Structural Basis For Dimerization And DNA Recognition Of A Lateral Organ Boundaries Domain Protein

The Lateral Organ Boundary Domain(LBD)proteins regulate plant pattern formation by precisely coordinating temporal and spatial developmental programs.It was well documented that these proteins are involved in a large number of developmental and metabolic processes,such as meristem programming,leaf patterning,inflorescence morphogenesis,pollen development,lateral root formation and so on.More recently,genetic studies have revealed that functional diversity of the LBD family exceeds lateral organ boundaries formation by defining plant regeneration,photomorphogenesis,disease susceptibility.LBD proteins are plant-specific and are widely distributed in plants.In model plant Arabidopsis,the LBD family contains 43 members,whereas in rice(Oryza sativum),35 members are identified,And a total of 44 LBD genes were also discovered in maize genome.The LOB domain is a highly conserved functional domain located at the N-terminus of LBD proteins and has been shown to be closely related to the dimerization and DNA-binding properties of LBD proteins.We determined the crystal structure of the N-terminal LOB domain of Triticum turgidum L.Ramosa2(termed TtRa2 LD).This is the first 3D structure of LOB domain and The structure was solved to a resolution of 1.88 ? by single-wavelength anomalous dispersion(SAD)using the natively bound zinc atoms.TtRa2 LD has unique structure folding characteristics.The crystal structure shows that a dimeric TtRa2 LD was crystallized in space group P21 21 21 which contains two subunits per asymmetric unit.It mainly fold into five ? helices and are composed of five distinct modules : a compact zinc-finger domain,a GAS domain composed of two ? helices(?2 and ?3)which are perpendicular to each other,and an ?-helical coiled coil Leucine-zipper element(?5)which is connected with GAS domain through ?4 and DPVYG motif.The Zinc finger of TtRa2 LD belongs to a new class of Cys4-type zinc finger.The Zinc finger is composed of an alpha helix(?1)with two extended random coil segment at its both ends.It has no sequencial and structural homology with the known Cys4-type zinc finger protein but is structurally similar to the Zn2/Cys6 zinc finger motif of GAL4.The conformation of the Zinc finger was stabilized by tetrahedral coordination of a Zinc ion with four conserved cysteine residues: Cys28,Cys31,Cys38 and Cys42.The DNA binding assays and analysis of circular dichroism(CD)spectra reveal that the reduction of DNA binding activity accompanied by an alteration of the secondary structures as increasing concentration of EDTA,confirming that Zn2+ is essential for keeping Zinc finger configuration for efficient DNA binding.The hydrophobic interaction of the coiled coil is the main force of TtRa2 LD dimerization and the salt bridge interections and the ?-? stacking at the C-terminus are not necessary to maintain its dimerization.Bimolecular fluorescence complementation(Bi FC)assays and DNA binding experiments demonstrated that TtRa2 LD could form homodimer under the normal physiological environment and the coiled-coil mediated dimerization is crucial for the DNA binding activity of TtRa2 LD.the GAS motif folds into two alpha helices(?2 and ?3)which are linked by an central highly conserved Gly64.Helix ?4 is linked to ?5 through a conserved loop,DPVYG motif.The perpendicular conformation between ?4 and ?5 is stabilized by a set of hydrogen-bonding interactions of the residues on the loop and contributes to the formation of TtRa2 LD dimer.The GAS motif,?4 and DPVYG motif are all involved in stabilizing the spatial structure of TtRa2 LD.Val62 or Asn67 in one monomer was hydrogen bonded with Asn 67' or Val62' in another,respectively.Arg94 and Glu90 at ?4 of one subunit also form two salt bridges with Glu90' and Arg94' of ?4',stabilizing the TtRa2 LD dimer.Point mutation experiments demonstrated that the salt bridging between the two ?4 helices of TtRa2 LD dimer is closely related to its dimer stability and DNA binding activity,which may play an important role in precisely controlling the relative position of the two zinc finger motifs in the TtRa2 LD dimer.While the interaction between the GAS motifs in the TtRa2 LD dimer has a certain degree of flexibility.Based on the three-dimensional structure of LOB domain,the Docking model of LOB domain-DNA complex was established by molecular dynamics simulation and further validated by point mutation experiments.According to the modelled structure,the flexible loop downstream of ?1 in zinc finger motif of TtRa2 LD,which contains Leu34,Arg35,Arg36 and Lys37,was permeated into the major groove of its targeted DNA and thereby played a key role in recognizing and binding to the LBD motifs.the point mutation experiments showed that the recognition and binding of TtRa2 LD to its targeted DNA were mainly dependent on the two key residues: Arg35 and Arg36,and the Lys37 also partly contributed to its DNA bindig activity.There were also significant differences in the DNA binding activity of TtRa2 LD to DNA substrates which contained two palindromic LBD motifs spaced by different length of nucleotides.Specifically,TtRa2 LD could preferably bind to the DNA substrates spaced by four or six nucleotides.But when the space was shortened to one nucleotide or lengthened to eight or twelve nucleotides,the binding capacity of TtRa2 LD to them would be significantly reduced.These results showed that the recognition and binding of TtRa2 LD to its target DNA was restricted by different spacing between two palindromic LBD motifs,but had a certain degree of flexibility.