Role of ligand binding in structural organization of add A-riboswitch aptamer: a molecular dynamics simulation

The specific binding of ligands is the first step of gene expression or translation regulation by riboswitches. However, understanding the mechanism of the specific binding is still difficult because the tertiary structures of the riboswitch aptamers are available almost only for ligand-bound state at present. In this paper we hope to give some insights into this problem through the studies of the role of ligand-aptamer interaction in the structural organization of add A-riboswitch aptamer, based on the crystal structure of the ligand-bound aptamer. We use all-atom molecular dynamics to simulate the behaviors of the aptamer in ligand-bound, free and mutated states by Amber force field. The results show that the correct paring of the ligand adenine with the nucleotide U74 in the binding pocket is crucial to stabilizing the conformations of the ligand-bound aptamer, especially the helix P1 connecting the expression platform. Our results also suggest that both the nucleotide U74 and U51 may be the key sites of the ligand recognition but the former has much higher probability as the initial docking site. This is in agreement with previous experimental results.     

Molecular dynamics simulation study of the binding of purine bases to the aptamer domain of the guanine sensing riboswitch

Riboswitches are a novel class of genetic control elements that function through the direct interaction of small metabolite molecules with structured RNA elements. The ligand is bound with high specificity and affinity to its RNA target and induces conformational changes of the RNA''s secondary and tertiary structure upon binding. To elucidate the molecular basis of the remarkable ligand selectivity and affinity of one of these riboswitches, extensive all-atom molecular dynamics simulations in explicit solvent (≈1 μs total simulation length) of the aptamer domain of the guanine sensing riboswitch are performed. The conformational dynamics is studied when the system is bound to its cognate ligand guanine as well as bound to the non-cognate ligand adenine and in its free form. The simulations indicate that residue U51 in the aptamer domain functions as a general docking platform for purine bases, whereas the interactions between C74 and the ligand are crucial for ligand selectivity. These findings either suggest a two-step ligand recognition process, including a general purine binding step and a subsequent selection of the cognate ligand, or hint at different initial interactions of cognate and noncognate ligands with residues of the ligand binding pocket. To explore possible pathways of complex dissociation, various nonequilibrium simulations are performed which account for the first steps of ligand unbinding. The results delineate the minimal set of conformational changes needed for ligand release, suggest two possible pathways for the dissociation reaction, and underline the importance of long-range tertiary contacts for locking the ligand in the complex.     

Loop–loop interaction in an adenine-sensing riboswitch: A molecular dynamics study

Riboswitches are mRNA-based molecules capable of controlling the expression of genes. They undergo conformational changes upon ligand binding, and as a result, they inhibit or promote the expression of the associated gene. The close connection between structural rearrangement and function makes a detailed knowledge of the molecular interactions an important step to understand the riboswitch mechanism and efficiency. We have performed all-atom molecular dynamics simulations of the adenine-sensing add A-riboswitch to study the breaking of the kissing loop, one key tertiary element in the aptamer structure. We investigated the aptamer domain of the add A-riboswitch in complex with its cognate ligand and in the absence of the ligand. The opening of the hairpins was simulated using umbrella sampling using the distance between two loops as the reaction coordinate. A two-step process was observed in all the simulated systems. First, a general loss of stacking and hydrogen bond interactions is seen. The last interactions that break are the two base pairs G37-C61 and G38-C60, but the break does not affect the energy profile, indicating their pivotal role in the tertiary structure formation but not in the structure stabilization. The junction area is partially organized before the kissing loop formation and residue A24 anchors together the loop helices. Moreover, when the distance between the loops is increased, one of the hairpins showed more flexibility by changing its orientation in the structure, while the other conserved its coaxial arrangement with the rest of the structure.     

