Identifying ligand-binding interfaces from NMR chemical shift perturbation data is a cornerstone of structural biology, yet it remains hampered by subjective thresholding and the loss of directional information in conventional scalar metrics. Traditional methods rely on empirical weighting factors that lack physical universality and ignore critical parameters like exchange-induced line broadening. Here, we introduce PALI (principal component analysis for ligand interactions), an objective and PCA-based framework designed to standardize multivariate NMR analysis. By implementing Z-score standardization, PALI replaces arbitrary constants with a rigorously data-driven statistical framework, preserving the multidimensionality of spectral changes. We demonstrate that PALI effectively filters stochastic noise and identifies binding hotspots, including features such as intermediate exchange that are often obscured in conventional 1D plots. Validation across diverse systems, from structured proteins to complex dynamic assemblies and intrinsically disordered regions/proteins, proves that PALI provides a robust, reproducible, and automated solution for interaction mapping. PALI is freely available as a Web-based dashboard, bridging the gap between advanced multivariate statistics and routine structural biology workflows.
Min June Yang, Joonhyeok Choi, Hyeonjun Lee et al.· Analytical Chemistry· 0 citations
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-constrained peptides that regulate immune signaling and stress adaptation. However, how HSL3 distinguishes these structurally constrained peptides from linear signaling molecules remains unknown. Here we report near-atomic resolution cryo-EM structures of HSL3 in apo and CTNIP448-70-bound states at ∼2.6 Å, using Arabidopsis CTNIP4 as a representative family member, revealing the mechanism 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 employs 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 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: CTNIP451-70 supported both rapid ROS production and sustained seedling growth inhibition, whereas the shorter CTNIP454-70 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.
