Joseph E. Wedekind

MESSENGER RNA EDITING IN MAMMALS: NEW MEMBERS OF THE APOBEC FAMILY SEEKING ROLES IN THE FAMILY BUSINESS

Alteration of mRNA sequence through base modification mRNA editing frequently generates protein diversity. Several proteins have been identified as being similar to C-to-U mRNA editing enzymes based on their structural domains and the occurrence of a catalytic domain characteristic of cytidine deaminases. In light of the hypothesis that these proteins might represent novel mRNA editing systems that could affect proteome diversity, we consider their structure, expression and relevance to biomedically significant processes or pathologies.

Crystal structure of a lead-dependent ribozyme revealing metal binding sites relevant to catalysis.

The leadzyme is a small RNA motif that catalyzes a site-specific, Pb 2+-dependent cleavage reaction. As such, it is an example of a metal-dependent RNA enzyme. Here we describe the X-ray crystallographic structure of the leadzyme, which reveals two independent molecules per asymmetric unit. Both molecules feature an internal loop in which a bulged purine base stack twists away from the helical stem. This kinks the backbone, rendering the phosphodiester bond susceptible to cleavage. The independent molecules have different conformations: one leadzyme copy coordinates Mg2+, whereas the other binds only Ba2+ or Pb2+. In the active site of the latter molecule, a single Ba2+ ion coordinates the 2´-OH nucleophile, and appears to mimic the binding of catalytic lead. These observations allow a bond cleavage reaction to be modeled, which reveals the minimal structural features necessary for catalysis by this small ribozyme.

Nanostructures of APOBEC3G Support a Hierarchical Assembly Model of High Molecular Mass Ribonucleoprotein Particles from Dimeric Subunits

Human APOBEC3G (hA3G) is a cytidine deaminase that restricts human immunodeficiency virus (HIV)-1 infection in a vif (the virion infectivity factor from HIV)-dependent manner. hA3G from HIV-permissive activated CD4+ T-cells exists as an inactive, high molecular mass (HMM) complex that can be transformed in vitro into an active, low molecular mass (LMM) variant comparable with that of HIV-non-permissive CD4+ T-cells. Here we present low resolution structures of hA3G in HMM and LMM forms determined by small angle x-ray scattering and advanced shape reconstruction methods. The results show that LMM particles have an extended shape, dissimilar to known cytidine deaminases, featuring novel tail-to-tail dimerization. Shape analysis of LMM and HMM structures revealed how symmetric association of dimers could lead to minimal HMM variants. These observations imply that the disruption of cellular HMM particles may require regulation of protein-RNA, as well as protein-protein interactions, which has implications for therapeutic development.

The Structural Basis for Recognition of the PreQ0 Metabolite by an Unusually Small Riboswitch Aptamer Domain.

Riboswitches are RNA elements that control gene expression through metabolite binding. The preQ1 riboswitch exhibits the smallest known ligand-binding domain and is of interest for its economical organization and high affinity interactions with guanine-derived metabolites required to confer tRNA wobbling. Here we present the crystal structure of a preQ1 aptamer domain in complex with its precursor metabolite preQ0. The structure is highly compact with a core that features a stem capped by a well organized decaloop. The metabolite is recognized within a deep pocket via Watson-Crick pairing with C15. Additional hydrogen bonds are made to invariant bases U6 and A29. The ligand-bound state confers continuous helical stacking throughout the core fold, thus providing a platform to promote Watson-Crick base pairing between C9 of the decaloop and the first base of the ribosome-binding site, G33. The structure offers insight into the mode of ribosome-binding site sequestration by a minimal RNA fold stabilized by metabolite binding and has implications for understanding the molecular basis by which bacterial genes are regulated.

Comparison of a preQ1 riboswitch aptamer in metabolite-bound and free states with implications for gene regulation.

Riboswitches are RNA regulatory elements that govern gene expression by recognition of small molecule ligands via a high affinity aptamer domain. Molecular recognition can lead to active or attenuated gene expression states by controlling accessibility to mRNA signals necessary for transcription or translation. Key areas of inquiry focus on how an aptamer attains specificity for its effector, the extent to which the aptamer folds prior to encountering its ligand, and how ligand binding alters expression signal accessibility. Here we present crystal structures of the preQ1 riboswitch from Thermoanaerobacter tengcongensis in the preQ1-bound and free states. Although the mode of preQ1 recognition is similar to that observed for preQ0, surface plasmon resonance revealed an apparent KD of 2.1 ± 0.3 nm for preQ1 but a value of 35.1 ± 6.1 nm for preQ0. This difference can be accounted for by interactions between the preQ1 methylamine and base G5 of the aptamer. To explore conformational states in the absence of metabolite, the free-state aptamer structure was determined. A14 from the ceiling of the ligand pocket shifts into the preQ1-binding site, resulting in “closed” access to the metabolite while simultaneously increasing exposure of the ribosome-binding site. Solution scattering data suggest that the free-state aptamer is compact, but the “closed” free-state crystal structure is inadequate to describe the solution scattering data. These observations are distinct from transcriptional preQ1 riboswitches of the same class that exhibit strictly ligand-dependent folding. Implications for gene regulation are discussed.

Structural and mechanistic studies of enolase

The high-resolution structure of yeast enolase cocrystallized with its equilibrium mixture of substrate and product reveals the stereochemistry of substrate/product binding and therefore the groups responsible for acid/base catalysis and stabilization of the enolate intermediate. Expression and characterization of site-specific mutant forms of the enzyme have confirmed the roles of amino acid side chains in the catalysis of the first and second steps of the reaction. Coordination of both required magnesium ions to the carboxylate of the substrate/product indicates a role for these cations in stabilization of the intermediate.

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 Mg2+ concentration and pH, both ligand-free aptamers adopt similar pre-folded state ensembles that differ in their ligand-mediated folding. Structure-based Gō-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.

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. WIREs RNA 2012, 3:369–384. doi: 10.1002/wrna.114.

APOBEC3G subunits self-associate via the C-terminal deaminase domain.

Human APOBEC3G (hA3G) is a cytidine deaminase active on HIV single-stranded DNA. Small angle x-ray scattering and molecular envelope restorations predicted a C-terminal dimeric model for RNA-depleted hA3G in solution. Each subunit was elongated, suggesting that individual domains of hA3G are solvent-exposed and therefore may interact with other macromolecules even as isolated substructures. In this study, co-immunoprecipitation and in-cell quenched fluorescence resonance energy transfer assays reveal that hA3G forms RNA-independent oligomers through interactions within its C terminus. Residues 209–336 were necessary and sufficient for homoligomerization. N-terminal domains of hA3G were unable to multimerize but remained functional for Gag and viral infectivity factor (Vif) interactions when expressed apart from the C terminus. These findings corroborate the small angle x-ray scattering structural model and are instructive for development of high throughput screens that target specific domains and their functions to identify HIV/AIDS therapeutics.

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 biosynthesis of the essential tRNA base queuosine. We report the first preQ1-II riboswitch structure at 2.3 Å resolution, which uses a novel fold to achieve effector recognition at the confluence of a three-way-helical junction flanking a pseudoknotted ribosome-binding site (RBS). The results account for preQ1-II-riboswitch-mediated translational control, and expand the known repertoire of ligand binding modes utilized by regulatory RNAs.