Structural analysis of a class III preQ1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics.

PreQ1-III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HLout-type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ1-III riboswitch aptamer forms a HLout-type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ1 binds tightly (KD,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3' RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (kdock ∼ 0.6 s(-1)) and undocking (kundock ∼ 1.1 s(-1)). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs.

Molecular mechanism for preQ1-II riboswitch function revealed by molecular dynamics.

Riboswitches are RNA molecules that regulate gene expression using conformational change, affected by binding of small molecule ligands. A crystal structure of a ligand-bound class II preQ1 riboswitch has been determined in a previous structural study. To gain insight into the dynamics of this riboswitch in solution, eight total molecular dynamic simulations, four with and four without ligand, were performed using the Amber force field. In the presence of ligand, all four of the simulations demonstrated rearranged base pairs at the 3' end, consistent with expected base-pairing from comparative sequence analysis in a prior bioinformatic analysis; this suggests the pairing in this region was altered by crystallization. Additionally, in the absence of ligand, three of the simulations demonstrated similar changes in base-pairing at the ligand binding site. Significantly, although most of the riboswitch architecture remained intact in the respective trajectories, the P3 stem was destabilized in the ligand-free simulations in a way that exposed the Shine-Dalgarno sequence. This work illustrates how destabilization of two major groove base triples can influence a nearby H-type pseudoknot and provides a mechanism for control of gene expression by a fold that is frequently found in bacterial riboswitches.

Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold.

PreQ1 riboswitches regulate genes by binding the pyrrolopyrimidine intermediate preQ1 during the biosynthesis of the essential tRNA base queuosine. We report what is to our knowledge the first preQ1-II riboswitch structure at 2.3-Å resolution, which uses a previously uncharacterized fold to achieve effector recognition at the confluence of a three-way helical junction flanking a pseudoknotted ribosome-binding site. The results account for translational control mediated by the preQ1-II riboswitch class and expand the known repertoire of ligand-binding modes used by regulatory RNAs.

Single transcriptional and translational preQ1 riboswitches adopt similar pre-folded ensembles that follow distinct folding pathways into the same ligand-bound structure.

Riboswitches are structural elements in the 5' untranslated regions of many bacterial messenger RNAs that regulate gene expression in response to changing metabolite concentrations by inhibition of either transcription or translation initiation. The preQ1 (7-aminomethyl-7-deazaguanine) riboswitch family comprises some of the smallest metabolite sensing RNAs found in nature. Once ligand-bound, the transcriptional Bacillus subtilis and translational Thermoanaerobacter tengcongensis preQ1 riboswitch aptamers are structurally similar RNA pseudoknots; yet, prior structural studies have characterized their ligand-free conformations as largely unfolded and folded, respectively. In contrast, through single molecule observation, we now show that, at near-physiological Mg(2+) concentration and pH, both ligand-free aptamers adopt similar pre-folded state ensembles that differ in their ligand-mediated folding. Structure-based Go-model simulations of the two aptamers suggest that the ligand binds late (Bacillus subtilis) and early (Thermoanaerobacter tengcongensis) relative to pseudoknot folding, leading to the proposal that the principal distinction between the two riboswitches lies in their relative tendencies to fold via mechanisms of conformational selection and induced fit, respectively. These mechanistic insights are put to the test by rationally designing a single nucleotide swap distal from the ligand binding pocket that we find to predictably control the aptamers' pre-folded states and their ligand binding affinities.

A transition-state interaction shifts nucleobase ionization toward neutrality to facilitate small ribozyme catalysis.

One mechanism by which ribozymes can accelerate biological reactions is by adopting folds that favorably perturb nucleobase ionization. Herein we used Raman crystallography to directly measure pK(a) values for the Ade38 N1 imino group of a hairpin ribozyme in distinct conformational states. A transition-state analogue gave a pK(a) value of 6.27 ± 0.05, which agrees strikingly well with values measured by pH-rate analyses. To identify the chemical attributes that contribute to the shifted pK(a), we determined crystal structures of hairpin ribozyme variants containing single-atom substitutions at the active site and measured their respective Ade38 N1 pK(a) values. This approach led to the identification of a single interaction in the transition-state conformation that elevates the base pK(a) > 0.8 log unit relative to the precatalytic state. The agreement of the microscopic and macroscopic pK(a) values and the accompanying structural analysis supports a mechanism in which Ade38 N1(H)+ functions as a general acid in phosphodiester bond cleavage. Overall the results quantify the contribution of a single electrostatic interaction to base ionization, which has broad relevance for understanding how RNA structure can control chemical reactivity.

FVIII proteins with a modified immunodominant T-cell epitope exhibit reduced immunogenicity and normal FVIII activity.

