Structural and Biochemical
Characterization of Linear Dinucleotide Analogues Bound to the c-di-GMP-I
Aptamer
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Abstract
The cyclic dinucleotide c-di-GMP regulates lifestyle
transitions in many bacteria, such as the change from a free motile
state to a biofilm-forming community. Riboswitches that bind this
second messenger are important downstream targets in this bacterial
signaling pathway. The breakdown of c-di-GMP in the cell is accomplished
enzymatically and results in the linear dinucleotide pGpG. The c-di-GMP-binding
riboswitches must be able to discriminate between their cognate cyclic
ligand and linear dinucleotides in order to be selective biological
switches. It has been reported that the c-di-GMP-I riboswitch binds
c-di-GMP 5 orders of magnitude better than the linear pGpG, but the
cause of this large energetic difference in binding is unknown. Here
we report binding data and crystal structures of several linear c-di-GMP
analogues in complex with the c-di-GMP-I riboswitch. These data reveal
the parameters for phosphate recognition and the structural basis
of linear dinucleotide binding to the riboswitch. Additionally, the
pH dependence of binding shows that exclusion of pGpG is not due to
the additional negative charge on the ligand. These data reveal principles
that, along with published work, will contribute to the design of
c-di-GMP analogues with properties desirable for use as chemical tools
and potential therapeutics