CN107904217A - The protein structures of Pyricularia oryzae mitogen-activated protein kinase Mps1 and its application in fungicide target - Google Patents
The protein structures of Pyricularia oryzae mitogen-activated protein kinase Mps1 and its application in fungicide target Download PDFInfo
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Abstract
Protein structures the invention discloses Pyricularia oryzae mitogen-activated protein kinase Mps1 and its application in fungicide target, the protein structures of the Pyricularia oryzae mitogen-activated protein kinase Mps1 are by two molecular compositions, by the protein structures for parsing Pyricularia oryzae mitogen-activated protein kinase Mps1, based on structure prediction its there may be self-interaction, further experiments prove that its there are itself c-terminus nonkinase domain (Mps1401‑415) and its own kinase domain (Mps11‑360) between interaction, and related experiment confirms the c-terminus nonkinase domain (Mps1 of Mps1401‑415) significant inhibitory action is respectively provided with to the phosphorylation level of Mps1, this is provided the foundation based on the space structure of Mps1 for the later stage further to provide Structural Guidelines to the Mps1 micromolecular inhibitors with greater activity by the protein steric structure of the c-terminus nonkinase domain of Mps1 designing, screening and optimizing as fungicide target to carry out correlative study.
Description
Technical field
The present invention relates to molecular biology and gene engineering technology field, and in particular to a kind of Pyricularia oryzae mitogen is lived
Change the protein structures of protein kinase Mps1 and its application in fungicide target.
Background technology
Rice is one of cereal crops main in the world, and the population that the whole world there are about 50% is used as staple food using rice.So
And the rice blast as caused by Pyricularia oryzae (Magnaporthe oryzae) infects rice is each rice producing region in the world generally sends out
One of raw, very harmful fungal disease, and restrict the important threat of rice safety production and grain security supply.According to report
Road, the Rice Yield Loss Caused as caused by rice blast about 10%~30%, tremendous influence is generated safely to world food every year
(Skamnioti and Gurr,2009).At present, the disease-resistant variety of Pyricularia oryzae can be effective against due to not yet selecting,
The prevention of rice blast is still based on chemical agent, but due to long-term, a large amount of and unscientific uses of fungicide, Pyricularia oryzae
The report to develop immunity to drugs to fungicide is commonplace, therefore has the green bactericide target compared with high specific to Pyricularia oryzae
Excavation and with greater activity green bactericide develop it is extremely urgent.
Since protein kinase is widely present in eucaryote, and it take part in the process of many vital movements, it is considered that,
Protein kinase (Protein kinase) can carry out correlative study as drug targets.Because of the amino that protein kinase is main
Sour residue is close, topological structure is similar, the key amino acid of catalytic site is guarded, so it is respectively provided with higher guarantor in structure
Keeping property.At present, the kinase protein of existing many mammals and its crystal structure of compound are successfully parsed, such as extracellular signal
Regulatory protein kinases (ExtracelluLar-signal reguLated protein kinase, ERK), c-Jun amino terminals
The albumin crystal structure of the kinases such as kinases (C-Jun N-terminal kinase, JNK) and p38, and p38 respectively with MAPK
The albumin crystal structure of inhibitor BIRB796 and Sorafenib, ERK2 and MAPK inhibitor rosiglitazone compounds
(Wilson et al.,1996；Pargellis et al.,2002；Simard et al.,2009；Gelin et al.,
2015), therefore, had been obtained for using protein kinase as drug targets in mammal extensively and in-depth study, and mesh
The preceding small molecular protein kinase inhibitor that has been related to has obtained promoting and application (Wu et al., 2015) in clinic.
