Methionine‐aromatic Interaction as a Tool to Enhance Protein Stability

Nonbonded aromatic interactions play a significant role in stabilizing a folded protein structure(1, 2). Apart from well‐studied aromatic‐aromatic interactions in proteins, preferential proximity of sulfur containing cysteine and methionine sidechains and aromatic rings is also observed in protein hydrophobic cores and at the interfaces of protein‐protein complexes. Methionine‐aromatic motifs are examples of such S‐aromatic motifs that provide additional stabilization at a longer distance range over pure hydrophobic interactions(3). Although the idea of preferential association of aromatic amino acids has been successfully implemented for de novo design of peptides and proteins, the additional stability provided by met‐aromatic motif over pure hydrophobic interaction and its potential significance in designing a stabler protein fold has not been examined before. The present work shows the relevance of S‐aromatic interaction in increasing thermodynamic stability of a protein fold using Small Ubiquitin‐Like Modifier (SUMO) protein. We found that a highly conserved methionine‐phenylalanine motif in the core is a unique signature of SUMO but absent in other homologous Ubiquitin‐like (Ubl) protein folds. Using biophysical measurements and solution NMR based experiments, we found that the specific ‘up’ conformation between the methionine‐phenylalanine pair is critical to SUMO’s β‐grasp fold stability and its function(4). Given the additional stability provided by met‐aromatic motif over classical hydrophobic interaction, we engineered new S‐aromatic motifs in SUMO by replacing phenylalanine with other naturally occurring aromatic amino acids. Combining all atom MD simulations and solution NMR studies, we identify the energetically preferred interaction geometry of different met‐aromatic motifs inside the core. We found that the engineered SUMOs with increasing S‐aromatic interaction strength enhances the overall protein stability. Overall, our results reveal the potential role of S‐aromatic interactions as a design tool in enhancing thermodynamic stability of a protein fold. References 1. Chatterjee, K. S., Tripathi, V., and Das, R. (2019) A conserved and buried edge‐to‐face aromatic interaction in small ubiquitin‐like modifier (SUMO) has a role in SUMO stability and function. J. Biol. Chem. 294 , 6772–6784 2. Serrano, L., Bycroft, M., and Fersht, A. R. (1991) Aromatic‐aromatic interactions and protein stability. J. Mol. Biol. 218 , 465–475 3. Valley, C. C., Cembran, A., Perlmutter, J. D., Lewis, A. K., Labello, N. P., Gao, J., and Sachs, J. N. (2012) The Methionine‐aromatic Motif Plays a Unique Role in Stabilizing Protein Structure. J. Biol. Chem. 287 , 34979–34991 4. Chatterjee, K. S., and Das, R. (2021) An “up” oriented methionine‐aromatic structural motif in SUMO is critical for its stability and activity. J. Biol. Chem. 297 , 100970