An "up" oriented methionine-aromatic structural motif in SUMO is critical for its stability and activity.

ABSTRACT

Protein structural bioinformatic analyses suggest preferential associations between methionine and aromatic amino acid residues in proteins. Ab initio energy calculations highlight a conformation-dependent stabilizing interaction between the interacting sulfur-aromatic molecular pair. However, the relevance of buried methionine-aromatic motifs to protein folding and function is relatively unexplored. The Small Ubiquitin-Like Modifier (SUMO) is a ß-grasp fold protein and a common posttranslational modifier that affects diverse cellular processes, including transcriptional regulation, chromatin remodeling, metabolic regulation, mitosis, and meiosis. SUMO is a member of the Ubiquitin-Like (UBL) protein family. Herein, we report that a highly conserved and buried methionine-phenylalanine motif is a unique signature of SUMO proteins but absent in other homologous UBL proteins. We also detect that a specific "up" conformation between the methionine-phenylalanine pair of interacting residues in SUMO is critical to its ß-grasp fold. The noncovalent interactions of SUMO with its ligands are dependent on the methionine-phenylalanine pair. MD simulations, NMR, and biophysical and biochemical studies suggest that perturbation of the methionine-aromatic motif disrupts native contacts, modulates noncovalent interactions, and attenuates SUMOylation activity. Our results highlight the importance of conserved orientations of Met-aromatic structural motifs inside a protein core for its structure and function.