Research shows, is primarily present in Pyricularia oryzae by the signal level of 3 element compositions of MAPKKK, MAPKK and MAPK
Unicom road, and cascade reaction is formed by top-down phosphorylation, and the MAPK after being phosphorylated by with it
The transcription factor interaction of trip regulates and controls the pathogenicity of Pyricularia oryzae, and then influences pathogenic (the Hamel et of Pyricularia oryzae
al.,2012).Mps1 (Slt2-MAPK) is the mitogen work that a kind of growth and development to Pyricularia oryzae plays important regulating and controlling effect
Change protein kinase, the function of being participated in Pyricularia oryzae mainly includes adjusting the integrality of cell membrane, reply unsuitable environmental condition
Response and regulation and control pathogenicity etc., wherein, Mps1 be to the integrality of cell membrane, the intyrusive of spore it is required,
Missing Mps1 can cause Pyricularia oryzae appresorium to penetrate Cuticle and cause to infect failure (Xu et al., 1998).
In protein kinase research, although having using MAPK as drug targets in mammal to carry out correlative study
Report, but mitogen-activated protein kinase as drug targets and is carried out into correlative study not yet in plant pathogenic fungi
Appear in the newspapers；Mps1 is as one of mitogen-activated protein kinase common in Pyricularia oryzae, the cause of its phenotype and Pyricularia oryzae
Characteristic of disease is directly related, can also be used as drug targets in theory and carries out related inhibitor or biocide molecules design in Pyricularia oryzae
The research of aspect.
The information for being disclosed in the background section is merely intended to understanding of the increase to the general background of the present invention, without answering
It has been the prior art well known to persons skilled in the art when being considered as recognizing or implying the information structure in any form.
The content of the invention
It is an object of the invention to provide a kind of protein crystal of Pyricularia oryzae mitogen-activated protein kinase Mps1
Structure and its application in fungicide target, relevant grind is designed so as to be based on target for the later stage and carry out related biocide molecules
Study carefully and provide Structural Guidelines.
To achieve the above object, the present invention provides a kind of albumen of Pyricularia oryzae mitogen-activated protein kinase Mps1
Matter crystal structure, the protein structures are by two molecular compositions.
In another embodiment, described two molecules are molecule A and molecule B to above-mentioned protein structures, described
The amino acid sequence of molecule A and molecule B are identical, i.e. SEQ ID NO:1, NCBI gene numbering is MGG_04943.
Above-mentioned protein structures in another embodiment, in Glu450 in molecule A c-terminuses and molecule B
Tyr234 and Val235, Ser258, Arg260 and Ala261 in Glu452 and molecule B in molecule A c-terminuses, molecule A carboxylics
The Leu457 and lle199 in molecule B in cardinal extremity is interacted by forming hydrogen bond respectively.
Above-mentioned protein structures in another embodiment, in Gly456 in molecule A c-terminuses and molecule B
Ser202, the Thr186 in Asp458 and molecule B in molecule A c-terminuses, in the Leu449 and molecule B in molecule A c-terminuses
Tyr264 interacted respectively by forming sat linkage.
Above-mentioned protein structures in another embodiment, molecule A c-terminus nonkinase domains (Mps1401 -415) and molecule B kinase domains (Mps11-360) between exist interaction, specifically, molecule A c-terminus nonkinase domains
On the FXF binding sites of molecule B.
Above-mentioned protein structures in another embodiment, the molecule A c-terminuses nonkinase domain
Mps1401-415Amino acid composition be：NDLEAELAGGLDQRR, i.e. SEQ ID NO:2.
In another embodiment, the FXF binding sites are promoted to divide above-mentioned protein structures by Pyricularia oryzae
Thr186, lle199, Ser202, Tyr234, Val235, Ser258 in former activated protein kinase Mps1 protein structures,
The hydrophobic amino acids such as Arg260, Ala261 and Tyr264 form.
In another embodiment, molecule A c-terminus nonkinase domains are to rice blast for above-mentioned protein structures
The phosphorylation level of bacterium mitogen-activated protein kinase Mps1 itself is inhibited.
Present invention also offers application of the above-mentioned protein structures in fungicide target.
In another embodiment, the FXF binding sites can be as the target site of fungicide for above application.
