Structural insights into ligand recognition by a sensing domain of the cooperative glycine riboswitch - PubMed (original) (raw)
Structural insights into ligand recognition by a sensing domain of the cooperative glycine riboswitch
Lili Huang et al. Mol Cell. 2010.
Abstract
Glycine riboswitches regulate gene expression by feedback modulation in response to cooperative binding to glycine. Here, we report on crystal structures of the second glycine-sensing domain from the Vibrio cholerae riboswitch in the ligand-bound and unbound states. This domain adopts a three-helical fold that centers on a three-way junction and accommodates glycine within a bulge-containing binding pocket above the junction. Glycine recognition is facilitated by a pair of bound Mg(2+) cations and governed by specific interactions and shape complementarity with the pocket. A conserved adenine extrudes from the binding pocket and intercalates into the junction implying that glycine binding in the context of the complete riboswitch could impact on gene expression by stabilizing the riboswitch junction and regulatory P1 helix. Analysis of riboswitch interactions in the crystal and footprinting experiments indicates that adjacent glycine-sensing modules of the riboswitch could form specific interdomain interactions, thereby potentially contributing to the cooperative response.
Copyright © 2010 Elsevier Inc. All rights reserved.
Figures
Figure 1. Sequence and Structure of the V. cholerae Riboswitch
(A) Secondary structure schematics of the glycine riboswitch with tandem sensing domain arrangement. Nucleotides conserved in ≥95 and ≥75 % sequences are in red and blue, respectively. (B) Crystal structure-based schematic of the VCII RNA fold. The bound glycine is in red. Dashes and circles indicate Watson-Crick and non-canonical base pairs. Key tertiary stacking interactions are shown as blue dashed lines. (C) Overall crystal structure of VCII RNA in a ribbon representation. (D) Zoomed-in view of the three-way junction. Green spheres depict Mg2+ cations. (E) Intercalation of A33 into the junctional region. The RNA is shown in stick representation with color scheme of atoms (nitrogen in blue, oxygen in red, phosphorus in yellow, and carbon in arbitrary colors). Putative hydrogen bonds are shown with black dashed lines. (F) Superposition of the three-way and four-way junctions of the glycine (light pink) and SAM-I (light blue) riboswitches, respectively. The root mean square deviation (RMSD) is 1.48 Å.
Figure 2. Glycine Binding Pocket of VCII RNA and Recognition of Glycine
(A) Overall view of the glycine-binding pocket. The C31 to G38 and A64 to G72 segments are in blue and light blue, respectively. Hydrated Mg2+ cations are in green with coordination bonds shown in stick representation. (B) Base triples in the binding pocket and putative hydrogen bonds (dashed lines) contributing to glycine recognition. (C) Refined _2∣F_o_∣_-∣F_c_∣ electron density map contoured at 1σ level (pink) and superposed with the refined model of the binding pocket of the glycine-bound structure. Green map shows omit _∣F_o_∣_-∣F_c_∣ map (3σ level) calculated prior to the addition of glycine. (D) The same view as (C) for the binding pocket in the unbound state. (E) Superposition of the binding pockets of glycine-bound (salmon) and unbound (blue) states. Arrow shows the distance between phosphorus atoms of C66 and A33 in the glycine-bound structure. (F) Surface view inside of the glycine-bound pocket, with bound glycine in red and a pair of Mg2+ cations in green. The helix above J3/3a-J3a/3 is omitted for clarity.
Figure 3. Glycine Binding to RNAs determined by ITC
(A) Summary of ITC-based parameters for single and tandem RNAs. (B) Integrated fitted heat plots of glycine binding to VCII RNA and mutants. Control: FMN riboswitch sensing domain. (C) Effect of magnesium concentration on glycine binding to VCII RNA. Experimental data for 40 mM MgCl2 and integrated plots for all MgCl2 concentrations are shown in top and bottom panels, respectively. (D) Cation effects on glycine binding to VCII RNA. (E) Glycine binding to VCI RNA. Values in parentheses depict RNA refolding temperature. Representative raw data at 20 mM MgCl2 and 75 °C refolding temperature are shown in top panel. (F) Glycine binding to VCI-II RNA.
Figure 4. Intermolecular Interactions in the Asymmetric Unit of the VCII RNA structure
(A) Two VCII RNA molecules (light blue and orange) in the asymmetric unit. Contact areas are indicated. (B) Split-up view of the interacting VCII RNAs in surface representation. Green spheres indicate Mg2+ cations located in the riboswitch interface. Nucleotide analog interference sites (Kwon and Strobel, 2008) which cannot be explained by the structure of the individual VCII RNA are shown in magenta. (C) Zoomed-in view of the J3b/3a-J3a/3b region. (D) A-minor interactions between J3b/3a-J3a/3b region and P1.
Figure 5. Ribonuclease V1 and T2 Probing of Interdomain Interactions
(A-F**)** Projections of weak and strong nuclease cleavage reductions (light and dark red) and enhancement (green) on the secondary RNA structures are shown on top and the corresponding gels are on the bottom. T1 and −OH designates RNase T1 and alkaline ladders, respectively. NR, no reaction. Cleavage reduction values (mean ± standard deviation from at least 3 gels) in the presence of 200-fold excess of glycine are shown on the secondary RNA structures at the top of panels B and D. Note that the 5′ and 3′ regions of RNAs have not been analyzed.
Figure 6. Likely Interdomain Interaction in V. cholerae Glycine Riboswitch
(A) Projection of intermolecular interactions found in the VCII crystallographic dimer and NAIM sites (Mandal and Breaker, 2004) on the secondary structure of natural tandem riboswitch. Domain I is shown in cyan and domain II in black. Green circles indicate NAIM sites which cannot be explained by the structure of the individual VCII RNA. Orange shading shows areas that correspond to the regions involved in interdomain interactions in the crystallographic dimer of VCII RNA. (B)-(C) Proposed interdomain interactions in the tandem natural riboswitch.
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