Crystal structures of the phosphorylated BRI1 kinase domain and implications for brassinosteroid signal initiation

Abstract

Brassinosteroids, which control plant growth and development, are sensed by the membrane receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1). Brassinosteroid binding to the BRI1 leucine-rich repeat (LRR) domain induces heteromerisation with a SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK)-family co-receptor. This process allows the cytoplasmic kinase domains of BRI1 and SERK to interact, trans-phosphorylate and activate each other. Here we report crystal structures of the BRI1 kinase domain in its activated form and in complex with nucleotides. BRI1 has structural features reminiscent of both serine/threonine and tyrosine kinases, providing insight into the evolution of dual-specificity kinases in plants. Phosphorylation of Thr1039, Ser1042 and Ser1044 causes formation of a catalytically competent activation loop. Mapping previously identified serine/threonine and tyrosine phosphorylation sites onto the structure, we analyse their contribution to brassinosteroid signaling. The location of known genetic missense alleles provide detailed insight into the BRI1 kinase mechanism, while our analyses are inconsistent with a previously reported guanylate cyclase activity. We identify a protein interaction surface on the C-terminal lobe of the kinase and demonstrate that the isolated BRI1, SERK2 and SERK3 cytoplasmic segments form homodimers in solution and have a weak tendency to heteromerise. We propose a model in which heterodimerisation of the BRI1 and SERK ectodomains brings their cytoplasmic kinase domains in a catalytically competent arrangement, an interaction that can be modulated by the BRI1 inhibitor protein BKI1.

Keywords: Arabidopsis thaliana; brassinosteroid receptor; growth control; hormone signaling; plant development; protein crystallography; protein phosphorylation; receptor kinase.

Figure 1

Overall structure of the active BRI1 kinase domain and features of the nucleotide binding site.(a) Schematic overview of the BRI1 kinase domain constructs used in this study with construct borders included. The JM, KD and CT segments have been previously assigned using a BRI1 homology model (Jaillais et al., 2011b). (b) Ribbon diagram of the BRI1^865–1160 kinase domain. The N-lobe (residues 865–956) is shown in light-blue, the hinge region (residues 957–959) in orange, the activation loop (residues 1027–1056) in yellow and the C-lobe (960–1160) in dark-blue, respectively. Four phosphorylation sites present in the structure are highlighted in bonds representation (with phosphorus coloured in cyan). (c) View of the adenine nucleotide binding pocket in BRI1 occupied by the non–hydrolysable ATP analogue AppNHp (gray, in bonds representation). The two Mn²⁺ ions are highlighted as magenta spheres, residues contacting the nucleotide are shown in yellow (in bonds representation). Hydrogen-bonding interactions of AppNHp with BRI1 are denoted as dotted lines (in black). The gatekeeper Tyr956 in the back pocket of the binding site makes a hydrogen-bond with Glu927, which in turn salt-bridges to Lys911 to keep the kinase domain in its active conformation. The genetic alleles bri1-1 (Ala909–Thr) and bri1-115 (Gly1048–Asp) in close proximity to the nucleotide binding site are highlighted as green spheres. (d) A complex structure with ATP (gray, in bonds representation) identifies the γ-phosphate of the nucleotide facing outwards, away from the catalytic Asp1009. An omit 2F_o–F_c electron density map contoured at 1.5 σ is shown alongside (blue mesh). A similar non-catalytic conformation of the nucleotide has been observed in a SERK3–AppNHp complex (in yellow, bonds representation, Protein Data Bank identifier (PDB-ID): 3uim).(e) The BRI1–ADP complex with ADP in grey (in bonds representation). Hydrogen-bonding interactions of the adenine base and ribose with the BRI1 hinge region (in orange) main chain atoms and with two water molecules (red spheres) are shown as dotted lines (in grey). An omit 2F_o–F_c electron density map contoured at 1.5 σ is shown alongside (blue mesh).

Figure 2

BRI1 does not contain a guanylate cyclase domain and has no detectable guanylate cyclase activity. (a) Ribbon diagram of the BRI1 kinase domain (in blue) and with AppNHp (gray, in bonds representation) and two Mn²⁺ ions (magenta spheres) bound in the active site. The GC domain fragment previously used in guanylate cyclase activity assays (Kwezi et al., 2007) is shown in yellow, the putative catalytic GC motif in red (residues 1071–1084). Asp1038 and 1087 suggested to be involved in magnesium ion coordination are depicted in bonds representation. (b) High-performance liquid chromatography (HPLC) analysis of nucleotide products in BRI1 activity assays. (Top panel) Elution profile showing the retention times for different adenine nucleotides. (Bottom panel) HPLC analysis after incubating 10 μm BRI1^865–1160 with 5 mm ATP for 0 (black line), 10 (blue) and 20 (red) min, respectively. (c) (Top panel) Elution profile showing the retention times for different guanine nucleotides. (Bottom panel) HPLC analysis after 10 μm BRI1^865–1160 with 5 mm GTP for 10 min (black line) and over-night (red line), respectively.