Compared with prior art, the present invention has the advantages that：
1) present invention parses Pyricularia oryzae mitogen-activated protein kinase Mps1 protein structures, and
Carry out relevant analysis based on resulting structures, meanwhile, further verified by related experiment, find its c-terminus nonkinase knot
Structure domain has very strong inhibitory action to the phosphorylation level of itself, and being based on target for the later stage carries out related biocide molecules design
Relevant research provides Structural Guidelines, has great importance；
2) protein structures of the invention by parsing Pyricularia oryzae mitogen-activated protein kinase Mps1, are based on
Structure prediction its there may be self-interaction, and further experiments prove that its there are itself c-terminus nonkinase structure
Domain (Mps1401-415) and its own kinase domain (Mps11-360) between interaction, and related experiment confirms Mps1's
C-terminus nonkinase domain (Mps1401-415) inhibitory action is respectively provided with to the phosphorylation level of Mps1, this is further passes through
The protein steric structure of the c-terminus nonkinase domain of Mps1 has greater activity designing, screening and optimizing to Mps1
Micromolecular inhibitor provides Structural Guidelines, and carries out correlation as fungicide target based on the space structure of Mps1 for the later stage
Research provides the foundation.
Brief description of the drawings
Fig. 1 is the protein steric structure of Pyricularia oryzae mitogen-activated protein kinase Mps1 according to the present invention；
Fig. 2 is to measure Pyricularia oryzae mitogen-activated protein kinase by yeast-two hybrid technique according to the present invention
There are the experimental result of self-interaction analysis in vitro by Mps1；
Fig. 3 is to measure Pyricularia oryzae mitogen-activated protein kinase by Immunoprecipitation according to the present invention
There are the experimental result of self-interaction analysis in Pyricularia oryzae body by Mps1；
Fig. 4 is Pyricularia oryzae mitogen-activated protein kinase Mps1 and its truncate phosphorylation level according to the present invention
Analysis experiment.
Embodiment
Below in conjunction with the accompanying drawings, the embodiment of the present invention is described in detail, it is to be understood that the guarantor of the present invention
Protect scope and from the limitation of embodiment.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, it is unless otherwise specified, commercially commercially available.
In order to inquire into whether Pyricularia oryzae mitogen-activated protein kinase Mps1 can carry out phase as drug targets
The research in terms of inhibitor is closed, the present invention has parsed the protein crystal knot of Pyricularia oryzae mitogen-activated protein kinase Mps1
Structure, and based on structural analysis Mps1 there may be self-interaction, meanwhile, by carry out external and internal related experiment into
One step confirms there is interaction between the c-terminus nonkinase domain of Mps1 and its own kinase domain, and c-terminus
Nonkinase domain is respectively provided with significant inhibitory action to the phosphorylation level of Mps1.
1 Mps1 structure elucidations of embodiment
The Mps1 protein crystal diffraction datas being collected into early period, the method replaced using homolgous molecule carry out together it
Source models, and has successfully parsed the protein structures of Mps1, in acquisitionHigh-resolution protein structures
In, by Fig. 1 it can be found that resulting structures are by two molecular compositions, and Mps1 c-terminus nonkinase domain (test into
One step is demonstrate,proved, and it is specifically to be made of 15 amino acid finally to confirm it) it is mutually close with the substrate binding site of kinases in its structure,
Speculate the Mps1 c-terminus nonkinase domains (Mps1 being made of 15 amino acid401-415) may be with kinase domain
(Mps11-360) between exist interaction, the Mps1 protein structures parsed are further analyzed, find its exist from
Body interacts, and the interface that interacts be concentrated mainly on the C-terminal region (c-terminus nonkinase structural region) of Mps1 molecules A with
There is interaction, and the Glu450 in molecule A c-terminuses between the interaction zone of the kinases FXF binding sites of molecule B
With the Tyr234 and Val235 in molecule B, Ser258, Arg260 in Glu452 and molecule B in molecule A c-terminuses and
Ala261, the Leu457 and lle199 in molecule B in molecule A c-terminuses are interacted by forming hydrogen bond respectively；
In addition, the Ser202 in Gly456 in molecule A c-terminuses and molecule B, in the Asp458 and molecule B in molecule A c-terminuses
Thr186, the Leu449 and Tyr264 in molecule B in molecule A c-terminuses are interacted by forming sat linkage respectively.
