Sandbox Reserved 933

This Sandbox is Reserved from 01/04/2014, through 30/06/2014 for use in the course "510042. Protein structure, function and folding" taught by Prof Adrian Goldman, Tommi Kajander, Taru Meri, Konstantin Kogan and Juho Kellosalo at the University of Helsinki. This reservation includes Sandbox Reserved 923 through Sandbox Reserved 947.

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Introduction

Figure 1. Schematic presentation LEAFY regulatory roles in controlling floral organ identity (A) and the ABC model in Arabidopsis thaliana (B).

FLORICAULA/LEAFY (FLO/LFY) genes encode a plant specific transcription factor family that controlling floral fate of reproductive phase. In the plant model system Arabidopsis thaliana , ‘’LFY’’ also acts upstream of floral homeotic genes to modulate organ identity. LFY activates the organ identity genes by binding to promoter regions of floral organ identity genes. LFY can directly bind to the promoter to APELATA1 (AP1), while co-regulators UNUSUAL FLORAL ORGANS (UFO) and WUSCHEL (WUS) are required for increment of binding affinity to promoter regions of APELATA3 (AP3) and AGAMOUS (AG), respectively. The exact mechanism how LFY binds to these promoters has yet to be well elucidated until the first structure report about LFY-pAP1 and LFY-pAG (ref 5). Among land plants, FLO/LFY homologs share a highly conserved DNA binding region that a hypothesis claimed substitution in this domain might result in the functional divergence (ref 6). Recently, a new study (ref 7) provided new insights of structural basis of LEAFY evolution by changing DNA binding activity.

spinqualitylabelsall models Structure of LFY binding with AP1 and AG promoter region (PDB entry: 2VY1/2VY2) Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural Basis of LEAFY binding Figure 2. Site specific recognition of LFY protein at major (A) and minor (B) grooves. Assembly of PDB entry 2VY1 were obtained from PISA server and further visualized by Pymol. Red arrows marked site specific hydrogen bonds. General information about the structure The LFY gene encodes a 424 amino acids protein that containing two domains. The N-terminal domain of LFY has been proved mediating homodimerization (ref) and it is also thought to be responsible for transcriptional activation (ref). The C-terminal consensus is highly conserved among land species and functioning as DNA-binding domain. Two DNA-protein binding structure for LEAFY were first published by Hame et al. 2008 (ref). These two structures include a recombinant C-terminal domain of LEAFY expressed by Escherichia coli strain RosettaBlue (DE3) and a short nucleotide structure from AP1 or AG promoter region. Final models of LFY-pAP1 and LFY-pAG were solved at 2.1 Å and 2.3 Å by X-ray diffraction and deposited as PDB entry 2VY1/2VY2. Site specific DNA recognition is conducted by a HTH-like motif The general structure of LEAFY DNA binding domain consists 2 β strands at the beginning followed by 7 α helices. A helix-turn-helix (HTH) motif can be found between α2 and α3 helices, which is recruited to the major groove of the binding DNA. There are two amino acid at this motif, Asn 291 on α2 and Lys 307 on α3 directly mediate site specific recognition with two guanines at the DNA strand. These two recognition sites were further validated by electrophoresis mobility shift assay (EMSA): mutation at either Asn 291 or Lys 307 dramatically decrease binding affinity to pAP1. In the minor groove, site specific recognition is conducted by Arg 237, which is at the beginning of this structure. Arabidopsis intermediate mutant lfy-4 (P240L) and lfy-5 (T244M) were located near this site and validate the function in planta. The super position of specific recognition sites is summaries at right figure produced by PyMol. DNA binding required cooperative dimerization Figure 3. Three residues mediate homodimerization of LFY dimerization at pAP1 site. Assembly of PDB entry 2VY1 were obtained from PISA server and further visualized by Pymol. Transcription factors tent to form homodimer or heterodimer to increase the binding specificity and affinity. Experimental evidence indicates a potential LFY dimer on the binding site. Crystal structure proved that LFY can form dimers at both pAP1 and pAG sites. The binding affinity of LFY protein dimer binds increased by 90-fold compared to the first LFY monomer in EMSA assay. Detailed structure revealed that the contact of two dimerized protein is mediated by three residues located at on helix (α7) and one loop (loop12) at the other protein. Hydrogen bonds can be formed between Asp 280 and His 387/Arg 390 are essential for this dimeriation. The detailed interaction is shown in the right figure produced by Pymol. Mutation in any of these three amino acids abolished the binding in EMSA assay. Recently, another experiment showing that despite these three residues, the entire N-terminal consensus is critical important for stabilizing the homodimerization, where strong physical interaction can be found by GST-pull down, Y2H and BiFC experiment at in vitro, in vivo and in planta level.