Mutagenesis-based optimal design of plant peptide phytosulfokine for enhanced biological activity

Recognition of phytosulfokine (PSK), a sulfated pentapeptide, by its receptor PSKRs is crucial in regulating plant growth, development, and reproduction. However, designing highly active PSK remains a formidable challenge due to the lack of understanding of the structure-property relationship, structural dynamics, and the binding characteristics of PSK. Here, with a combined theoretical and experimental approach, we have investigated the binding dynamics of key interactions between PSK and AtPSKR1^LRR to reveal the molecular mechanism of PSK recognition. Our molecular dynamics simulations and free energy perturbation calculations demonstrate that the sulfated tyrosines (PSK^sY1 and PSK^sY3) are indispensable for forming stable PSK-AtPSKR1^LRR complex, while the alanine substitution at PSK^Q5 site is rather tolerated. Furthermore, two promising PSK peptide analogs (PSK^Q5A and PSK^Q5K) with enhanced biological activity have been designed through <i>in silico</i> mutagenesis studies and <i>in vivo</i> experiments. They have a strong promoting effect (20 % enhancement) on stimulating root development compared with the wild-type PSK treatment. This work offers an effective strategy to design new peptide-based drugs for facilitating plant growth and consequent crop productivity, potentially benefiting efforts to address the global food crisis.