Mutagenesis and free energy calculations to optimize the ProRgpB inhibitor loop and identify variants with higher affinity for Porphyromonas gingivalis RgpB.

Arginine-specific gingipain B (RgpB), a key cysteine protease from Porphyromonas gingivalis, is associated with several systemic diseases. It is synthesized as a zymogen bound to a propeptide inhibitor that blocks its catalytic activity until activation in the extracellular environment. To identify inhibitory peptide variants with enhanced affinity, mutants of the propeptide inhibitory loop were designed. A total of 52 mutants were generated and, for each model, four independent molecular dynamics replicas were performed, followed by binding free energy calculations using MM/GBSA. Most variants exhibited more favorable binding affinities than the wild-type (WT) loop (-109.8 ± 4.9 kcal mol-1). Among them, V126K (-128.1 ± 5.6 kcal mol-1), E131D (-120.6 ± 8.2 kcal mol-1), and N132R (-135.8 ± 4.6 kcal mol-1) emerged as promising, with the first two showing statistically significant improvements using ANOVA followed by Tukey's post-hoc test (p = 0.001). Thermodynamic integration calculations were consistent with increased binding affinity for the selected variants. Thermodynamic integration calculations further confirmed a significant increase in the relative binding affinity for the selected variants compared to the wild type. These results provide a solid basis for future in vitro validation and establish an in silico framework to accelerate the rational design of propeptide-based inhibitors and the development of novel therapeutic approaches targeting RgpB.