Tetracycline Determines the Conformation of Its Aptamer at Physiological Magnesium Concentrations

Synthetic riboswitches are versatile tools for the study and manipulation of biological systems. Yet, the underlying mechanisms governing its structural properties and regulation under physiological conditions are poorly studied. We performed spectroscopic and calorimetric experiments to explore the folding kinetics and thermodynamics of the tetracycline-binding aptamer, which can be employed as synthetic riboswitch, in the range of physiological magnesium concentrations. The dissociation constant of the ligand-aptamer complex was found to strongly depend on the magnesium concentration. At physiological magnesium concentrations, tetracycline induces a significant conformational shift from a compact, but heterogeneous intermediate state toward the completely formed set of tertiary interactions defining the regulation-competent structure. Thus, the switching functionality of the tetracycline-binding aptamer appears to include both a conformational rearrangement toward the regulation-competent structure and its thermodynamic stabilization.     

Structural study of an RNA aptamer for a Tat protein complexed with ligands.

An RNA aptamer for an HIV Tat protein has been isolated by the in vitro SELEX method. The RNA aptamer binds to the Tat protein 50-100 times more strongly than native TAR RNA does. Here, we have investigated the structure of the RNA aptamer complexed with ligands, partial peptide fragments of the Tat protein or argininamide, by multidimensional 1H/13C/15N NMR. It is strongly suggested that two U:A:U base triples are formed in the RNA aptamer upon binding of ligands. Specific hydrogen bonds between arginine side chains of ligands and guanine bases located adjacent to the base triples are identified. On the basis of many intramolecular and intermolecular NOEs, a structural model of the complex has been constructed.     

An adaptable pentaloop defines a robust neomycin-B RNA aptamer with conditional ligand-bound structures

Aptamers can be highly specific for their targets, which implies precise molecular recognition between aptamer and target. However, as small polymers, their structures are more subject to environmental conditions than the more constrained longer RNAs such as those that constitute the ribosome. To understand the balance between structural and environmental factors in establishing ligand specificity of aptamers, we examined the RNA aptamer (NEO1A) previously reported as specific for neomycin-B. We show that NEO1A can recognize other aminoglycosides with similar affinities as for neomycin-B and its aminoglycoside specificity is strongly influenced by ionic strength and buffer composition. NMR and 2-aminopurine (2AP) fluorescence studies of the aptamer identified a flexible pentaloop and a stable binding pocket. Consistent with a well-structured binding pocket, docking analysis results correlated with experimental measures of the binding energy for most ligands. Steady state fluorescence studies of 2AP-substituted aptamers confirmed that A16 moves to a more solvent accessible position upon ligand binding while A14 moves to a less solvent accessible position, which is most likely a base stack. Analysis of binding affinities of NEO1A sequence variants showed that the base in position 16 interacts differently with each ligand and the interaction is a function of the buffer constituents. Our results show that the pentaloop provides NEO1A with the ability to adapt to external influences on its structure, with the critical base at position 16 adjusting to incorporate each ligand into a stable pocket by hydrophobic interactions and/or hydrogen bonds depending on the ligand and the ionic environment.     

Molecular dynamics simulation of the induced‐fit binding process of DNA aptamer and L‐argininamide

Aptamers are rare functional nucleic acids with binding affinity to and specificity for target ligands. Recent experiments have lead to the proposal of an induced‐fit binding mechanism for L ‐argininamide (Arm) and its binding aptamer. However, at the molecular level, this mechanism between the aptamer and its coupled ligand is still poorly understood. The present study used explicit solvent molecular dynamics (MD) simulations to examine the critical bases involved in aptamer‐Arm binding and the induced‐fit binding process at atomic resolution. The simulation results revealed that the Watson‐Crick pair (G10‐C16), C9, A12, and C17 bases play important roles in aptamer‐Arm binding, and that binding of Arm results in an aptamer conformation optimized through a general induced‐fit process. In an aqueous solution, the mechanism has the following characteristic stages: (a) adsorption stage, the Arm anchors to the binding site of aptamer with strong electrostatic interaction; (b) binding stage, the Arm fits into the binding site of aptamer by hydrogen‐bond formation; and (c) complex stabilization stage, the hydrogen bonding and electrostatic interactions cooperatively stabilize the complex structure. This study provides dynamics information on the aptamer‐ligand induced‐fit binding mechanism. The critical bases in aptamer‐ligand binding may provide a guideline in aptamer design for molecular recognition engineering.     

