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Evolution of DNA binding domain of LEAFY

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. ^[1][2]. In the plant model system Arabidopsis thaliana , ‘’LFY’’ also acts upstream of floral homeotic genes to modulate floral organ identity. ^[3] LFY activates the downstream genes by binding to promoter regions. LFY can directly bind to the promoter to APELATA1 (AP1), while co-regulators UNUSUAL FLORAL ORGANS (UFO) ^[4] and WUSCHEL (WUS)^[5] are required for increment of binding affinity to promoter regions of APELATA3 (AP3) and AGAMOUS (AG), respectively (Figure 1). The exact mechanism how LFY recognizing and binding to these promoters has yet to be elucidated until the first structure report about two DNA-protein complex： and ^[6] . 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^[7] . Recently, a new structure about LFY homolog in mosses provided new insights of structural basis of how LEAFY evolved by changing DNA binding activity^[8]. In this page, we will focus on the structural basis about LEAFY evolution by DNA binding.

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 Contents 1 Structural Basis of LEAFY binding 1.1 General information about the structure 1.2 Site specific DNA recognition is conducted by a HTH-like motif 1.3 DNA binding required cooperative dimerization 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 [1][2]. 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. 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 /. Site specific DNA recognition is conducted by a HTH-like motif 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. The general structure of LEAFY DNA binding domain consists 2 at the beginning followed by 7 . A (HTH) motif can be found between α2 and α3 helices, which is recruited to the of the binding DNA. There are two amino acid at this motif, on α2 and on α3 directly mediate site specific recognition with 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 , 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[1]. The super position of specific recognition sites is summaries at figure 2. DNA binding required cooperative dimerization 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 and 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 located at on helix () and one loop () at the other protein. Hydrogen bonds can be formed between and / are essential for this dimeriation. The detailed interaction is shown in figure 3 produced by Pymol. Mutation in any of these three amino acids abolished the binding in EMSA assay. Recently, another experiment showing that besides 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 [9] .

LEAFY Evolution

Figure 4. A summary of LEAFY evolution by substation on three different types of DNA binding motif. Position 312 and 345 are critical importance in determining type I or type II binding motif (AtLFY and PpLFY). In algae, LFY binds to the type III motif is largely because amino acid substitution disrupt the interface of dimer (TsLFY). In this figure, 3D structures were visualized by Pymol with assembly files of 2VY1 and 4BHK calculated by PISA. Lower 2D diagrams present how different LFY interact with three types of binding motifs. Information in the diagram were summarized from Hames et al. 2008, Sayou et al. 2014 and further visualized by keynote.

Different from other transcription factor families, LFY and its homologs retains as a single copy gene in almost all land plants. This brought a new entry point that how LFY evolved to control different developmental processes in other plant lineages. LFY homologs in mosses have been reported controlling cell division, through binding to a different motif rather than the one that was found in Arabidopsis. Multiple alignment and a new method by systematic evolution of ligands by exponential enrichment (SELEX) of LFY and its homologs revealed that at specific position, few amino acids were substituted from angiosperms to algae to bind to specific motifs. Such motifs can be further categorized into three subgroups: Type I (angiosperm), type II (mosses) and type III (algae), which probably result in a diverged functionality. Crystal structure of DNA-protein complex composed of a moss LFY homolog from Physicomitrella patens and its binding sites further validated this hypothesis. In Arabidopsis, site specific recognition of LFY is conducted by two residues located at a HTH motif. Interestingly, another residue 312D was found in the HTH motif mediating direct site specific recognition to a cytosine at the major groove.(figure 4, PpLFY) In contrast, 312 was occupied by a histidine and bond to a residue R345 from α7 in Arabidopsis, and the DNA binding motif at the position is a guanine instead of a cytosine (figure 4, AtLFY). This structural difference finally result in a difference between type I and type II binding motif. Additionally, there are no difference in dimerization in binding type I and II motif, because three important residues mediating cooperative binding are not changed. In different from type I & II binding, LFY homologs in algea bind to the type III binding motif by a unique structure. Switching of His 387 to a Ser in the three critical cooperative binding site dramatically change the dimerization surface (figure 4, KsLFY). Here is a movie that showing how the LFY evolved. Surprisingly, a LFY homolog in hornwort, shares a promiscuous binding habit, that it can bind all three types of binding motifs. This phenomenon can be explained at structural. At 312 position, LFY homolog in N. aenigmaticus harbors a glutamine, which is identical to type algae LFY. However, 345C and 387H indicate the similar binding properties with type I and II. This finding give new insights about a single copy gene evolution, through a promiscuous intermediate. An interesting movie showing this progress very well: https://www.youtube.com/watch?v=Yvk3ond-WHk.

Reference

1. ↑ ^{1.0 1.1 1.2} Weigel, D., Alvarez, J., David R., Yanofsky, M.F. & Meyerowitz, E.M. 1992. LEAFY controls floral meristem identity in Arabidopsis. Cell 69 :843-859, http://dx.doi.org/10.1016/0092-8674(92)90295-N.
2. ↑ ^{2.0 2.1} Coen, E.S., Romero, J.M., Doyle, S., Elliot, R., Murphy, G. & Carpenter, R. 1990. Floricaula: a homeotic gene required for flower development in Antirrhinum majus. Cell 63: 1311–1322 http://dx.doi.org/10.1016/0092-8674(90)90426-F
3. ↑ Irish, V. F. 2010. The flowering of Arabidopsis flower development. The Plant Journal, 61: 1014–1028. http://dx.doi.org/10.1111/j.1365-313X.2009.04065.x
4. ↑ Chae, E., Tan, Q.K., Hill, T.A. & Irish, V.F. 2008. An Arabidopsis F-box protein acts as a transcriptional co-factor to regulate floral development. Development 135:1235-45 http://dx.doi.org/10.1242/dev.015842
5. ↑ HONG, R.L., HAMAGUCHI, L., BUSCH, M.A. and WEIGEL, D. 2003. Regulatory elements of the floral homeotic gene AGAMOUS identified by phylogenetic footprinting and shadowing. The Plant Cell 15: 1296-1309. http://dx.doi.org/10.1105/tpc.009548
6. ↑ Hames, C., Ptchelkine, D., Grimm, C., Thevenon, E., Moyroud, E., Gérard, F. Martiel, J.L., Benlloch, R., Parcy, F. & Müller, C.W. 2008. Structural basis for LEAFY floral switch function and similarity with helix-turn-helix proteins. EMBO Journal 27:2628-2637. http://dx.doi.org/10.1038/emboj.2008.184
7. ↑ MAIZEL, A., BUSCH, M.A., TANAHASHI, T., PERKOVIC, J., KATAO, M., HASEBE, M. and WEIGEL, D. (2005). The floral regulator LEAFY evolves by substitutions in the DNA binding domain. Science 308: 260-263. http://dx.doi.org/10.1126/science.1108229
8. ↑ Sayou, C., Monniaux, M., Nanao, M.H., Moyroud, E., Brockington, S.F., Thévenon, E., Chahtane, H., Warthmann, N., Melkonian, M., Zhang, Y., Wong, G., Weigel, D., Parcy, F. and Dumas, R. 2014. A Promiscuous Intermediate Underlies the Evolution of LEAFY DNA Binding Specificity Science 343: 645-648 http://dx.doi.org/10.1126/science.1248229
9. ↑ Siriwardana, N. S. & Lamb, R. S. 2012. A conserved domain in the N-terminus is important for LEAFY dimerization and function in Arabidopsis thaliana. The Plant Journal 71: 736–749. http://dx.doi.org/10.1111/j.1365-313X.2012.05026.x8