PPI-hotspotID for detecting protein–protein interaction hot spots from the free protein structure

Experimental detection of residues critical for protein–protein interactions (PPI) is a time-consuming, costly, and labor-intensive process. Hence, high-throughput PPI-hot spot prediction methods have been developed, but they have been validated using relatively small datasets, which may compromise their predictive reliability. Here, we introduce PPI-hotspot ID , a novel method for identifying PPI-hot spots using the free protein structure, and validated it on the largest collection of experimentally confirmed PPI-hot spots to date. We explored the possibility of detecting PPI-hot spots using (i) FTMap in the PPI mode, which identifies hot spots on protein–protein interfaces from the free protein structure, and (ii) the interface residues predicted by AlphaFold-Multimer. PPI-hotspot ID yielded better performance than FTMap and SPOTONE, a webserver for predicting PPI-hot spots given the protein sequence. When combined with the AlphaFold-Multimer-predicted interface residues, PPI-hotspot ID yielded better performance than either method alone. Furthermore, we experimentally verified several PPI-hotspot ID -predicted PPI-hot spots of eukaryotic elongation factor 2. Notably, PPI-hotspot ID can reveal PPI-hot spots not obvious from complex structures, including those in indirect contact with binding partners. PPI-hotspot ID serves as a valuable tool for understanding PPI mechanisms and aiding drug design. It is available as a web server ( https://ppihotspotid.limlab.dnsalias.org/ ) and open-source code ( https://github.com/wrigjz/ppihotspotid/ ).