Protonation states of the key active site residues and structural dynamics of the glmS riboswitch as revealed by molecular dynamics

Abstract

The glmS catalytic riboswitch is part of the 5'-untranslated region of mRNAs encoding glucosamine-6-phosphate (GlcN6P) synthetase (glmS) in numerous gram-positive bacteria. Binding of the cofactor GlcN6P induces site-specific self-cleavage of the RNA. However, the detailed reaction mechanism as well as the protonation state of the glmS reactive form still remains elusive. To probe the dominant protonation states of key active site residues, we carried out explicit solvent molecular dynamic simulations involving various protonation states of three crucial active site moieties observed in the available crystal structures: (i) guanine G40 (following the Thermoanaerobacter tengcongensis numbering), (ii) the GlcN6P amino/ammonium group, and (iii) the GlcN6P phosphate moiety. We found that a deprotonated G40(-) seems incompatible with the observed glmS active site architecture. Our data suggest that the canonical form of G40 plays a structural role by stabilizing an in-line attack conformation of the cleavage site A-1(2'-OH) nucleophile, rather than a more direct chemical role. In addition, we observe weakened cofactor binding upon protonation of the GlcN6P phosphate moiety, which explains the experimentally observed increase in K(m) with decreasing pH. Finally, we discuss a possible role of cofactor binding and its interaction with the G65 and G1 purines in structural stabilization of the A-1(2'-OH) in-line attack conformation. On the basis of the identified dominant protonation state of the reaction precursor, we propose a hypothesis of the self-cleavage mechanism in which A-1(2'-OH) is activated as a nucleophile by the G1(pro-R(p)) nonbridging oxygen of the scissile phosphate, whereas the ammonium group of GlcN6P acts as the general acid protonating the G1(O5') leaving group.

FIGURE 1

Structure of the glmS riboswitch from Thermoanareobacter tengcongensis. (A) Rear and front views of the three-dimensional structure of the glmS riboswitch. Double-helical stems are shown in different colors, base stacking modules are in yellow, and unstructured parts and GNRA tetraloop are in gray. (B) The sequence and secondary structure of the glmS riboswitch. The colors of structural elements match those in panel A. Base pairs are annotated using standard classification.

SCHEME 1

Structure and atom numbering of the cofactor GlcN6P.

FIGURE 2

Stereo view of key nucleotides in the glmS riboswitch active site taken from the first snapshot of the G40/GlcN⁺6P²⁻ simulation, showing a stabilized reactive in-line attack conformation of the nucleophile A-1(O2') and proper binding of GlcN6P by a specific hydrogen bond network (T. tengcongensis numbering). See supplemental Fig. S10 for enlarged but non-stereo version of this figure.

FIGURE 3

Proposed mechanisms for glmS riboswitch self-cleavage. (A) The conserved G40 is deprotonated and acts as the general base while GlcN6P acts as the general acid. (B) Glucosamine-6-phosphate acts both as the general base accepting a proton from the A-1(O2') nucleophile via two tightly bound waters and as the general acid transferring this proton to the leaving G1(O5') oxygen. Red arrows denote electron flow during the reaction.

FIGURE 4

(A) The superimposed snapshots of G40/GlcN⁺6P²⁻ simulation taken at each nanosecond demonstrate flexibility of P1, P3, P3.1 and P4 stems and rigidity of the pseudoknot core. (B) The thermal B-factors of the backbone atoms of each residue calculated from MD simulation (for comparison with X-ray B-factors see Supplementary Materials). The coloring of the stripes at the top of the plot matches the colors of stems (shown above).

FIGURE 5

The front and rear view of the oblique interaction of G128|A127|A104|A105|A106 purine stack with minor groove of the P2.1 stem.

FIGURE 6

The RMSDs of the selected active site region: (A) Time dependence of the RMSD from the initial crystal structure calculated for three different regions of the glmS riboswitch active site as depicted in (B) for the green line, (C) for the red line and (D) for the black line. The specific atoms used for the RMSD calculation are shown in stick representation. The lowest RMSD of ~0.5 Å is characteristic for structures with no geometrical changes compared to the starting X-ray geometries. RMSD values above ~0.5 Å indicate changes from the starting crystal structure (for detailed description see Supplementary Materials).

FIGURE 7

Time evolution of key hydrogen bonds in our MD simulations of the glmS riboswitch, documenting cofactor binding and stability of the active site architecture. (A) Glucosamine-6-phosphate bound in the glmS active site with its hydrogen bonding network. (B) Time evolution of the hydrogen bonds of panel A. (C) Part of the active site including the scissile phosphate and two neighboring guanines with their network of hydrogen bonds. (D) Time evolution of the hydrogen bonds of panel C (the G65(N2)…G1(pro-R_p) distance marked in red is shown in panel A).

FIGURE 8

Proposed mechanism for glmS riboswitch self-cleavage based on presented MD simulations. Simultaneously with the nucleophilic attack the G1(pro-R_p) non-bridging oxygen acts as the general base accepting proton from A-1(O2') nucleophile and the GlcN6P acts as the general acid to donate its proton to the leaving oxygen G1(O5'). Red arrows denote electron flow during the reaction.