2 Mps1 self-interactions of embodiment are analyzed：
In order to verify that the c-terminus nonkinase domain of Mps1 that embodiment 1 sees in the structure may swash with its own
There is interaction between enzyme domains, now it is verified with being divided using yeast-two hybrid technique and Immunoprecipitation
Analysis.
1 yeast two-hybrid, specifically includes following related reagent and step：
Reagent：Yeast double miscellaneous kit, fission yeast auxotrophic strain AH109 (being purchased from Clontech companies)；
Carrier：PGADT7, pGBKT7 (are purchased from Clontech companies)；
Step：
(1) the saccharomycete AH109 frozen -80 DEG C of early period is cultivated in line, and picking single bacterium colony is inoculated in containing 50mL YPDA liquid
In the 200mL sterilizing triangular flasks of body culture medium, the shake culture under the conditions of 30 DEG C of 250rpm, until OD600＞ 1.5；
(2) the above-mentioned 200 μ L of culture of quantitative in 300mL YPDA fluid nutrient mediums under the conditions of 30 DEG C of 250rpm into
Row shakes training, to OD600Reach 0.5 or so；
(3) it is transferred in sterilized 500mL centrifugal barrels, 5min is centrifuged under the conditions of 5000rpm, abandon supernatant, adds 50mL
Sterile water by precipitate suspension get up；Then 5min is centrifuged under the conditions of 5000rpm again, will be precipitated with 1.5mL × TE/LiAc
Suspend, be spare competent cell；
(4) added in the EP pipes of 10mL：20 μ L ds cDNA, 6 μ L pGADT7-Mps11-360(0.5 μ g/ μ L), 5 μ g
PGBKT7/bait Plasmid DNA (total amount is no more than 10 μ L), 20 μ L Denatured herring testes carrier DNA；
50 μ L herring testes carrier DNA are gone to in the EP pipes of 1.5mL the pre-degeneration 20min under the conditions of 100 DEG C, are stood
It is placed on ice, DNA is changed into single-stranded.Again plus this DNA is repeated once when cotransformation system；
(5) plus the above-mentioned competent yeast cells got ready of 600 μ L are in above-mentioned pipe, gentle to mix；2.5mL is added again
PEG/LiAc solution is with pipe, gently mixing.Training 45min is shaken under the conditions of 30 DEG C of 200rpm；
(6) DMSO of 160uL is added in pipe, is fully mixed；Water-bath 20min, abundant at interval of 10min under the conditions of 42 DEG C
Mix 1 time；
(7) 700g centrifuges 5min；Supernatant is abandoned, is suspended and precipitated with 3mL YPD Plus Liquid Medium, in 30 DEG C
Training 90min is shaken under the conditions of 200rpm；
(8) 700g centrifuges 5min；Supernatant is abandoned, is got up with suitable 0.9%NaCl by suspension is precipitated；
(9) cotransformation product is equably tiled respectively onto amino acid-deficient culture medium SDO, TDO, QDO tablet；
(SDO：SD/-Leu-Trp, two lack culture medium；TDO：SD/-His-Leu-Trp, three lack culture medium), general 150 μ L/ tablets
(90mm), is cultivated in 30 DEG C of constant incubators to growing bacterium colony, then selects that growth is vigorous, and bacterium colony of the diameter more than 2mm is coated with
(QDO on to QDO tablets：SD/-Ade-His-Leu-Trp, four lack culture medium), 3-15d is cultivated in 30 DEG C of constant incubators,
Eugonic bacterium colony is applied to 4 containing X- α-gal again to lack on tablet SD/-Ade-His-Leu-Trp/X- α-gal, is seen
Examine color change situation.
By the yeast two-hybrid assay of Fig. 2 the result shows that：Mps1 kinase domains Mps11-360With its own nonkinase work(
Can domain Mps1401-415In the presence of direct interaction, Mps1 is further demonstrated that1-360With Mps1401-415It is to exist mutually in vitro
Effect.
