Hyaluronan-Arginine Enhanced Interaction Emerges from Distinctive Molecular Signature with Improved Electrostatics and Side-Chain Specificity

Hyaluronan, a sugar polymer found outside the plasma membrane, is a critical component of the extracellular matrix (ECM) scaffold. Hyaluronan's length and moderate negative charge aid in ECM entanglement and solubility. This allows it to effectively foster gel-like conditions, which are critical for supporting and protecting cells in various tissues. As a recognized ligand for multiple proteins, understanding its interaction with various amino acids helps elucidate its biological functions. This study employs NMR and molecular dynamics simulations (MD) to explore a potential general molecular mechanism for hyaluronan-protein interactions. We examine the binding of short arginine, lysine, or glycine oligopeptides to hyaluronan polysaccharides, using chemical shift perturbation (CSP) to detect changes in hyaluronan's 1H NMR spectra upon peptide titration. CSP helps to identify concrete binding regions and offers insights into the relative interaction strength of the peptides. We also use nuclear Overhauser effect (NOE) measurements to identify a potentially stable hyaluronan-peptide complex. However, the lack of NOE signals does not support the formation of strong and defined complexes. The combination of a positive CSP signal and negative NOE suggests a dynamic binding mode without stable complex formation, which MD further supports. Arginine, a positively charged amino acid, exhibits the strongest binding to the negatively charged hyaluronan, with the similarly charged lysine coming in distant second, highlighting the significance of electrostatics and side-chain specificity in these interactions. Hyaluronan's carboxyl and amide groups drive its interaction with the peptides, further stabilized by hydrogen bonds. Our findings elucidate hyaluronan-protein interaction patterns, typically involving arginine, and highlight arginine's crucial role in hyaluronan recognition by proteins.