Understanding the nuclease activity of Xanthomonas albilineans Cas2 via its solution structure and dynamics

Abstract

The CRISPR-Cas system confers prokaryotes an adaptive immunity. The system integrates a small genetic fragment from invader into the host CRISR-locus as a memory of infection. Distinctively, each types of the system carries different organizations of CRISPR-associated proteins (Cas), which are featured by their roles in certain steps of the immunity - acquisition, expression, and interference. Among various Cas proteins, Cas2 is the most conserved protein that functions as a scaffold cooperating with Cas1 during the acquisition step. Although studies have demonstrated the intrinsic metal-dependent nuclease activity of Cas2, the unnecessary role of its nuclease activity for proper acquisition process arises questions to the possession and mechanisms of its activity. Moreover, especially in type I-C, all of known Cas2 crystal structures are catalytically inactive conformational states, which makes even more ambiguous to understand the procedures for its nuclease activity to take place. Thus, in this study, we have investigated the solution state of Xanthomonas albilineans Cas2 (XaCas2) to provide direct mechanistic evidences for its nuclease activity. Based on NMR experiments, residual dipolar couplings (RDC) analysis have resulted that XaCas2 adopts an inactive conformation in solution

and, the NMR relaxation data indicates that the active site and hinge regions possess highly dynamical motion as for the driving force of conformational change. In conclusion, the results suggest that XaCas2 is thermodynamically stable as inactive conformation in solution, and the forces from highly dynamic regions allow catalytically active conformation when encounter genetic substrates and preferred metal ions. Taken together, the intrinsic function of Cas2 can be explained by a dynamic equilibrium of conformational states that serves as a scaffold and as a nuclease on demand.