2 co-immunoprecipitation experiments, specifically include following related reagent and step：
Reagent：It is conventional reagent；
Carrier：YIP101, YIP105 and GTN (preservation of this laboratory)；
Step：
(1) with this laboratory it is basic experimental technique structure fusion up to carrier (label of fusion for GFP or 3 ×
Flag), and by sequencing verified；
(2) fusion expression vector is transformed into Pyricularia oryzae bacterial strain, screens the correct transformant of resistance, and carry out PCR
Confirm verification；
(3) total protein of candidate's transformant (containing fusion tag GFP or 3 × Flag) is extracted, is placed on ice, it is spare；
(4) Red anti-FLAG M2Afffinity Gel are fully mixed, in the EP pipes for drawing 40 μ L to 1.5mL immediately；
(5) 500 μ L TBS are added, shake 30s；30s is centrifuged under the conditions of 4 DEG C of 8200g；After slowly washing out supernatant, then
The TBS of 500 μ L is added, shakes 30s；30s is centrifuged under the conditions of 4 DEG C of 8200g；
(6) the above-mentioned protein samples got ready of 1mL are added into clean Afffinity Gel, are fully mixed；In 4 DEG C of bars
Under part, concussion mixes 2h；
(7) 30S is centrifuged under the conditions of 4 DEG C of 8200g, abandons supernatant, added the TBS of 500 μ L, gently mixed with pipette tips, this step
Suddenly it is repeated 3 times；
(8) TBS and 20 μ L 5 × protein loading buffer of 80 μ L is added into precipitation, is mixed；Sample becomes
Western blot experiments can be carried out after property；
(9) by analyzing the presence or absence of hybridising band and size in western blot experiments, to determine between protein
Interaction relationship.
By the co-immunoprecipitation experiment of Fig. 3 the result shows that：Mps1 its kinase domain Mps1 in Pyricularia oryzae body1-360
With nonkinase domain Mps1401-415There are interaction, further demonstrates that Mps11-360With Mps1401-415In rice blast thalline
It is inside the presence of interaction.
3 Mps1 phosphorylation levels of embodiment analyze (Western blot)
Reagent：It is conventional reagent；
Step：
(1) will advance purified Mps1WTAnd Mps11-360According to mass ratio it is equal in the case of carry out preparation of samples；Take
The above-mentioned ready protein samples of 80 μ L, add 20 μ L 5 × protein loading buffer, and boiling is boiled 5min, stood on ice
It is spare after 5min；
(2) SDS-PAGE protein adhesives, loading are prepared according to the formula in Takara catalogues；
(3) SDS-PAGE protein adhesive 15V electricity is turned over into night (attention：Cathode is put on, glue is in anode), and electric turn trough is put
In on blender, to avoid electroporation is burnt out；
(4) first with dye liquor Ponceau S solution dyes 0.5min or so, you can see the albumen gone on film；Outwell
After dye liquor, with 5% skimmed milk power (being prepared with 1 × TBS-T buffer) in room temperature close membrane 1h on shaking table；
(5) wash film 3 times with 1 × TBS-T buffer, rinse 5min every time, add primary antibody Phospho-p44/42MAPK
Antibody (CST, dilutes 1000 times with 1 × TBS-T buffer+5% skimmed milk powers and uses, 10 μ L antibody in 10mL TBS-T, in
1h is reacted at room temperature on shaking table；
(6) wash film 3 times with 1 × TBS-T buffer, rinse 5min every time, add secondary antibody anti-IgG (rabbit source antibody,
Abmart companies, dilute 10000 times of uses with 1 × TBS-T buffer+5% skimmed milk powers, 1uL are added in 10mL TBS-T
Anti-IgG) in reacting at room temperature 1h on shaking table；
(7) wash film 3 times with 1 × TBS-T buffer, rinse 5min every time, add fluorescent dye solution afterwards
Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific) are in 4 DEG C of conditions
Under wash film 5min；
(8) after film is wrapped, face-up, covered with X-ray, press mold 2min.X-ray is then taken out, in developer solution
Develop 2min or so, and tap water rinses, and is then fixed 2min, and according to the presence or absence of hybridising band and size, to determine protein
The level of phosphorylation.
