Structural basis of the specific interactions of GRAS family proteins

Abstract

The plant-specific GAI-RGA-and-SCR (GRAS) family of proteins function as transcriptional regulators and play critical roles in development and signalling. Recent structural studies have shed light on the molecular functions at the structural level. The conserved GRAS domain comprises an α-helical cap and α/β core subdomains. The α-helical cap mediates head-to-head heterodimerization between SHR and SCR GRAS domains. This type of dimerization is predicted for the NSP1-NSP2 heterodimer and DELLA proteins such as RGA and SLR1 homodimers. The α/β core subdomain possesses a hydrophobic groove formed by surface α3- and α7-helices and mediates protein-protein interactions. The groove of the SHR GRAS domain accommodates the zinc fingers of JKD, a BIRD/IDD family transcription factor, while the groove of the SCL7 GRAS domain mediates the SCL7 homodimerization.

Keywords: BIRD family; DELLA protein; GRAS domain; IDD family; SAM-dependent methyltransferase; Transcription cofactor; Transcription factor; Zinc finger; α/β protein.

Figure 1

Sequence alignment of GRAS proteins. Domain map of GRAS protein. The domain map of SCR is shown as an example. GRAS domains comprise five designated regions, LHRI, VHIID, LHRII, PYRE and SAW. These historically designated regions do not necessarily correspond to structural/functional subdomains.

Figure 2

Analytical ultracentrifugation (AUC) of GRAS domains. (A) The SCL5 GRAS domain exists as a monomer. The AUC sedimentation velocity analysis shows an estimated molecular mass of 46.6 ± 3.5 kDa, suggesting a monomer in solution (calculated mass 48.1 kDa). (B) The SCL3 GRAS domain exists as a homodimer. The analysis shows an estimated molecular mass of 106.3 ± 1.6 kDa, suggesting a homodimer in solution (calculated mass 50.8 kDa). (C) The SHR and SCR GRAS domains exist as a heterodimer. The analysis shows a monodispersed state with an estimated molecular mass of 105.4 ± 1.4 kDa, suggesting a heterodimer in solution (calculated mass 89.2 kDa).

Figure 3

GRAS domain structure. (A) the GRAS domain structure of SHR. The GRAS domain comprises an α‐helical cap and α/β core subdomains. The colour codes are cyan for α‐ and 3₁₀‐helices and magenta for β‐strands. (B) As in (A), but for the GRAS domain structure of SCR. (C) As in (A), but for the GRAS domain structure of SCL7. The current SCL7 structure lacks the peptide segment (a red dotted line) linking for α3‐ and α4‐helices. The segment of SHR forms αA‐helix and that of SCR forms ηA‐helix in the SHR‐SCR complex (see A,B).

Figure 4

Topology of GRAS domain. GRAS domain comprises α‐helical cap (pink) and α/β core (blue and cyan). The SHR GRAS domain contains fourteen α‐helices (α1–α13 and αA), four 3₁₀‐helices (η0–η3) and nine β‐strands (β1–β9). Compared with the α/β folds of SAM‐MTs, the α/β core of the GRAS domain possesses an additional β6‐α13‐β7 segment (cyan).

Figure 5

Cavity of the GRAS domains. (A) The SHR GRAS domain has a large cavity (grey). The cavity of the SHR GRAS domain is located at the central region enclosed by αA helix (cyan), β2–α7 loop (magenta), β4–α9 loop (green) and α11–α12 loop (orange), and occupies a large internal space (1529 ų). (B) The active site cavity of Mycobacterium marinum fatty acid O‐methyltransferase (FAMT), a member of the SAM‐MT family. The cavity contains bound SAH (magenta) and substrate (green) molecules in the cavity (1309 ų).

Figure 6

GRAS domain heterodimer, SHR‐SCR. (A) Ribbon diagram of the Arabidopsis SHR‐SCR GRAS domain heterodimer. SHR comprises the N‐terminal strap (in red), α‐helical cap (magenta) and α/β core (blue) subdomain; SCR consists of the α‐helical cap (green) and α/β core (turquoise). The α/β core subdomains contain GRAS‐specific segments, β6–α13–β7(cyan in SHR and light blue in SCR). (B) Domain and motif organizations of SHR and SCR GRAS domains.

Figure 7

GRAS domain homodimer, SCL7‐SCL7. (A) Ribbon diagram of the rice SCL7 GRAS domain homodimer. The GRAS domain comprises the α‐helical cap (magenta/green) and α/β core (blue/cyan). The homodimer is formed with the contact between the α/β core subdomains. (B) The dimer interface comprises α4‐ and α7‐helices forming a groove that accommodates α12 helix from the other protomer. At the groove, α12 helix primarily contacts with α6‐ and α7‐helices.

Figure 8

BIRD/IDD recognition by the SHR‐SCR heterodimer. (A) BIRD/IDD transcription factors contain four conserved zinc fingers (ZFs) at the N‐terminal regions, with the first two (ZF1 and ZF2) of the C₂H₂ type ZF and the next two (ZF3 and ZF4) of the C₂ HC type. ZF1–3 mediates specific binding to DNAs of target genes. (B) The structure of the JKD‐SHR‐SCR complex. The SHR GRAS domain comprises the helical cap (magenta) and the α/β core (blue) and also the SCR GRAS domain comprises the helical cap (green) and the α/β core (cyan). The JKD ZF3 and ZF4 modules (orange) directly bound to SHR of the SHR‐SCR heterodimer. The groove formed by α4‐ and α7‐helies of the α/β core accommodates the ZF4 α‐helix.

Figure 9

The SHR‐binding motif. (A) The SHR‐binding motif found in the α‐helix of ZF4 of JKD. The conserved residues IT and AF are located the nearly the same helix surface forming the interface with SHR on docking into the SHR groove. (B) Sequential alignment of the ZF4 zinc fingers among IDD family proteins. The SHR‐binding motif is conserved in 13 members (IDD1–13) of the BIRD/IDD family of transcription factors. Sequence alignment of the ZF4 zinc fingers in Arabidopsis thaliana IDDs. Highly conserved residues (more than 80%) and relatively conserved residues (60–80%) are filled in pink and yellow, respectively, while the conserved Cys or His residues which are essential for coordinating zinc ions are filled in green. In the JKD‐SHR‐SCR ternary complex 12, JKD/IDD10 residues whose side chain atoms form hydrogen bonds with SHR residues, residues whose main chain atoms form hydrogen bonds with SHR residues and residues involved in hydrophobic interactions with SHR are marked with filled circle (cyan), open circle (cyan) and triangle (red), respectively.

Figure 10

The BIRD/IDD‐SHR‐SCR bound to DNA. A model of the JKD‐SHR‐SCR complex bound to DNA. The JKD ZF1–3 bound to DNA to read out the sequence and the ZF3 and ZF4 bound to SHR of the SHR‐SCR complex.