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 β-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 β-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.
Highlights
Sulfide (DMS) and isolated aromatic rings have indicated two major “up” and “down” conformations where the “up” orientation is energetically more favorable over “down” conformations by 2 to 3 kcal/mol [9, 12] (Fig. 1A)
Compared with other Ubiquitin-like protein sequences, we found that the conserved methionine is unique for Small Ubiquitin-like Modifier (SUMO) and absent for other Ubiquitin-like homologous folds (Fig. S1)
In Ubiquitin or NEDD8, which has a structurally similar β grasp fold, methionine is replaced by an isoleucine residue
Summary
Sulfide (DMS) and isolated aromatic rings have indicated two major “up” and “down” conformations where the “up” orientation is energetically more favorable over “down” conformations by 2 to 3 kcal/mol [9, 12] (Fig. 1A). Even slight perturbations of the Met-aromatic motif by replacing methionine with conserved aliphatic residues result in reduced stability, altered structure, and impaired function. A methionine residue (M82) interacts with a phenylalanine (F66) aromatic ring in human SUMO1 and SUMO2 (Fig. 1, A and B). For M82I-SUMO1, the emission maxima shifted by 6 nm (522 nm–516 nm), suggesting alteration from the wt-SUMO1 packing due to disruption of the Met-aromatic interaction (Fig. 2D).
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