ORIGINAL PAPER
Computational insight into novel molecular recognition mechanism of different bioactive GAs and the Arabidopsis receptor GID1A
Hongxia Duan&Dongling Li&Hongchen Liu&
Desheng Liang&Xinling Yang
Received:20March2013/Accepted:31July2013
#Springer-Verlag Berlin Heidelberg2013
Abstract Gibberellin(GA)is an essential plant hormone and plays a significant role during the growth and development of the higher plants.The molecular recognition mode between GA and receptor Arabidopsis thaliana GIBBERELLIN INSENSI-TIVE DW ARF1A(AtGID1A)was investigated by molecular docking and dynamics simulations to clarify the selective per-ceived mechanism of different bioactive GA molecules to AtGID1A.The6-COOH group of GA,especially itsβconfig-uration,was found to be an indispensable pharmacophore group for GA recognition and binding to AtGID1A.Not only does a strong salt bridge interaction between the6β-COOH group of GA and Arg244of AtGID1A play a
very important role in the GA recognition of the receptor,but also an indirect water bridge interaction between the pharmacophore group6β-COOH of GA and the residue Tyr322of AtGID1A is essential for the GA binding to the receptor.The site-directed residues mutant modeling study on the receptor-binding pocket con-firmed that the mutations of Arg244and Tyr322decreased the GA binding activity due to the disappearances of the salt bridge and the hydrogen bond interaction.The3β-OH group of GA was well known to be necessary for the GA bioactivity due to its forming a unique hydrogen bond with Tyr127of AtGID1A.In addition,the hydrophobic interaction between GA and AtGID1A was considered a necessary factor to lock the GA active conformation and stabilize the GA-GID1A complex structure.The novel molecular recognition mode will be beneficial in elucidating the GA regulation function on the growth and development of the higher plants.
Keywords AtGID1A.GA.Molecular recognition mechanism.Residue mutation.SAR
Introduction
Gibberellin(GA),which is a well-known tetracyclic diterpenoid phytohormone,participates in control of higher plant throughout the life cycle process.GA may be responsible for a wide range of plant growth responses,including seed germination,stem elongation,leaf expansion,induction of flowering,and pollen
maturation[1].The famous agriculture‘Green Revolution’in the1960s was reported to relate to the alterations in GA biosyn-thesis or its signaling transduction processes[2–4].This was because GA induces the dwarfing traits in rice and wheat and further develops their high-yielding varieties.In addition,GA also results in the promotion of growth in a variety of fruit crops, an increase in the sugar yield from sugarcane,and the stimula-tion of the barley-malting process in the beer-brewing industry [5].Therefore,GA exerts a great influence not only in agricul-ture production but also in commercial application.
Since the first discovery of GA3in the Gibberella fujikuroi fungus[6],136GAs have been identified from different plant, fungi and bacteria at present[7].However,only a few,such as GA3,GA4,GA1,and GA7,function as bioactive plant hor-mones(Fig.1)[8].The others are either their precursors or degradation products in the GA biosynthesis and signaling transduction processes[9].Currently,the empirical rule on the structure-activity relationship(SAR)of GA suggests that6-COOH was important for the GA biological activity in the dwarf pea,rice,and barley[10].The3β-OH group and a lactone ring between C4and C10were confirmed to be necessary for the biological activity of GA in some
dwarf Electronic supplementary material The online version of this article
(doi:10.1007/s00894-013-1971-0)contains supplementary material,
which is available to authorized users.
