The Impact of Second-Shell Nucleotides on Ligand Specificity in Cyclic Dinucleotide Riboswitches

Ligand
specificity is an essential requirement for all riboswitches.
Some variant riboswitches utilize a common structural motif, yet through
subtle sequence differences, they are able to selectively respond
to different small molecule ligands and regulate downstream gene expression.
These variants discriminate between structurally and chemically similar
ligands. Crystal structures provide insight into how specificity is
achieved. However, ligand specificity cannot always be explained solely
by nucleotides in direct contact with the ligand. The cyclic dinucleotide
variant family contains two classes, cyclic-di-GMP and cyclic-AMP-GMP
riboswitches, that were distinguished based on the identity of a single
nucleotide in contact with the ligand. Here we report a variant riboswitch
with a mutation at a second ligand-contacting position that is promiscuous
for both cyclic-di-GMP and cyclic-AMP-GMP despite a predicted preference
for cyclic-AMP-GMP. A high-throughput mutational analysis, SMARTT,
was used to quantitatively assess thousands of sites in the first-
and second-shells of ligand contact for impacts on ligand specificity
and promiscuity. In addition to nucleotides in direct ligand contact,
nucleotides more distal from the binding site, within the J1/2 linker
and the terminator helix, were identified that impact ligand specificity.
These findings provide an example of how nucleotides outside the ligand
binding pocket influence the riboswitch specificity. Moreover, these
distal nucleotides could be used to predict promiscuous sequences.