Structural basis of HSL3/NUT receptor kinase-mediated recognition of a disulfide-constrained phytocytokine

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, which are disulfide-constrained peptides that regulate immune signaling and stress adaptation. However, how HSL3 distinguishes these structurally constrained peptides from linear signaling peptides remains unknown. Here, we report near-atomic-resolution cryo-electron microscopy structures of HSL3 in the apo and CTNIP4^48-70-bound states at ∼2.6 Å, using Arabidopsis CTNIP4 as a representative family member, thereby revealing the structural basis of disulfide-constrained peptide recognition. The conserved CTNIP motif occupies a negatively charged pocket in the C-terminal region of HSL3 through a combination of polar contacts, hydrogen bonds, salt bridges, and van der Waals interactions. The receptor uses a two-step recognition mechanism-electrostatic steering followed by motif anchoring-that enables rapid ligand capture, consistent with the dynamic nature of stress signaling. Notably, an N-glycan at Asn449 directly contacts CTNIP4, revealing an active role for glycosylation 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 CTNIP4^51-70 as the minimal active fragment that supports both rapid ROS production and sustained seedling growth inhibition, whereas the shorter CTNIP4^54-70 variant retained the ability to induce ROS production but failed to trigger long-term seedling growth inhibition. Structure-guided coevolutionary analysis across plant lineages revealed patterns of both conservation and variation at the receptor-ligand interface, which exhibit evolutionary flexibility while preserving core recognition features. These conserved features, together with receptor glycosylation and evolutionary plasticity, enable specificity in peptide signaling, with implications for the engineering of stress-resilient crops.