Mechanism of Ligand Discrimination by the NMT1 Riboswitch

Abstract

Riboswitches are conserved functional domains in mRNA that almost exclusively exist in bacteria. They regulate the biosynthesis and transport of amino acids and essential metabolites such as coenzymes, nucleobases, and their derivatives by specifically binding small molecules. Due to their ability to precisely discriminate between different cognate molecules as well as their common existence in bacteria, riboswitches have become potential antibacterial drug targets that could deliver urgently needed antibiotics with novel mechanisms of action. In this work, we report the recognition mechanisms of four oxidization products (XAN, AZA, UAC, and HPA) generated during purine degradation by an RNA motif termed the NMT1 riboswitch. Specifically, we investigated the physical interactions between the riboswitch and the oxidized metabolites by computing the changes in the free energy on mutating key nucleobases in the ligand binding pocket of the riboswitch. We discovered that the electrostatic interactions are central to ligand discrimination by this riboswitch. The relative binding free energies of the mutations further indicated that some of the mutations can also strengthen the binding affinities of the ligands (AZA, UAC, and HPA). These mechanistic details are also potentially relevant in the design of novel compounds targeting riboswitches.

Figure 1:. Structural details of the NMT1 riboswitch and ligand molecules studied in our work.

(A) A cartoon representation of the tertiary structure of the NMT1 riboswitch. The different structural motifs (stems: P1, P2a, and P2b; and junctions: J1 and J2) are uniquely colored and labeled. (B) A zoomed-view of the ligand binding pocket of the NMT1 riboswitch. Several key nucleotides and the ligand are shown in sticks. The metal ions (white) and water molecules (red) are shown by spheres. (C) The chemical structure of each of the four ligands (XAN, AZA, UAC, and HPA) studied in our work.

Figure 2:. Non-bonded interaction energy analysis.

(A) The total electrostatic and van der Waals interaction energies of different ligands (XAN, AZA, UAC, and HPA) with the NMT1 riboswitch, and (B) the energy contribution of each nucleotide in the binding pocket of the XAN-NMT1 complex. The standard error of the mean associated with the averaged non-bonded interaction energies are shown as error bars. See also Figure S8.

Figure 3:. Structural insights from MD simulations of the ligand-NMT1 complexes.

For each complex, shown is an initial structure (before MD) and an MD-derived structure highlighting the representative conformation based on the dominant conformational cluster from the respective MD simulation. Selected RNA nucleotides and each ligand are represented as sticks. The interaction patterns of the ligands with the key nucleotides in the binding pocket of the NMT1 riboswitch are shown with dotted lines in the same color as the ligands, and the remaining interactions are shown by white dotted lines. The metal ions and water molecules are shown in white and red spheres, respectively. The fractional occupancies of these interactions are shown in Figure S6.

Figure 4:. Energetics of riboswitch mutations in each ligand-riboswitch complex.

The relative binding free energy (ΔΔG) of mutations in the NMT1 riboswitch when it is bound to each ligand (XAN, AZA, UAC, and HPA). We calculated the error by propagating the standard error of the mean associated with the averaged ΔG_comp and ΔG_free, and the binding free energies are reported in kilocalories per mole (kcal/mol).

Figure 5:. Structural changes in the XAN-NMT1 complexes on mutations.

In panels (A) through (F), we show the interaction pattern of the ligand XAN with the nucleotides in the ligand binding pocket of the riboswitch after A6G, G10A, G35A, A39G, U40C, and U41C mutations, respectively. The fractional occupancies of these interactions are shown in Figure S13. The interactions between XAN and the riboswitch are highlighted by green dotted lines. The ions and water molecules are depicted by white and red spheres, respectively.

Figure 6:. Structural changes in the ligand-riboswitch complexes on mutations.

Structural insights from the (A) AZA-NMT1, (B) UAC-NMT1, and (C) HPA-NMT1 complexes upon mutations. The fractional occupancies of the interactions depicted are reported in Figures S18, S23, and S28.