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 in silico mutagenesis studies and in vivo 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.