H.Duan(*)
:D.Li:H.Liu:X.Yang
Department of Applied Chemistry,College of Science,China
Agricultural University,Beijing,People’s Republic of China100193
e-mail:hxduan@cau.edu
D.Liang
College of Chemistry and Chemical Engineering,Graduate
University of Chinese Academy of Sciences,Beijing100049,
People’s Republic of China
DOI10.1007/s00894-013-1971-0
plants [11,12].However,how to function for these pharmacophore groups of GA is not clear at a molecular level.A number of GA binding proteins (GBPs)were identified as some GA receptor candidates in an early stage of the GA perception study [13].These soluble GBPs in the pea and cucumber hypocotyls were used to evaluate the GA binding activity by the stem elongation assay experiment [14].Until 2005,GIBBERELLIN INSENSITIVE DWARF1(GID1)in rice was first determined successfully as a GA receptor by Ueguchi et al.[15].Subsequently,three GID1homologs,in-cluding AtGID1A,B,and C,were found in Arabidopsis thaliana [16–19].It was very exciting that the crystal structures of GA 3/GA 4-AtGID1A-DELLA were successfully determined by Murase et al.in 2008[20].The complex structures of Oryza sativa GID1(OsGID1)co-crystallized with GA 3and GA 4were reported almost at the same time [21].The successful hunt for the GA receptor not only reveals a new insight into how GAwas recognized by GID1A,but also enhances the molecular level understanding of the GA signaling transduction pathway.There-fore,a new model of the GA signal transduction pathway was proposed based on the reported crystal structure GA 3-AtGID1A-DELLA [20].GA,as an ‘allosteric inducer,’induces a GID1A conformational change in the N-terminal helical swit
ch and then promotes a conformational transition of the N-terminal of the downstream DELLA protein to further enhance the binding interaction between GID1A and DELLA.Recently,this model was refined such that the GID1A-DELLA binding also leads to a conformational change in the C-terminal GRAS domain of DELLA to enhance its GRAS domain recognized by the F-box protein,which promotes polyubiquitination by SCFSL Y1/GID2[22].Even though there are a number of studies on the interaction of GID1-DELLA to be report-ed,the studies focusing on the molecular interaction mode between active or inactive GAs and the receptor GID1are still limited.
Although the crystal structures of two receptors AtGID1A and OsGID1bound with GA 3and GA 4were both reported,respectively,there are only a few residue-mutant studies on OsGID1.The residue mutations at S127A,S123A,D250A,and V246A in OsGID1were recently produced and their binding activities to GAs were examined in vitro.The obtained results showed that these four mutants only retained a low or moderate binding activity to GA 4[21].The other mutation studies on the variants F27L,I133L,I133V ,and L330I in OsGID1indicated to be their lower affinity and specificity for active and inactive GAs [23].The latest mutant studies on S123A,Y134F,S198A,and Y329F in OsGID1revealed that these residue mutations decreased the binding activity to GA 4[24].Therefore,these conserved residues in OsGID1were indicated to be important for the GA binding
a
b c
Fig.1Chemical structure of the bioactive and inactive GAs.a GAs of different functional groups on C6;b GAs of different functional groups at C3;c GAs of different functional groups at C13
affinity.To the best of our knowledge,there are no studies on the residue mutation in AtGID1A and their influence on the binding affinity with GAs.
With the crystal structure determination on the GA receptor AtGID1A,therefore,the SAR of the GA molecule were investigated by molecular docking and dynamics simulations to clarify the key pharmacophore character of active and inactive GAs.Meanwhile,the site-directed mutant modeling study on some key residues in AtGID1A was performed with the aid of the molecular simulation technology to explore the importance of the conserved residues in AtGID1A to recog-nize the active GA.These studies will be favorable to eluci-date the molecular mechanism of GAs perception to the receptor AtGID1A and further discover some novel GA-like active molecules based on the receptor AtGID1A structure.
Materials and methods
Materials
As shown in Fig.1[7],the active and inactive GA molecules GA3/GA3Me,GA7/GA7Me,GA4/3-epi-GA4/GA9and GA1 were selected to investigate the SAR of the GA compounds and their molecular recognition mechanism based on the target AtGID1A.According to the pharmacophore group characteristics,these GA molecules were divided into three sample groups,namely,6-COOH(a),3-OH(b),and13-OH (c).The IC50of all of the selected GAs were shown in Table1, which were determined by observing the competitive inhibi-tion affinity between the tritiated16,17-dihydro-GA4and various bioactive GAs binding to the receptor[25,26].The structure of AtGID1A was retrieved from the RCSB Protein Data Bank(PDB:2ZSH)[20].Methods
Molecular docking
Molecular docking calculations were carried out using a Surflex-dock algorithm in the Sybyl7.3software package on the Linux platform[27].The suitable putative pose of ligand called protomol was generated rapidly by means of the Ham-merhead scoring function with a surface-based molecular similarity method[28–30].In our study,the automatic mode was adopted to generate an ideal protomol in the active site of the receptor.All hydrogen atoms and MMFF94charges were added to the receptor in our
molecular simulation.The docked conformations of all GA molecules were generated and opti-mized based on the GA3conformation extracted from the reported crystal complex GA3-AtGID1A-DELLA.A series of AtGID1A mutants were produced using the Biopolymer module and their energy minimization were performed by the MMFF94force field and MMFF94charges.All of the other parameters were defined as their default ones.
