hanging drop method.