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.

配体（ligand）特异性是所有核糖开关（riboswitch）的核心必要条件。部分变异核糖开关共享保守的结构基序，却可通过细微的序列差异，选择性识别不同的小分子配体并调控下游基因表达。这类变异核糖开关可精准区分结构与化学性质高度相似的配体。晶体结构研究为阐明配体特异性的调控机制提供了关键视角。然而，配体特异性并非总能仅通过与配体直接接触的核苷酸得以解释。环二核苷酸变异家族包含两类：环二鸟苷单磷酸（cyclic-di-GMP）核糖开关与环腺苷鸟苷单磷酸（cyclic-AMP-GMP）核糖开关，二者最初仅通过与配体直接接触的单个核苷酸的碱基种类加以区分。本研究报道一种位于第二配体接触位点发生突变的核糖开关：尽管理论上偏好结合环腺苷鸟苷单磷酸，但其对环二鸟苷单磷酸与环腺苷鸟苷单磷酸均表现出结合混杂性。本研究采用高通量突变分析技术SMARTT，对配体接触的第一、第二壳层的数千个位点开展定量分析，以评估其对配体特异性与结合混杂性的影响。除与配体直接接触的核苷酸外，本研究还鉴定出结合位点远端的核苷酸——位于J1/2连接区与终止子螺旋（terminator helix）内的核苷酸——同样可影响配体特异性。该研究结果为配体结合口袋以外的核苷酸如何调控核糖开关特异性提供了典型范例。此外，这些远端核苷酸可用于预测具有结合混杂性的核糖开关序列。