An integrated approach of NMR experiments and MD simulations visualizes structural dynamics of a cyclic multi-domain protein.

Abstract

Cyclization can stabilize the structure of proteins, as previously demonstrated in single-domain proteins. Although Lys48-linked polyubiquitin, a multi-domain protein, is also known to be cyclized in human cells, the structural effects of cyclization remain unclear. Here, we examined the impact of cyclization on the structural stability and dynamics of cyclic Lys48-linked diubiquitin (Ub2 ). As expected, cyclization increased the thermal stability of Ub2 and its resistance to proteolytic digestion, indicating that cyclization stabilized the structure of Ub2 . Furthermore, cyclization repressed the interdomain motion in Ub2 , but cyclic Ub2 still exhibited microsecond conformational exchange in NMR relaxation dispersion experiments. A series of long coarse-grained (CG) MD simulations visualized how cyclization slowed down the intrinsic nanosecond open-closed domain motion of Ub2 to microseconds. Thus, CG-MD analysis helped to explain the unexpected NMR relaxation results, thereby facilitating characterization of the structural stabilization of cyclic Ub2 .