Motif-guided identification of KRAS-interacting proteins

Background: KRAS has long been a major challenge in drug discovery due to its critical role in cancer progression and its reputation as an “undruggable” protein. KRAS mediates downstream signaling pathways through protein-protein interactions, but many of its interaction partners remain unidentified.

Results: We developed a workflow combining computational predictions and experimental validations to identify potential KRAS-interacting proteins based on its known interaction modes. Using experimentally determined KRAS-containing protein complexes, we identified 17 KRAS-interacting motifs. These motifs were employed as queries in our in-house protein-protein interaction prediction tool, PPI-Miner, to search the human protein structure database for similar fragments. Among the 78 predicted potential KRAS-interacting proteins, 10 were selected for experimental validation, including BRAF (a known interacting protein used as a positive control) and KRpep-2d (a peptide that binds KRAS, also serving as a control).

Biolayer interferometry assays of the predicted motifs revealed that 4 of the 10 proteins exhibited binding affinities to KRAS, with GRB10 showing the strongest interaction. Further validation confirmed the interaction between the GRB10 RA-PH domain and KRAS through immunofluorescence and co-immunoprecipitation experiments.

Conclusions: This study highlights the utility of our workflow in identifying potential KRAS-interacting proteins. These findings enhance our understanding of KRAS-driven tumor mechanisms and provide insights for developing targeted therapeutic strategies.