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Structural insights into amino acid binding and gene control by a lysine riboswitch

Abstract

In bacteria, the intracellular concentration of several amino acids is controlled by riboswitches1,2,3,4. One of the important regulatory circuits involves lysine-specific riboswitches, which direct the biosynthesis and transport of lysine and precursors common for lysine and other amino acids1,2,3. To understand the molecular basis of amino acid recognition by riboswitches, here we present the crystal structure of the 174-nucleotide sensing domain of the Thermotoga maritima lysine riboswitch in the lysine-bound (1.9 ångström (Å)) and free (3.1 Å) states. The riboswitch features an unusual and intricate architecture, involving three-helical and two-helical bundles connected by a compact five-helical junction and stabilized by various long-range tertiary interactions. Lysine interacts with the junctional core of the riboswitch and is specifically recognized through shape-complementarity within the elongated binding pocket and through several direct and K+-mediated hydrogen bonds to its charged ends. Our structural and biochemical studies indicate preformation of the riboswitch scaffold and identify conformational changes associated with the formation of a stable lysine-bound state, which prevents alternative folding of the riboswitch and facilitates formation of downstream regulatory elements. We have also determined several structures of the riboswitch bound to different lysine analogues5, including antibiotics, in an effort to understand the ligand-binding capabilities of the lysine riboswitch and understand the nature of antibiotic resistance. Our results provide insights into a mechanism of lysine-riboswitch-dependent gene control at the molecular level, thereby contributing to continuing efforts at exploration of the pharmaceutical and biotechnological potential of riboswitches.

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Figure 1: Overall structure and long-range tertiary interactions of the lysine-bound T. maritima riboswitch.
Figure 2: Structure and interactions in the junctional region of the lysine riboswitch.
Figure 3: Interactions of lysine analogues with the riboswitch.
Figure 4: Probing lysine riboswitch tertiary structure.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates of the X-ray structures of the lysine riboswitch bound to ligands have been deposited in the RCSB Protein Data Bank under the following accession codes: lysine, 3DIL; AEC, 3DIG; l-4-oxalysine, 3DJ0; homoarginine, 3DIQ; and iminoethyl-l-lysine, 3DIR. Codes for other structures are: free state, 3DIS; [Ir(NH3)6]3+-soaked, 3DIO; Cs+-soaked, 3DIM; Tl+-soaked, 3DJ2; Mn2+-soaked, 3DIY; K+-anomalous, 3DIX; and Mg2+-free form, 3DIZ.

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Acknowledgements

We thank personnel of beamline X29 at the Brookhaven National Laboratory and beamlines 24-ID-C/E at the Advanced Photon Source, Argonne National Laboratory, funded by the US Department of Energy. We thank O. Ouerfelli for the synthesis of iridium hexamine. D.J.P. was supported by funds from the National Institutes of Health.

Author Contributions L.H. crystallized the T. maritima lysine riboswitch; A.S. determined the structures and was assisted by L.H. during refinement; A.S. and L.H. performed biochemical experiments; and A.S. and D.J.P. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Alexander Serganov or Dinshaw J. Patel.

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Serganov, A., Huang, L. & Patel, D. Structural insights into amino acid binding and gene control by a lysine riboswitch. Nature 455, 1263–1267 (2008). https://doi.org/10.1038/nature07326

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