Molecular dynamic simulations
Molecular dynamics(MD)simulations on the complex GA3-GID1A-DELLA and its mutants were performed using the GROMACS4.0.5package[31].The G43a1force field was used to calculate the protein energy.The topology files and force field parameters of GA3were generated using the PRODRG program[32].The whole complex was solvated with explicit solvent SPC water and was neutralized by adding 10NA+ions to replace the corresponding water molecules. The steepest descent and conjugated gradient methods were used for the energy minimization of each system.The refer-ence temperature was fixed at300K and all bonds were constrained with LINCS[33].A long-range electrostatics
Table1Binding scores and bio-activity IC50of different GAs for the receptor
a.IC50of cucumber hypocotyls GBP for different GA molecules (Yalpani et al.[26])
b.IC50of AtGID1A for different GA molecules in Arabidopsis (Nakajima et al.[16])GAs ScoreΔG(kcal mol−1)IC50(M)pIC50Ref.
6-COOH GA310.63−14.445×10−6(3×10−5)  5.30(4.52)a(b) GA3Me  5.46−7.42>5×10−4<3.30a
GA710.92−14.845×10−87.30a
GA7Me  5.99−8.14>5×10−4<3.30a
reported6-epi-GA38.14−11.06–––
6-nor-GA38.21−11.16–––
3-OH GA411.29−15.343×10−7(5×10−8)  6.52(7.30)b(a) GA99.21−12.51>3×10−4(5×10−5)  3.52(4.30)b(a)
3-epi-GA48.85−12.02>3×10−4(5×10−4)  3.52(3.30)b(a) 13-OH GA710.92−14.845×10−87.30a GA310.63−14.443×10−5(5×10−6)  4.52(5.30)b(a)
GA411.29−15.343×10−7(5×10−8)  6.52(7.30)b(a)
GA110.92−14.843×10−5(5×10−6)  4.52(5.30)b(a)
was handled using PME during the whole simulation process [34].The energy minimization of the whole systems was first subjected to a position restrained simulations for 100ps.Then,a full molecular dynamics simulation was performed for 10ns on each system with the NPT canonical ensemble.The coor-dinates of the whole system were written to the trajectory file at 2ps intervals.
