Sandbox Reserved 933

Structural Basis of LEAFY binding

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 /.

Site specific DNA recognition is conducted by a HTH-like motif

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. The detail site specific recognition is summaries at right figure produced by PyMol.

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 three residues located at one helix (α7) and one loop (loop 12) at the other protein. Hydrogen bonds can be formed between Asn 290 and His 387/Arg 390 are essential for this dimeriation. Mutation in any of these three amino acids abolished the binding. 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.