The phosphoric acid of body protein test result indicates that, is truncated to the Mps1 and Mps1 of purifying by the Western blot of Fig. 4
Change level is analyzed, in molal quantity Mps1 under the same conditionsWTAnd Mps11-360Can be by general phospho-AB
P-p44/42 is detected, shows the Mps1 that purifying obtainsWTThere is tyrosine phosphorylation ability, but Mps11-360Band it is notable
Compare Mps1WTIt is significantly broadening, so as to show the c-terminus nonkinase domain (Mps1 of Mps1401-415) to the phosphorylation of Mps1 itself
State has higher inhibitory action.
It is foregoing to the present invention specific exemplary embodiment description be in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can be much changed
And change.The purpose of selecting and describing the exemplary embodiment is that explain that the certain principles of the present invention and its reality should
With so that those skilled in the art can realize and utilize the present invention a variety of exemplaries and
Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.
Sequence table
<110>China Agricultural University
<120>The protein structures of Pyricularia oryzae mitogen-activated protein kinase Mps1 and its in fungicide target
Application
<130> P172043DD1F
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 415
<212> PRT
<213> Magnaporthe oryzae（SEQ ID NO：1）
<400> 1
Met Ser Asp Leu Gln Gly Arg Lys Ile Phe Lys Val Phe Asn Gln Asp
1 5 10 15
Phe Ile Val Asp Glu Arg Tyr Thr Val Thr Lys Glu Leu Gly Gln Gly
20 25 30
Ala Tyr Gly Ile Val Cys Ala Ala Val Asn Asn Gln Thr Ser Glu Gly
35 40 45
Val Ala Ile Lys Lys Val Thr Asn Val Phe Ser Lys Lys Ile Leu Ala
50 55 60
Lys Arg Ala Leu Arg Glu Ile Lys Leu Leu Gln His Phe Arg Gly His
65 70 75 80
Arg Asn Ile Thr Cys Leu Tyr Asp Met Asp Ile Pro Arg Pro Asp Asn
85 90 95
Phe Asn Glu Thr Tyr Leu Tyr Glu Glu Leu Met Glu Cys Asp Leu Ala
100 105 110
Ala Ile Ile Arg Ser Gly Gln Pro Leu Thr Asp Ala His Phe Gln Ser
115 120 125
Phe Ile Tyr Gln Ile Leu Cys Gly Leu Lys Tyr Ile His Ser Ala Asn
130 135 140
Val Leu His Arg Asp Leu Lys Pro Gly Asn Leu Leu Val Asn Ala Asp
145 150 155 160
Cys Glu Leu Lys Ile Cys Asp Phe Gly Leu Ala Arg Gly Phe Ser Val
165 170 175
Asp Pro Glu Glu Asn Ala Gly Tyr Met Thr Glu Tyr Val Ala Thr Arg
180 185 190
Trp Tyr Arg Ala Pro Glu Ile Met Leu Ser Phe Gln Ser Tyr Thr Lys
195 200 205
Ala Ile Asp Val Trp Ser Val Gly Cys Ile Leu Ala Glu Leu Leu Gly
210 215 220
Gly Arg Pro Phe Phe Lys Gly Arg Asp Tyr Val Asp Gln Leu Asn Gln
225 230 235 240
Ile Leu His Ile Leu Gly Thr Pro Asn Glu Glu Thr Leu Ser Arg Ile
245 250 255
Gly Ser Pro Arg Ala Gln Glu Tyr Val Arg Asn Leu Pro Phe Met Ala
260 265 270
Lys Lys Pro Phe Pro Thr Leu Phe Pro Asn Ala Asn Pro Asp Ala Leu
275 280 285
Asp Leu Leu Asp Arg Met Leu Ala Phe Asp Pro Ser Ser Arg Ile Ser
290 295 300
Val Glu Gln Ala Leu Glu His Pro Tyr Leu His Ile Trp His Asp Ala
305 310 315 320
Ser Asp Glu Pro Asp Cys Pro Thr Thr Phe Asn Phe Asp Phe Glu Val
325 330 335
Val Glu Asp Val Gly Glu Met Arg Lys Met Ile Leu Asp Glu Val Tyr
340 345 350
Arg Phe Arg Gln Leu Val Arg Thr Ala Pro Gly Ala Gly Gly His Gly
355 360 365
Ala Pro His Ala Pro Gln Val Pro Ile Pro Ala Gly Ala Gly Gln Gly
370 375 380
Gln Trp Lys Ala Glu Asp Pro Arg Pro Gln Glu Tyr Val Gly Gln Met
385 390 395 400
Asn Asp Leu Glu Ala Glu Leu Ala Gly Gly Leu Asp Gln Arg Arg
405 410 415
<210> 2
<211> 15
<212> PRT
<213> Magnaporthe oryzae（SEQ ID NO：2）
<400> 2
Asn Asp Leu Glu Ala Glu Leu Ala Gly Gly Leu Asp Gln Arg Arg
1 5 10 15
Claims (10)
- A kind of 1. protein structures of Pyricularia oryzae mitogen-activated protein kinase Mps1, it is characterised in that the egg White matter crystal structure is by two molecular compositions.
