Coiled-coil motif in LBD16 and LBD18 transcription factors are critical for dimerization and biological function in arabidopsis

ABSTRACT

The LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD) gene family members encode a class of plant-specific transcription factors that play important roles in many different aspects of plant growth and development. The LBD proteins contain a conserved LOB domain harboring a Leu zipper-like coiled-coil motif, which has been predicted to mediate protein-protein interactions among the LBD family members. Dimerization of transcription factors is crucial for the modulation of their DNA-binding affinity, specificity, and diversity, contributing to the transcriptional regulation of distinct cellular and biological responses. Our various molecular and biochemical experiments with genetic approaches on LBD16 and LBD18, which are known to control lateral root development in Arabidopsis, demonstrated that the conserved Leu or Val residues in the coiled-coil motifs of these transcription factors are critical for their dimerization as well as the transcriptional regulation to display their biological functions during lateral root formation. We further showed that beside the coiled-coil motif, the carboxyl-terminal region in LBD18 acts as an additional dimerization domain. These findings provide a molecular framework for the homo- and hetero-dimerization of the LBD family proteins for displaying their distinct and diverse biological functions in plants.

The LBD proteins are plant-specific transcription factors which are characterized by the presence of a highly conserved N-terminal DNA-binding domain known as LOB domain, and are involved in a plethora of the growth and development in plants.^1,2 The LOB domain is approximately 100 amino acids in length and contains a four-Cys motif with CX₂CX₆CX₃C spacing, a Gly-Ala-Ser (GAS) block, and a predicted Leu-zipper-like coiled-coil motif with LX₆LX₃LX₆L spacing.^3-5 Various regions of some LBD proteins have been functionally characterized for nuclear targeting, identifying the DNA binding activity in the LOB domain, and identifying the transcription-activating domains in the C-terminal region, showing that the LBD proteins act as transcription factors.^6-10 Based on the LOB domain structure, 42 members of the Arabidopsis LBD gene family have been divided into two major classes, class I (LOB, LBD1 to 33, LBD35, and LBD36) and class II (LBD37 to 42).^1,4 The class I proteins harbor an LOB domain similar to that in the LOB protein, whereas the class II proteins are less similar to the class I proteins but share a conserved amino acid sequence outside the LOB protein.

In many transcription factors, protein dimerization plays a critical role in the modulation of their DNA-binding affinity, specificity, and diversity, which contributes to transcriptional regulation for initiating a sequence of regulatory events leading to a particular cellular and biological response. Several LBD proteins have been reported to form homodimers and/or heterodimers with other LBD family members, and the coiled-coil motif has been proposed to mediate the dimerization among the class I LBD proteins.^5,11-15 However, the role of the coiled-coil motif in the dimerization of LBD proteins has not been assessed experimentally yet. Thus we adopted biochemical and genetic approaches to verify this prediction with LBD16 and LBD18 that have been genetically and molecularly well characterized in controlling lateral root development in Arabidopsis.¹⁶ Our molecular modeling for the coiled-coil motif in LBD16 and LBD18 showed that the two coiled-coil motifs from each monomer form a Leu zipper-like conformation in both LBD16 and LBD18, although LBD16 is not a typical Leu zipper.¹⁶ The involvement of the coiled-coil motifs in the dimerization of LBD16 and LBD18 was first evaluated by determining the effects of mutations in the conserved Leu, Val, or Ile residues into Pro residue on protein-protein interactions using the bimolecular fluorescence complementation assay.¹⁶ The increasing number of mutations in the conserved amino acid residues in the coiled coil resulted in a significant reduction in the homodimerization of LBD16 and LBD18, indicating that the coiled-coil motif plays an important role in the dimerization of these LBD proteins. We also used other molecular assays for determining protein-protein interactions, such as firefly luciferase complementation imaging, GST pull-down, and coimmunoprecipitation assays, demonstrating that the conserved Leu or Val residues in the coiled-coil motif are critical for protein-protein interactions in LBD18. The biological role of the coiled coil motif-mediated dimerization in lateral root formation was confirmed by showing that the reduced lateral root density of lbd16 or lbd18 is rescued to the wild-type levels by overexpressing wild-type LBD16 or LBD18, but is not rescued by overexpressing LBD16^Q or LBD18^Q harboring quadruple mutations in the conserved Leu, Val, or Ile residues in the coiled coil. Consistent with this, we further found that in those transgenic Arabidopsis, the expression of EXP14, a direct target of LBD18, is not activated by LBD18^Q, whereas EXP14 expression is normally induced by the wild-type LBD18. As transient gene expression assays with Arabidopsis protoplasts showed that LBD18^Q still exhibits the transcription-activating capability just like that of the wild-type LBD18, the inability of LBD18^Q in activating EXP14 expression is not due to the transactivation capability lost by quadruple mutations. These results together demonstrated that the homodimerization of LBD18 is crucial for transcriptional regulation via binding to the promoters of the target genes to display its biological function. We further identified the C-terminal domain beyond the coiled coil in LBD18 as an additional dimerization domain that matches with the region having transcription-activating function. As the class II LBD proteins and a few class I LBD proteins do not contain the predicted coiled-coil, this C-terminal region may serve as a protein-protein interaction domain for the LBD proteins lacking coiled coils.

Previous studies have shown that some LBD proteins can form homodimers as well as heterodimers with specific LBD proteins, whereas other LBD proteins can exclusively form heterodimers.^5,10-13,15 These observations indicate that the dimerization specificity of the LBD proteins are regulated by the unique amino acid sequences in the coiled coils. The dimerization specificity of the basic region-leucine zipper (B-ZIP) proteins have been extensively studied.¹⁷ The genome-wide dimerization properties of Homo sapiens, Drosophila melanogaster, and Arabidopsis thaliana B-ZIP motifs have been investigated based on the structural properties of the dimeric α-helical leucine zipper coiled coil structure.^18-21 Based on the structural rules, Arabidopsis B-ZIPs are predicted to form, almost exclusively, homodimers or quasihomodimers, dimers between two paralogs.²¹ Many Arabidopsis B-ZIP leucine zippers are predicted to be eight or more heptads in length, whereas the four or five heptads are typically found in H. sapiens B-ZIP leucine zippers.²¹ The homodimerization property of many Arabidopsis B-ZIP proteins has been proposed to be due to long leucine zippers with asparagine placed in the “a” position of the different heptads.²¹ All LBD proteins tested for protein-protein interactions were shown to form heterodimers in addition to homodimer formation in the case of LBD10 and LBD25.^13,15 Heterodimerization was shown to be essential for nuclear localization of these LBD proteins in Arabidopsis protoplasts.^13,15 As four heptads were identified in the Leu-zipper-like coiled coil motifs in LBD16 and LBD18,¹⁶ smaller heptads in the LBD proteins as well as other structural features could contribute to the dimerization specificity. Intensive research is required for understanding the forces governing the dimerization of the LBD proteins and for determining the three dimensional structures of the LBD dimers bound to DNA, to elucidate the dimerization specificity of the LBD proteins.

Funding Statement

This study was supported by grants from the Next-Generation BioGreen 21 Program (PJ01104701), RDA, Republic of Korea and Mid-career Researcher Program (2016R1A2B4015201) and Basic Research Laboratory (2017R1A4A1015620) through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology of Korea to J. Kim.

Abbreviations

B-ZIP

basic region-leucine zipper

EXP

expansin

LBD

lateral organ boundaries domain

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.