Modulation of Protein Flexibility with Changes in Sequence and Complexation State of Ubiquitin Family Proteins

Abstract

Ubiquitin (Ub) and small ubiquitin-related modifier (SUMO) proteins are structurally homologous molecular tags which regulate diverse cellular processes in eukaryotes. Here, we provide a conceptual basis to understand how nature harnesses protein flexibility, a molecular descriptor, to modulate the diversity of macromolecular interactions and cellular responses seen for ubiquitin family proteins. We hypothesize that the flexibility of ubiquitin family proteins is modulated by both changes in protein sequence and complexation with other proteins. To validate our hypothesis, we developed computational measures of protein flexibility, directional and overall spring constants, from microsecond molecular dynamics simulations and examined these measures for ubiquitin and SUMO proteins in free form and within complexes. Our results show ubiquitin to be stiffer than SUMO proteins and comparable stiffness for SUMO1 and SUMO2 proteins. Complexation with an E2 enzyme which attaches the ubiquitin tag (UBCH5A) to target proteins in the ubiquitylation cascade increases the intrinsic stiffness of ubiquitin. In contrast, complexation with a motif that recognizes ubiquitin tagged molecular cargo (TSG101 UEV domain) decreases intrinsic ubiquitin stiffness. Directional spring constants for ubiquitin (along N-C and 48-C directions), SUMO2 (N-C direction) and SUMO1 (N-C direction) show excellent correlations with spring constants extracted from single-molecule force spectroscopy pulling experiments and we provide experimentally testable predictions for stiffness in other directions. Our computational framework is transferable and can be applied to study subtle changes in protein flexibility as a function of sequence and complexation state in topologically similar proteins and structural motifs. Finally, our computational framework offers detailed insights on the nature of the underlying energy landscape sampled by proteins in molecular dynamics simulations.