Riboswitch structure: an internal residue mimicking the purine ligand

The adenine and guanine riboswitches regulate gene expression in response to their purine ligand. X-ray structures of the aptamer moiety of these riboswitches are characterized by a compact fold in which the ligand forms a Watson–Crick base pair with residue 65. Phylogenetic analyses revealed a strict restriction at position 39 of the aptamer that prevents the G39–C65 and A39–U65 combinations, and mutational studies indicate that aptamers with these sequence combinations are impaired for ligand binding. In order to investigate the rationale for sequence conservation at residue 39, structural characterization of the U65C mutant from Bacillus subtilis pbuE adenine riboswitch aptamer was undertaken. NMR spectroscopy and X-ray crystallography studies demonstrate that the U65C mutant adopts a compact ligand-free structure, in which G39 occupies the ligand-binding site of purine riboswitch aptamers. These studies present a remarkable example of a mutant RNA aptamer that adopts a native-like fold by means of ligand mimicking and explain why this mutant is impaired for ligand binding. Furthermore, this work provides a specific insight into how the natural sequence has evolved through selection of nucleotide identities that contribute to formation of the ligand-bound state, but ensures that the ligand-free state remains in an active conformation.     

Tertiary contacts control switching of the SAM-I riboswitch

Riboswitches are non-coding RNAs that control gene expression by sensing small molecules through changes in secondary structure. While secondary structure and ligand interactions are thought to control switching, the exact mechanism of control is unknown. Using a novel two-piece assay that competes the anti-terminator against the aptamer, we directly monitor the process of switching. We find that the stabilization of key tertiary contacts controls both aptamer domain collapse and the switching of the SAM-I riboswitch from the aptamer to the expression platform conformation. Our experiments demonstrate that SAM binding induces structural alterations that indirectly stabilize the aptamer domain, preventing switching toward the expression platform conformer. These results, combined with a variety of structural probing experiments performed in this study, show that the collapse and stabilization of the aptamer domain are cooperative, relying on the sum of key tertiary contacts and the bimodal stability of the kink-turn motif for function. Here, ligand binding serves to shift the equilibrium of aptamer domain structures from a more open toward a more stable collapsed form by stabilizing tertiary interactions. Our data show that the thermodynamic landscape for riboswitch operation is finely balanced to allow large conformational rearrangements to be controlled by small molecule interactions.     

Dissecting the influence of Mg2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain

Long-range tertiary interactions determine the three-dimensional structure of a number of metabolite-binding riboswitch RNA elements and were found to be important for their regulatory function. For the guanine-sensing riboswitch of the Bacillus subtilis xpt-pbuX operon, our previous NMR-spectroscopic studies indicated pre-formation of long-range tertiary contacts in the ligand-free state of its aptamer domain. Loss of the structural pre-organization in a mutant of this RNA (G37A/C61U) resulted in the requirement of Mg²⁺ for ligand binding. Here, we investigate structural and stability aspects of the wild-type aptamer domain (Gsw) and the G37A/C61U-mutant (Gsw^loop) of the guanine-sensing riboswitch and their Mg²⁺-induced folding characteristics to dissect the role of long-range tertiary interactions, the link between pre-formation of structural elements and ligand-binding properties and the functional stability. Destabilization of the long-range interactions as a result of the introduced mutations for Gsw^loop or the increase in temperature for both Gsw and Gsw^loop involves pronounced alterations of the conformational ensemble characteristics of the ligand-free state of the riboswitch. The increased flexibility of the conformational ensemble can, however, be compensated by Mg²⁺. We propose that reduction of conformational dynamics in remote regions of the riboswitch aptamer domain is the minimal pre-requisite to pre-organize the core region for specific ligand binding.     

