Structural analysis of Notch-regulating Rumi reveals basis for pathogenic mutations

Area of Science:

• Biochemistry
• Developmental Biology
• Structural Biology

Background:

• Rumi O-glucosylates EGF repeats in proteins vital for metazoan development, including Notch.
• Rumi is essential for Notch signaling, and its dysregulation is implicated in human diseases.
• The precise mechanism by which Rumi selectively glucosylates serine residues within EGF repeats remains unclear.

Purpose of the Study:

• To elucidate the structural basis of Rumi's catalytic mechanism and substrate recognition.
• To understand how Rumi interacts with EGF repeats and donor substrates.
• To investigate the impact of disease-associated Rumi mutations on its enzymatic activity.

Main Methods:

• X-ray crystallography was used to determine the structures of Drosophila Rumi.
• Binary and ternary complexes of Rumi with folded EGF repeats and/or donor substrates were analyzed.
• Structural analysis focused on identifying Rumi's recognition of EGF motif signatures.

Main Results:

• Crystal structures revealed Rumi in complex with EGF repeats and donor substrates, offering mechanistic insights.
• Rumi recognizes specific structural signatures of the EGF motif, including the U-shaped consensus sequence (C-X-S-X-(P/A)-C) and a conserved hydrophobic region.
• Five cancer- and Dowling-Degos disease-associated Rumi mutations were found to cluster around the active site and impair Rumi's activity.

Conclusions:

• The study provides structural insights into Rumi's catalytic mechanism and substrate specificity.
• Loss of Rumi activity due to mutations may underlie certain human diseases, including cancers and Dowling-Degos disease.
• Mechanistic understanding of Rumi may aid in developing modulators of Notch signaling for therapeutic purposes.