Conformational Dynamics of DNA Binding and Cas3 Recruitment by the CRISPR RNA-Guided Cascade Complex

Bacteria
and archaea rely on CRISPR (clustered regularly interspaced
short palindromic repeats) RNA-guided adaptive immune systems for
sequence specific elimination of foreign nucleic acids. In Escherichia coli, short CRISPR-derived RNAs (crRNAs) assemble
with Cas (CRISPR-associated) proteins into a 405-kilodalton multisubunit
surveillance complex called Cascade (CRISPR-associated complex for
antiviral defense). Cascade binds foreign DNA complementary to the
crRNA guide and recruits Cas3, a trans-acting nuclease-helicase required
for target degradation. Structural models of Cascade have captured
static snapshots of the complex in distinct conformational states,
but conformational dynamics of the 11-subunit surveillance complex
have not been measured. Here, we use hydrogen–deuterium exchange
coupled to mass spectrometry (HDX-MS) to map conformational dynamics
of Cascade onto the three-dimensional structure. New insights from
structural dynamics are used to make functional predictions about
the mechanisms of the R-loop coordination and Cas3 recruitment. We
test these predictions in vivo and in vitro. Collectively, we show how mapping conformational dynamics onto static
3D-structures adds an additional dimension to the functional understanding
of this biological machine.