- 2. protein structures according to claim 1, it is characterised in that described two molecules are molecule A and molecule The amino acid sequence of B, the molecule A and molecule B are identical, i.e. SEQ ID NO:1.
- 3. protein structures according to claim 1, it is characterised in that the Glu450 in molecule A c-terminuses is with dividing Tyr234 and Val235 in sub- B, Ser258, Arg260 and Ala261 in Glu452 and molecule B in molecule A c-terminuses, The Leu457 and lle199 in molecule B in molecule A c-terminuses is interacted by forming hydrogen bond respectively.
- 4. protein structures according to claim 1, it is characterised in that the Gly456 in molecule A c-terminuses is with dividing Ser202 in sub- B, the Thr186 in Asp458 and molecule B in molecule A c-terminuses, Leu449 in molecule A c-terminuses with Tyr264 in molecule B is interacted by forming sat linkage respectively.
- 5. protein structures according to claim 1, it is characterised in that molecule A c-terminus nonkinase domains position In on the FXF binding sites of molecule B.
- 6. protein structures according to claim 5, it is characterised in that the molecule A c-terminuses nonkinase structure The amino acid in domain forms：NDLEAELAGGLDQRR, i.e. SEQ ID NO:2.
- 7. protein structures according to claim 5, it is characterised in that the FXF binding sites are by Pyricularia oryzae Thr186, lle199, Ser202, Tyr234, Val235 in mitogen-activated protein kinase Mps1 protein structures, Ser258, Arg260, Ala261 and Tyr264 hydrophobic amino acid form.
- 8. protein structures according to claim 5, it is characterised in that molecule A c-terminus nonkinase domains pair The phosphorylation level of Pyricularia oryzae mitogen-activated protein kinase Mps1 itself is inhibited.
- 9. according to application of the claim 5-8 any one of them protein structures in fungicide target.
- 10. application according to claim 9, it is characterised in that the FXF binding sites can be as the effect of fungicide Target site.
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| CN115043834A (en) * | 2022-05-12 | 2022-09-13 | 中国农业大学 | Naphthyridine amide compound and preparation method and application thereof |
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| JP2010220590A (en) * | 2009-03-25 | 2010-10-07 | National Institute Of Agrobiological Sciences | Screening method using new reporter gene |
| CN107090443A (en) * | 2016-02-18 | 2017-08-25 | 中国农业大学 | A kind of method for preparing Pyricularia oryzae mitogen-activated protein kinase MoMps1 crystal |
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| JP2010220590A (en) * | 2009-03-25 | 2010-10-07 | National Institute Of Agrobiological Sciences | Screening method using new reporter gene |
| CN107090443A (en) * | 2016-02-18 | 2017-08-25 | 中国农业大学 | A kind of method for preparing Pyricularia oryzae mitogen-activated protein kinase MoMps1 crystal |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115043834A (en) * | 2022-05-12 | 2022-09-13 | 中国农业大学 | Naphthyridine amide compound and preparation method and application thereof |
| CN115043834B (en) * | 2022-05-12 | 2023-06-06 | 中国农业大学 | Naphthyridinamide compound and preparation method and application thereof |
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