Figure 3

Structural basis for BRI1 dual-specificity kinase activity. (a) BRI1 and human IRAK-4 are closely related kinases. Structural superposition of the apo BRI1^865–1160 (blue C_α trace) and IRAK-4 (PDB-ID 2iob, in orange) kinase domains, sharing 35% sequence identity (RMSD is approximately 1.6 Å comparing 254 corresponding C_α atoms). (b) Arrangement of the gatekeeper tyrosine in human IRAK-4 and plant receptor-like kinases. Shown are C_α traces of the BRI1 (in blue), IRAK-4 (PDB-ID 2oib, orange) and SERK3 (PDB-ID 3uim, gray) kinase domains, with the gatekeeper tyrosine and the conserved Lys/Glu highlighted in bonds representation and interatomic interactions shown alongside (dotted lines). The kinase domains of BRI1 and SERK3 share 40% sequence identity and superimpose with a RMSD of approximately 1.5 Å comparing 264 corresponding C_α atoms. (c) Structural superposition of the BRI1 (blue) and IRAK-4 (orange) activation loops. The phosphorylation sites present in the BRI1 and IRAK-4 (PDB-ID 2oib) structures are shown alongside (in bonds representation). (d) Surface-exposed phosphorylation events in BRI1. Activation loop (in gold) Thr1039 and Ser1042 (in bonds representation) are in hydrogen-bond contact with His1040. The nearby Arg1062 and the phosphorylated Ser1060 are depicted in blue. (e) Structural superposition of the activation loop segments of BRI1 (in blue) and insulin receptor (orange, PDB-ID 1ir3, RMSD is approximately 2.5 Å comparing 253 corresponding C_α atoms). pSer1044 (blue, in bonds representation) in BRI1 binds to a positively charged surface pocket formed by BRI1 arginine residues 922, 1008 and 1032. Very similar interactions are made by insulin receptor Tyr1163 with arginine residues 1131 and 1155. (f) The P + 1 pocket in BRI1 (shown in blue, in bonds representation) perfectly superimposes with the corresponding segment in the classical Ser/Thr protein kinase A (in gold, PDB-ID 1jbp, RMSD is approximately 2.3 Å comparing 231 corresponding C_α atoms).

Figure 4

Phosphorylation sites in the BRI1 kinase domain. (a) Ribbon diagram of the BRI1 kinase domain, coloured according to Figure1(b). The known phosphorylation sites are highlighted by red spheres. They are grouped into three major clusters; the N-lobe of the kinase (in light-blue), the activation loop region (in yellow) and the kinase active site (in red). The region N-terminal of the first β-strand in BRI1 (residues 865–888) is highlighted in magenta. (b) Analogous N-terminal regions in BRI1 and the human kinase Nek7. Shown are C_α traces of BRI1 (in blue) and Nek7 (PDB-ID 2qwm, gold) with the BRI1 phosphorylation sites depicted as red spheres, and selected residues shown in bonds representation. Note that the area surrounding Thr872 in BRI1 is very similar in Nek7 and that the N-terminal region needs to unfold to allow for Thr880 and S887 to become phosphorylated. (c) Detailed view of the BRI1 kinase nucleotide binding site. pSer891 is located in the glycine-rich loop of BRI1 (residues 890–895, glycine residues shown as blue spheres). The gatekeeper Tyr956 is in hydrogen-bond contact with the critical Lys911/Glu927 pair and its phosphorylation is likely to inactivate BRI1.

Figure 5

Distribution of genetic alleles in the BRI1 kinase domain. (a) Ribbon diagram of BRI1, coloured according to Figure1(b). Genetic missense alleles are depicted as green spheres. (b, c) Surface representation of the BRI1 C-lobe in two different orientations. Residues affecting BKI1 binding (Ala1104, Leu1106) and the bri1-117 missense allele (Asp1139–Asn) are highlighted in orange.

Figure 6

Oligomeric state-analysis of individual domains in BRI1 and in SERKs. (a) Schematic overview of the SERK3 (in orange) and SERK2 (in yellow) kinase domain fragments with construct borders included. (b) SDS-PAGE analysis of the purified kinase domain fragments used in this study. (c) A 280 nm absorbance trace of an analytical size-exclusion chromatography on a Superdex 75 HR 10/30 column. Wild-type BRI1^814–1196 elutes as a homodimer, while deletion of either the JM and/or the CT region renders the resulting kinase domain apparently monomeric. Void (V₀) and total volume (V_t) are shown together with the elution volumes for molecular weight standards (A, aldolase, MW 158 000; B, conalbumin, MW 75 000; C, ovalbumin, MW 43 000; D, carbonic anhydrase, MW 29 000; E, ribonuclease A, MW 13 700, F, aprotinin, MW 6500). The calculated molecular weights are 42 800 (BRI1 JM–KD–CT), 39 000 (JM–KD), 37 000 (KD–CT) and 33 200 (KD). (d) Analytical size-exclusion chromatography of SERK2 and 3 kinase domain constructs performed as in (c). The calculated molecular weights are 41 700 (SERK3 JM–KD–CT), 33 200 (SERK KD), and 39 300 (SERK2 JM–KD–CT). (e) Analytical size-exclusion analysis of a mixture of BRI1 JM–KD–CT and SERK3 JM–KD–CT on a Superdex 200 HR 10/30 column. A, thyroglobulin, MW 669 000; B, ferritin MW 440 000; C, aldolase, MW 158 000; D, conalbumin, MW 75 000; E, ovalbumin, MW 43 000; F, carbonic anhydrase, MW 29 000). (f) Immunoblot of peak fractions from (e) using anti-BRI1 and anti-SERK3 antibodies. (g) Analytical ultracentrifugation of the isolated brassinolide-bound BRI1 ectodomain reveals its monomeric state. (h) Analytical ultracentrifugation of the BRI1–brassinolide–SERK1 ectodomain complex reveals a 1:1 stoichiometry. The purified BRI1 and SERK1 ectodomains are approximately 110 and 30 kDa, respectively.