Solution structure and dynamics of Xanthomonas albilineans Cas2 provide mechanistic insight on nuclease activity

Abstract

Cas2 protein in the CRISPR-Cas system functions as a scaffold for the acquisition of foreign DNA fragments, and as a nuclease against DNA and RNA substrates. Crystal structures of Cas2 have shown catalytically inactive conformational states that do not explain the mechanism of Cas2 nuclease activity. Here, we report that Xanthomonas albilineans Cas2 (XaCas2) assumes an inactive conformation in solution. Residual dipolar couplings and NMR relaxation, however, provide direct evidence on conformational dynamics at the predicted hinge region. Furthermore, XaCas2 transiently associates with metal ions for nuclease activity via highly mobile Asp8. Taken together, the dual function of Cas2 can be explained by a dynamic equilibrium of conformational states that serve as a scaffold or as a nuclease on demand.

Structural and functional analyses of Burkholderia pseudomallei BPSL1038 reveal a Cas-2/VapD nuclease sub-family

“…6a ). The cleavage of dsDNA is consistent with the studies reported for Spy_Cas2, Bha_Cas2 and Xal_Cas2 10 – 12 , Nonetheless, unlike the activities of all these known Cas2 dsDNA-targetting DNases that are divalent metal ion dependent, the activity of BPSL1038 was shown to be divalent ion independent (Fig. 6d ).…”

“…3 ) suggests a similar functionality. The D11 pair in BPSL1038 is 5.9 Å apart, and thus much closer compared to those identified in the Cas2 proteins that are in the range of 6.5–15 Å apart 10 , 12 . This makes it possible for BPSL1038 to coordinate with divalent metal ions such as Mg 2+ that are important for Cas2 nuclease activity.…”

“…1 , 3 ). As a result, BPSL1038 has a short and rigid β1-α1 turn compared to Cas2 proteins such as Bha_Cas2, Spy_Cas2 and Xal_Cas2 that are known to cleave dsDNA 10 – 12 (Fig. 3 ).…”

“…In order to chelate the divalent cation, the distance between these two aspartate residues was proposed to be a crucial determinant of catalytic activity, where if they are too far apart, it would not be possible to chelate the metal ion 10 . To date, all existing Cas2 and VapD structures are suggested to be in the catalytically inactive conformation due to the aspartate pair being 6.5–15 Å apart, a distance that is unable to coordinate with the divalent metal ion 10 , 12 .…”

“…The coordinate of 3 10 -helix α-3 10 1 of BPSL1038 was located at the region equivalent to loop β1-α1 in Cas2 proteins. A long β1-α1 loop was proposed as the DNA binding feature for the Bha_Cas2, Spy_Cas2 and Xal_Cas2 proteins 10 – 12 . However, BPSL1038 has a rather short turn connecting the β1 and α1 through α-3 10 1 (Fig.…”