Factor VIII (FVIII)-neutralizing antibodies (inhibitors) are a serious complication in hemophilia A (HA). The peptide FVIII2194-2213 contains an immunodominant HLA-DRA*01-DRB1*01:01 (DRB1*01:01)-restricted epitope recognized by CD4+ T-effector cells from HA subjects. The aim of this study was to identify amino acid substitutions to deimmunize this epitope while retaining procoagulant function and expression levels comparable to those of wild-type (WT) FVIII proteins. The shortest DRB1*01:01-binding peptide was FVIII2194-2205, and residues important for affinity were identified as F2196, M2199, A2201, and S2204. T-cell proliferation experiments with Ala-substituted FVIII2194-2205 peptides identified F2196A as a substitution that abrogated proliferation of clones specific for the WT sequence. T-cell clones that were stimulated by recombinant WT-FVIII-C2 (rWT-FVIII-C2) protein did not proliferate when cultured with rFVIII-C2-F2196A, indicating the immunogenic peptide includes a naturally processed T-cell epitope. Additional amino acid substitutions at F2196 and M2199 were evaluated by peptide-MHC class II (MHCII)-binding assays, T-cell proliferation assays, epitope prediction algorithms, and sequence homologies. Six B-domain-deleted (BDD)-FVIII proteins with substitutions F2196A, F2196L, F2196K, M2199A, M2199W, or M2199R were produced. Proliferation of T-cell clones and polyclonal lines in response to rBDD-FVIII-F2196K and rBDD-FVIII-M2199A was reduced compared with responses to WT-BDD-FVIII. The BDD-FVIII-F2196K sequence modification appears to be the most promising sequence variant tested here, due to its effectiveness at eliminating DRB1*01:01-restricted immunogenicity, low potential immunogenicity in the context of other MHCII alleles, expression level comparable to WT-BDD-FVIII, and retained procoagulant activity. These results provide proof of principle for the design of less immunogenic FVIII proteins targeted to specific subsets of HA patients.

ITC analysis of ligand binding to preQ1 riboswitches.

Riboswitches regulate genes by binding to small-molecule effectors. Isothermal titration calorimetry (ITC) provides a label-free method to quantify the equilibrium association constant, K(A), of a riboswitch interaction with its cognate ligand. In addition to probing affinity and specific chemical contributions that contribute to binding, ITC can be used to measure the thermodynamic parameters of an interaction (ΔG, ΔH, and ΔS), in addition to the binding stoichiometry (N). Here, we describe methods developed to measure the binding affinity of various preQ1 riboswitch classes for the pyrrolopyrimidine effector, preQ1. Example isotherms are provided along with a review of various preQ1-II (class 2) riboswitch mutants that were interrogated by ITC to quantify the energetic contributions of specific interactions visualized in the crystal structure. Protocols for ITC are provided in sufficient detail that the reader can reproduce experiments independently, or develop derivative methods suitable for analyzing novel riboswitch-ligand binding interactions.

Riboswitch structure in the ligand-free state.

Molecular investigations of riboswitches bound to small-molecule effectors have produced a wealth of information on how these molecules achieve high affinity and specificity for a target ligand. X-ray crystal structures have been determined for the ligand-free state for representatives of the preQ1-I, S-adenosylmethionine I, lysine, and glycine aptamer classes. These structures in conjunction with complimentary techniques, such as in-line probing, NMR spectroscopy, Förster resonance energy transfer, small-angle scattering, and computational simulations, have demonstrated that riboswitches adopt multiple conformations in the absence of ligand. Despite a number of investigations that support ligand-dependent folding, mounting evidence suggests that free-state riboswitches interact with their effectors in the sub-populations of largely prefolded states as embodied by the principle of conformational selection, which has been documented extensively for protein-mediated ligand interactions. Fundamental riboswitch investigations of the bound and free states have advanced our understanding of RNA folding, ligand recognition, and how these factors culminate in communication between an aptamer and its expression platform. An understanding of these topics is essential to comprehend riboswitch gene regulation at the molecular level, which has already provided a basis to understand the mechanism of action of natural antimicrobials.

Crystallographic analysis of small ribozymes and riboswitches.

Ribozymes and riboswitches are RNA motifs that accelerate biological reactions and regulate gene expression in response to metabolite recognition, respectively. These RNA molecules gain functionality via complex folding that cannot be predicted a priori, and thus requires high-resolution three-dimensional structure determination to locate key functional attributes. Herein, we present an overview of the methods used to determine small RNA structures with an emphasis on RNA preparation, crystallization, and structure refinement. We draw upon examples from our own research in the analysis of the leadzyme ribozyme, the hairpin ribozyme, a class I preQ(1) riboswitch, and variants of a larger class II preQ(1) riboswitch. The methods presented provide a guide for comparable investigations of noncoding RNA molecules including a 48-solution, "first choice" RNA crystal screen compiled from our prior successes with commercially available screens.

Base ionization and ligand binding: how small ribozymes and riboswitches gain a foothold in a protein world.

Genome sequencing has produced thousands of nonprotein coding (nc)RNA sequences including new ribozymes and riboswitches. Such RNAs are notable for their extraordinary functionality, which entails exquisite folding that culminates in biocatalytic or ligand-binding capabilities. Here we discuss advances in relating ncRNA form to function with an emphasis on base pK(a) shifting by the hairpin and hepatitis delta virus ribozymes. We then describe ligand binding by the two smallest riboswitches, which target preQ(1) and S-adenosyl-(l)-homocysteine, followed by an analysis of a second-messenger riboswitch that binds cyclic-di-GMP. Each riboswitch is then compared to a protein that binds the same ligand to contrast binding properties. The results showcase the breadth of functionality attainable from ncRNAs, as well as molecular features notable for antibacterial design.