AlphaFold3 predictions of novel GLI-SUFU interfaces identify binding-defective SUFU missense variants from medulloblastoma and Gorlin Syndrome patients.

A strong, direct and dynamic protein-protein interaction between GLI transcription factors and the tumor suppressor SUFU is a critical regulatory mechanism in Hedgehog (HH) signaling. Germline loss-of-function mutations in the SUFU gene predispose to medulloblastoma, the most frequent brain tumor in infants and young children, and are also found in patients with the rare, cancer-prone diseases Gorlin syndrome and Joubert syndrome. Limited structural information is available on the GLI-SUFU complex, however. As a result, most of the almost 700 SUFU missense mutations sequenced from afflicted patients are of unknown clinical significance. Here, using AlphaFold3 and other newly developed computational tools, we generated novel predicted structures of GLI-SUFU complexes, and of the homologous Ci-dSufu complex from Drosophila melanogaster . The predicted complexes revealed novel interfaces between GLI/Ci and SUFU. Using site-directed mutagenesis and binding assays, we validated key interface residues mediating the interaction between the novel GLI 'RSSL' motif and SUFU. Importantly, many of the amino acid residues in SUFU that are part of the predicted interfaces are mutated in familial medulloblastoma and Gorlin syndrome. Biochemical assays confirmed the importance of most of these residues for GLI-SUFU binding. These findings provide new structure-function insights into a key signaling complex mutated in cancer and other diseases. Structure prediction: Summary: Alphafold3 predicts a more extended interaction of the GLI1 transcription factor with the tumor suppressor SUFU than has been seen in published x-ray structures; these predictions are supported by structure-function experiments, including analysis of patient-derived missense variants of SUFU.