Crystal structure of the lysine riboswitch regulatory mRNA element

Abstract

Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8 angstroms resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding.

FIGURE 1.

A, the chemical structure of lysine and the antimicrobial compound AEC. The carbon atom designation is shown for lysine. B, secondary structure of the T. maritima lysine riboswitch, reflecting the tertiary structure of the RNA. Base-pairing interactions are shown using the nomenclature of Leontis and Westhof (42). Nucleotides shown in red are >90% conserved across phylogeny, and positions where mutations confer resistance to AEC are circled in blue (blue asterisks denote approximate positions). C, schematic diagram of the tertiary structure of the lysine riboswitch with each of the three stacks colored differently. Lysine is shown represented as van der Waals spheres. D, details of the hydrogen-bonding interactions between lysine and the RNA. The distances of the bonds between lysine and RNA are given in Ångströms.

FIGURE 2.

A, closeup of the lysine-binding pocket with the superposition of the free (orange) and bound (green; lysine in magenta) RNA. B, chemical modification of the T. maritima and B. subtilis lysine riboswitch emphasizing two key regions of each RNA that undergoes strong magnesium- and lysine-dependent changes. The full annotated gels are shown in supplemental Fig. S3. C, small angle x-ray data corresponding to free (EDTA, gray; magnesium, orange) and lysine-bound (green). The left panel shows the experimental electron pair distribution plot where the x-intercept reflects the most likely maximum intermolecular scattering distance (D_max). The right panel is a Kratky plot that reflects the extent of unfoldedness of the macromolecule (40). D, map of mutations (cyan) conferring AEC resistance onto the lysine riboswitch structure.