Structure reveals that BAK1 as a co-receptor recognizes the BRI1-bound brassinolide

Abstract

In animals, steroid hormones play critical roles in many vital physiological processes. Brassinosteroids (BRs) produced by plants have similar structures to the animal steroid hormones and play indispensable roles in plant growth and development 1 . BRs have now acknowledgedly become a sixth class of plant hormones. In contrast to animal steroid hormones that bind to nuclear receptors, BRs are recognized by the cell surface receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1) 2 and its homologs 3 . BRI1 belongs to a large family of plant leucine-rich repeat (LRR) receptor kinases (RKs), characterized by an extracellular ligand-sensing domain and an intracellular kinase domain. The extracellular domain of BRI1 contains 25 LRRs (BRI1LRR) and an island domain. Recognition of brassinolide (BL), the most active BR identified thus far, by BRI1LRR is through a surface pocket embedded in a 70-aa island (residues 587-656) domain 4 , 5 . BR binding greatly enhances BRI1 heteromerization with a second RK, BRI1-associated kinase 1 (BAK1, also known as SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 3, SERK3) 6 , 7 . The BR-induced BRI1-BAK1 stable association initiates a phosphorylation-mediated cascade transducing the extracellular steroid signal to transcriptional programs 1 , 3 . This process is accompanied with dissociation of a BRI1 kinase inhibitor 1 (BKI1) from the intracellular domain of BRI1 3 . Several models on BR-induced BRI1 activation have been proposed 1 , 4 , 5 , 8 . However, whether BAK1 acts as a regulator or co-receptor of BRI1 and how BR binding promotes BRI1-BAK1 interaction are not well understood. To test whether the extracellular domains of BRI1 and BAK1 are sufficient to form a BL-induced complex, we expressed the LRR domains of Arabidopsis BRI1 (residues 24-784, BRI1LRR) and BAK1 (residues 1-220, BAK1LRR) in insect cells, purified the proteins to homogeneity, and tested their interaction using a gel filtration assay. Unexpectedly, when assayed with a buffer at pH 8.0, the BRI1LRR and BAK1LRR proteins did not form a stable complex ( Figure 1A ). As binding of BL to BRI1 occurs in the extracellular acidic space, we reasoned that lowering pH may favor formation of BL-induced BRI1LRR-BAK1LRR complexes. In complete agreement with the prediction, the two proteins strongly interacted with each other at pH 4.0 in the presence of BL, as indicated by co-migration of the two proteins in gel filtration, which was dependent on the existence of BL ( Figure 1A ). These results indicate that the extracellular domains of BRI1 and BAK1 interact with each other in a BL- and pH-dependent manner. The complex was eluted at the position corresponding to a monomeric BRI1LRR-BAK1LRR ( ∼ 100 kD), suggesting that BL binding induces no homodimerization of the BRI1LRR-BAK1LRR complex. Figure 1 BAK1LRR recognizes the BRI1LRR-bound BL to form a pH-dependent BRI1LRR-BAK1LRR heterodimer. (A) BL induces BRI1LRR-BAK1LRR heterodimerization in solution at an acidic pH. Top panel, gel filtration profiles of BRI1LRR and BAK1LRR in the presence (red at pH 4.0 and green at pH 8.0) and absence (purple) of BL. The vertical and horizontal axes represent ultraviolet absorbance (λ = 280 nm) and elution volume (ml), respectively. BL, brassinolide. The 2nd, 3rd and 4th panels are Coomassie blue staining of the peak fractions following SDS-PAGE. Frame colors indicate their corresponding gel filtration runs shown on the top. The numbers shown on top of the gels indicate elution volume (ml). MM: molecular weight maker; the five bands from top to bottom indicate 112 kD, 66 kD, 45 kD, 35 kD and 25 kD, respectively. (B) Overall structure of BRI1LRR-BL-BAK1LRR shown in cartoon. Color codes are indicated. “N” and “C” represent the N- and C-terminus, respectively. (C) BAK1LRR binding induces no striking conformational changes in BRI1LRR. Shown in the figure is structural comparison of the BAK1LRR-free and BAK1LRR-bound BL-BRI1LRR. BL in BRI1LRR and BRI1LRR-BL-BAK1LRR is shown in yellow and cyan, respectively. (D) BAK1LRR recognizes the BRI1LRR-bound BL. Detailed interactions of BL with the N-terminal side of BAK1LRR. The side chains from BAK1LRR and BRI1LRR are shown in cream white and purple, respectively. Dashed red lines indicate hydrogen bonds. Two of the carbon atoms in BL are labeled. (E) Direct interaction between BRI1LRR and BAK1LRR. Residues from BAK1LRR and BRI1LRR are labeled in black and purple, respectively. Full size image We then solved the crystal structure of the BRI1LRR-BL-BAK1LRR complex at a resolution of 3.6 Å ( Supplementary information, Table S1 ). The BRI1LRR-organized complex is assembled with a 1:1:1 stoichiometry ( Figure 1B ). Supporting the gel filtration data, no homodimeric complexes were formed in the structure. Structural comparison showed that the conformations of BRI1LRR in the BAK1LRR-free and BAK1LRR-bound forms are markedly similar ( Figure 1C ), indicating that BAK1LRR binding induces no striking conformational changes in