Abstract
LATERAL ORGAN BOUNDARIES DOMAIN (LBD) proteins, a family of plant-specific transcription factors harboring a conserved Lateral Organ Boundaries (LOB) domain, are regulators of plant organ development. Recent studies have unraveled additional pivotal roles of the LBD protein family beyond defining lateral organ boundaries, such as pollen development and nitrogen metabolism. The structural basis for the molecular network of LBD-dependent processes remains to be deciphered. Here, we solved the first structure of the homodimeric LOB domain of Ramosa2 from wheat (TtRa2LD) to 1.9 Å resolution. Our crystal structure reveals structural features shared with other zinc-finger transcriptional factors, as well as some features unique to LBD proteins. Formation of the TtRa2LD homodimer relied on hydrophobic interactions of its coiled-coil motifs. Several specific motifs/domains of the LBD protein were also involved in maintaining its overall conformation. The intricate assembly within and between the monomers determined the precise spatial configuration of the two zinc fingers that recognize palindromic DNA sequences. Biochemical, molecular modeling, and small-angle X-ray scattering experiments indicated that dimerization is important for cooperative DNA binding and discrimination of palindromic DNA through a molecular calipers mechanism. Along with previously published data, this study enables us to establish an atomic-scale mechanistic model for LBD proteins as transcriptional regulators in plants.
Highlights
LATERAL ORGAN BOUNDARIES DOMAIN (LBD) proteins, a family of plant-specific transcription factors harboring a conserved Lateral Organ Boundaries (LOB) domain, are regulators of plant organ development
A DNA-binding study has shown that Arabidopsis LBD proteins, such as LOB, LBD4, and ASYMMETRIC LEAVES2 (AS2), bind a 6-bp consensus LBD-response element (LBD-RE, GCGGCG), which is conserved in the four-nucleotide core sequence CGGC [24]
The LBD family proteins consist of an N-terminal LOB domain and a variable C-terminal region that regulates downstream gene expression [1]
Summary
Because the C-terminal sequence includes five repetitions of hydrophobic amino acid residues, such as valine, isoleucine, and leucine, with six-residue intervals, and it has characteristics similar to a coiled-coil motif [20, 23]. A DNA-binding study has shown that Arabidopsis LBD proteins, such as LOB, LBD4, and ASYMMETRIC LEAVES2 (AS2), bind a 6-bp consensus LBD-response element (LBD-RE, GCGGCG), which is conserved in the four-nucleotide core sequence CGGC [24]. It is well-documented that LBD proteins are nuclear proteins, and the LBD-RE sequence is present in promoters of several target genes of LBD proteins [25,26,27,28,29,30]. The intricate assembly within and between the monomers determines the exact spatial arrangement of the two zinc fingers through which the palindrome sequences are recognized and bound These structural features were further confirmed by biochemical and biophysical studies. The structure, together with our molecular modeling and SAXS experiments, enables us to gain a deep insight into the structure-based mechanism for DNA-binding specificity in the LBD family of transcription factors
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