Folding of the SAM aptamer is determined by the formation of a K-turn-dependent pseudoknot

The S-adenosylmethionine (SAM) riboswitch is one of the most recurrent riboswitches found in bacteria and has three known different natural aptamers. The Bacillus subtilis yitJ SAM riboswitch aptamer is organized around a four-way junction which is characterized by the presence of a pseudoknot and a K-turn motif. By replacing the adenine involved in a Watson-Crick base pair at position 138 in the core region of the aptamer with the fluorescent analogue 2-aminopurine (2AP), we show that the ligand-induced reorganization of the aptamer strongly attenuates 2AP fluorescence. The fluorescence quenching process is specific to SAM on the basis of the observation that the structural analogue S-adenosylhomocysteine does not promote a similar effect. We find that the pseudoknot is important for the reorganization of the core domain and that the K-turn motif also has a marked influence on the core domain reorganization, most probably through its important role in pseudoknot formation. Finally, we show that SAM riboswitch ligand binding is facilitated by the L7Ae K-turn binding protein, which suggests that K-turn motifs may be protein anchor sites used by riboswitches to promote RNA folding.     

A water channel in the core of the vitamin B(12) RNA aptamer

BACKGROUND: The 3.0 A crystal structure of the vitamin B(12) RNA aptamer revealed an unusual tertiary structure that is rich in novel RNA structural motifs. Important details of the interactions that stabilize noncanonical base pairing and the role of solvent in the structure were not apparent owing to the limited resolution. RESULTS: The structure of the vitamin B(12) RNA aptamer in complex with its ligand has been determined at 2.3 A resolution by X-ray crystallography. The crystallographic asymmetric unit contains five independent copies of the aptamer-vitamin B(12) complex, making it possible to accurately define well-conserved features. The core of the aptamer contains an unusual water-filled channel that is buried between the three strands of an RNA triplex. Well-ordered water molecules positioned within this channel form bridging hydrogen bonds and stabilize planar base triples that otherwise lack significant direct base-base contacts. The water channel terminates at the interface between the RNA and the bound ligand, leaving a pair of water molecules appropriately positioned to hydrogen bond with the highly polarized cyanide nitrogen of vitamin B(12). Analysis of the general solvation patterns for each nucleotide suggests that water molecules are not precisely positioned, as observed in previous RNA duplex structures, but instead might adjust in response to the varying local environment. Unusual intermolecular base pairing contributes to the formation of three different dimerization contacts that drive formation of the crystal lattice. CONCLUSIONS: The structure demonstrates the important role of water molecules and noncanonical base pairing in driving the formation of RNA tertiary structure and facilitating specific interactions of RNAs with other molecules.     

Identification of a tertiary interaction important for cooperative ligand binding by the glycine riboswitch

The glycine riboswitch has a tandem dual aptamer configuration, where each aptamer is a separate ligand-binding domain, but the aptamers function together to bind glycine cooperatively. We sought to understand the molecular basis of glycine riboswitch cooperativity by comparing sites of tertiary contacts in a series of cooperative and noncooperative glycine riboswitch mutants using hydroxyl radical footprinting, in-line probing, and native gel-shift studies. The results illustrate the importance of a direct or indirect interaction between the P3b hairpin of aptamer 2 and the P1 helix of aptamer 1 in cooperative glycine binding. Furthermore, our data support a model in which glycine binding is sequential; where the binding of glycine to the second aptamer allows tertiary interactions to be made that facilitate binding of a second glycine molecule to the first aptamer. These results provide insight into cooperative ligand binding in RNA macromolecules.     

