Structural basis of cyclic phytocytokine recognition by the HSL3/NUT receptor

Plant receptor kinases perceive diverse peptide signals to coordinate stress responses and developmental programs. The HAESA-LIKE 3 (HSL3/NUT) receptor recognizes CTNIP/SCREW phytocytokines—disulfide-stabilized cyclic peptides that regulate immune signaling and stress adaptation. However, how HSL3 distinguishes these structurally constrained cyclic peptides from linear signaling molecules remains unknown. Here we report near-atomic resolution cryo-EM structures of HSL3 in apo and CTNIP4 48-70 -bound states at ∼2.6 Å, using Arabidopsis CTNIP4 as a representative family member, revealing distinct mechanisms for cyclic peptide recognition. The conserved CTNIP motif occupies a negatively charged pocket in HSL3’s C-terminal region through a combination of polar contacts, hydrogen bonds, salt bridges, and van der Waals interactions. The receptor employs a two-step recognition mechanism—electrostatic steering followed by motif anchoring—that enables rapid ligand capture and release, consistent with the transient nature of stress signaling. Notably, an N-glycan at Asn449 directly contacts the CTNIP4 peptide, establishing glycosylation as an active participant in ligand recognition. Structure-guided mutagenesis combined with reactive oxygen species (ROS) burst assays confirmed the functional importance of key binding interfaces. N-terminal truncation experiments revealed a minimal active fragment: CTNIP4 51–70 supported both rapid ROS production and sustained seedling growth inhibition, whereas the shorter CTNIP4 54–70 variant retained ROS activity but failed to trigger long-term seedling growth inhibition. Structure-guided coevolutionary analysis across plant lineages reveals patterns of both conserved and variable receptor–ligand interfaces, highlighting evolutionary flexibility while preserving core features of recognition. These conserved recognition principles, mediated by receptor glycosylation and evolutionary plasticity, enable specificity in peptide signaling with implications for engineering stress-resilient crops.