Results and discussion
The SAR study of GA based on the receptor AtGID1A All of the selected GA molecules were docked into the recep-tor AtGID1A to explore their molecular interaction mode and clarify the SAR of GAs.As shown in Fig.2,the docked results on GA 3indicated that the hydrogen bond interactions and the hydrophobic effect both cooperated for the signal molecule GA recognition of the binding pocket of AtGID1A.The docked conformation of GA 3was overlapped well in the crystal structure GA 3-AtGID1A-DELLA with only a 0.52Åroot mean squared deviation (RMSD).To further confirm the docked system stability,MD simulations on the docked sys-tem with GA 3and its crystal structure were performed in a water environment by the GROMACS program.Based on Fig.3a ,the simulation results indicated that the RMSD value of the C αbackbone in the docking system was almost the same to that of the crystal system with about 2.2Å.It was clear that the docked system GA 3-AtGID1A-DELLA was very stable verified by the MD simulations.The docked results demonstrated that the 3-OH group of GA 3formed
a
Fig.2The 2D diagram of the molecular interactions between GA 3and the receptor AtGID1A.The pink circles represent residues involved in the hydrogen bond interactions.The green circles represent residues involved in the hydrophobic interactions.Water molecules are represented by the aquamarine circles.Hydrogen bond interactions with the water molecules,amino acid main-chains,and amino acid side-chains are represented by the black ,green ,and blue dashed lines ,respectively,directed toward the electron
donor
Fig.3The molecular dynamics results of GA 3-GID1A-DELLA com-plex.a The RMSD value of the docked (blue)and crystal complex (red )obtained during the 10ns MD simulations.b The binding mode of GA 3with AtGID1A after the MD simulations
direct hydrogen bond to the phenolic hydroxyl group of Tyr127with a distance of2.95Å(O/O)in Fig.2.This corresponding distance became longer with3.70Å(O/O)after the simulations but was within the range of the hydrogen bond in Fig.3b.The6-COOH group of GA3not only formed a strong salt bridge interaction with a distance2.61Åto the guanidinium group of Arg244in the receptor,but it was also involved in the multiple hydrogen bond network with residues Ser116,Ser191and Tyr322of AtGID1
A in Fig.2.It is important to note that these key interactions between the6-COOH of GA3and AtGID1Awere conserved even after10ns MD simulations in Fig.3b.It was obvious that these interac-tions between the3-OH,6-COOH group of GA3and AtGID1A both contributed to the binding affinity between GA3and the receptor GID1A,which was in agreement with the previously reported results on the crystal structure[20]. However,as indicated in Fig.2,the13-OH group of GA3 formed a direct hydrogen bond with a negatively charged residue Asp243instead of Phe238in the crystal structure due to the hydrogen atom orientation changed in the13-OH group of GA3[20].After the simulations,this hydrogen bond interaction still existed but was changed to an indirect water bridge via HOH350in the Fig.3b.In addition,some important hydrophobic interactions were found between the alkyl group and the carbocyclic ring of GA3and the surrounding residues Ile126,Leu323,Val319,Val239,Ile24,Phe27,and Tyr31of AtGID1A in Fig.2,which was in agreement with the results reported by Murase et al.[20].
The importance of the6β-COOH group for the GA binding affinity
GA3and GA7were docked into the binding pocket of the receptor AtGID1A to explore the effect of the6-COOH group on the GA binding ability to the receptor.The6-COOH group of GA7was found to be similar to that of GA3and anchors the whole hormone molecule at the bottom of the binding pocket of
AtGID1A by forming some multiple hydrogen bonds with residues Ser116and Ser191and with Tyr322via two water molecules H2O400and H2O458.More importantly,the neg-ative charge on the6-COOH group of GA7was neutralized by the formation of a salt bridge with residue Arg244of AtGID1A.These docked results were consistent with the molecular interaction character described above between the 6-COOH group of GA3and the receptor AtGID1A.It was recently reported that the methylation of the6-COOH group in GA would reduce the GA binding affinity to GID1A[16, 35].To further clarify the influence of the6-COOH methyla-tion on the GA binding activity,GA3Me and GA7Me were also docked into the binding pocket of the protein AtGID1A. As indicated in Table1,the calculated results revealed that the scores were significantly decreased from10.63for GA3to 5.46for GA3Me.A similar change was observed for GA7and GA7Me with a score of10.92and5.99,respectively.These molecular simulation scores were in good agreement with their IC50values obtained from the GA-binding assay in vitro.As shown in Fig.4a,this was because the indirect water bridge interaction surrounding the6-COOH group in both GA3Me and GA7Me disappeared completely compared with GA3and GA7.Meanwhile,the multiple hydrogen bond interactions were weakened because of the methylation of
the Fig.4The hydrogen bond interactions between GAs and AtGID1A.a The hydrogen bond interaction between the6-COOH group of GA3 (cyan),GA3Me(magenta)and AtGID1A.The hydrogen bond between GA3,GA3Me and AtGID1A are represented by the black and magenta broken lines,respectively;b The hydrogen bond interactions between the 3-OH group of GA4(pink),GA9(green),3-epi-GA4(blue)and the key residues of the binding pocket of AtGID1A;c The hydrogen bond and electrostatic interactions between13-OH of GA3(cyan),GA7(orange) and AtGID1A

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