Engineering a structure switching mechanism into a steroid-binding aptamer and hydrodynamic analysis of the ligand binding mechanism

The steroid binding mechanism of a DNA aptamer was studied using isothermal titration calorimetry (ITC), NMR spectroscopy, quasi-elastic light scattering (QELS), and small-angle X-ray spectroscopy (SAXS). Binding affinity determination of a series of steroid-binding aptamers derived from a parent cocaine-binding aptamer demonstrates that substituting a GA base pair with a GC base pair governs the switch in binding specificity from cocaine to the steroid deoxycholic acid (DCA). Binding of DCA to all aptamers is an enthalpically driven process with an unfavorable binding entropy. We engineered into the steroid-binding aptamer a ligand-induced folding mechanism by shortening the terminal stem by two base pairs. NMR methods were used to demonstrate that there is a transition from a state where base pairs are formed in one stem of the free aptamer, to where three stems are formed in the DCA-bound aptamer. The ability to generate a ligand-induced folding mechanism into a DNA aptamer architecture based on the three-way junction of the cocaine-binding aptamer opens the door to obtaining a series of aptamers all with ligand-induced folding mechanisms but triggered by different ligands. Hydrodynamic data from diffusion NMR spectroscopy, QELS, and SAXS show that for the aptamer with the full-length terminal stem there is a small amount of structure compaction with DCA binding. For ligand binding by the short terminal stem aptamer, we propose a binding mechanism where secondary structure forms upon DCA binding starting from a free structure where the aptamer exists in a compact form.     

Structural basis of the highly efficient trapping of the HIV Tat protein by an RNA aptamer

An RNA aptamer containing two binding sites exhibits extremely high affinity to the HIV Tat protein. We have determined the structure of the aptamer complexed with two argininamide molecules. Two adjacent U:A:U base triples were formed, which widens the major groove to make space for the two argininamide molecules. The argininamide molecules bind to the G bases through hydrogen bonds. The binding is stabilized through stacking interactions. The structure of the aptamer complexed with a Tat-derived arginine-rich peptide was also characterized. It was suggested that the aptamer structure is similar for both complexes and that the aptamer interacts with two different arginine residues of the peptide simultaneously at the two binding sites, which could explain the high affinity to Tat.     

Structure determination and binding kinetics of a DNA aptamer-argininamide complex

The structure of a DNA aptamer, which was selected for specific binding to arginine, was determined using NMR spectroscopy. The sequence forms a hairpin loop, with residues important for binding occurring in the loop region. Binding of argininamide induces formation of one Watson-Crick and two non-Watson-Crick base pairs, which facilitate generation of a binding pocket. The specificity for arginine seems to arise from contacts between the guanidino end of the arginine and phosphates, with atoms positioned by the shape of the pocket. Complex binding kinetics are observed suggesting that there is a slow interconversion of two forms of the DNA, which have different binding affinities. These data provide information on the process of adaptive recognition of a ligand by an aptamer.     

Influence of ground-state structure and Mg2+ binding on folding kinetics of the guanine-sensing riboswitch aptamer domain

Riboswitch RNAs fold into complex tertiary structures upon binding to their cognate ligand. Ligand recognition is accomplished by key residues in the binding pocket. In addition, it often crucially depends on the stability of peripheral structural elements. The ligand-bound complex of the guanine-sensing riboswitch from Bacillus subtilis, for example, is stabilized by extensive interactions between apical loop regions of the aptamer domain. Previously, we have shown that destabilization of this tertiary loop-loop interaction abrogates ligand binding of the G37A/C61U-mutant aptamer domain (Gsw(loop)) in the absence of Mg(2+). However, if Mg(2+) is available, ligand-binding capability is restored by a population shift of the ground-state RNA ensemble toward RNA conformations with pre-formed loop-loop interactions. Here, we characterize the striking influence of long-range tertiary structure on RNA folding kinetics and on ligand-bound complex structure, both by X-ray crystallography and time-resolved NMR. The X-ray structure of the ligand-bound complex reveals that the global architecture is almost identical to the wild-type aptamer domain. The population of ligand-binding competent conformations in the ground-state ensemble of Gsw(loop) is tunable through variation of the Mg(2+) concentration. We quantitatively describe the influence of distinct Mg(2+) concentrations on ligand-induced folding trajectories both by equilibrium and time-resolved NMR spectroscopy at single-residue resolution.