Biochemical mapping of a ligand‐binding domain within Arabidopsis BAM1 reveals diversified ligand recognition mechanisms of plant LRR‐RKs

Abstract

SUMMARYLeucine-rich repeat receptor kinases (LRR-RKs) are the largest sub-family of transmembrane receptor kinases in plants. In several LRR-RKs, a loop-out region called an 'island domain', which intercepts the extracellular tandem LRRs at a position near the transmembrane domain, constitutes the ligand-binding pocket, but the absence of the island domain in numerous LRR-RKs raises questions about which domain recognizes the ligand in non-island domain LRR-RKs. Here, we used photoaffinity labeling followed by chemical and enzymatic digestion to show that BAM1, a CLV1/BAM-family LRR-RK whose extracellular domain comprises 22 consecutive LRRs, directly interacts with the small peptide ligand CLE9 at the LRR6-LRR8 region that is relatively distal from the transmembrane domain. Multiple sequence alignment and homology modeling revealed that the inner concave side of LRR6-LRR8 of CLV1/BAM-family LRR-RKs deviates slightly from the LRR consensus. In support of our findings, the clv1-4 mutant carries a missense mutation at the inner concave side of LRR6 of CLV1, and introduction of the corresponding mutation in BAM1 resulted in complete loss of ligand binding activity. Our results indicate that the ligand recognition mechanisms of plant LRR-RKs are more complex and diverse than anticipated.

Root-derived CLE glycopeptides control nodulation by direct binding to HAR1 receptor kinase

“…To confirm expression, HaloTag-fused proteins were specifically labelled using HaloTag TMR (Promega), separated by SDSpolyacrylamide gel electrophoresis, and visualized by a fluorescent image analyzer with a 523 nm excitation filter and a 580 nm emission filter. Photoaffinity labelling was performed as described 28 .…”

Plant stem cell maintenance by transcriptional cross-regulation of related receptor kinases

“…S1D). Furthermore, expression of a mutant BAM1 gene [G199E, equivalent to the clv1-4 dominant-negative allele (Shinohara et al, 2012)] that abolishes binding of the CLV3-related CLE9 peptide failed to restore rosette growth or fertility. Similarly, pBAM3:BAM3 complemented bam1 bam2 bam3 rosette size and leaf shape (not shown).…”

Complexity in Differential Peptide–Receptor Signaling: Response to Segonzac et al. and Mueller et al. Commentaries

“…Lee et al conducted binding and competition experiments using 125 I-Tyr-MCLV3p, not 125 I-Tyr-flg22, as was the case with Mueller et al (2012). Further molecular understanding will require biochemical, structural, and genetic mapping of the precise functional peptide-leucine-rich repeat interactions for each peptide ligand with FLS2 (Mueller et al, 2012a;Shinohara et al, 2012).…”