Conformational flexibility of adenine riboswitch aptamer in apo and bound states using NMR and an X-ray free electron laser
Riboswitches are structured cis-regulators mainly found in the untranslated regions of messenger RNA. The aptamer domain of a riboswitch serves as a sensor for its ligand, the binding of which triggers conformational changes that regulate the behavior of its expression platform. As a model system for understanding riboswitch structures and functions, the add adenine riboswitch has been studied extensively. However, there is a need for further investigation of the conformational dynamics of the aptamer in light of the recent real-time crystallographic study at room temperature (RT) using an X-ray free electron laser (XFEL) and femtosecond X-ray crystallography (SFX). Herein, we investigate the conformational motions of the add adenine riboswitch aptamer domain, in the presence or absence of adenine, using nuclear magnetic resonance relaxation measurements and analysis of RT atomic displacement factors (B-factors). In the absence of ligand, the P1 duplex undergoes a fast exchange where the overall molecule exhibits a motion at kex ~ 319 s−1, based on imino signals. In the presence of ligand, the P1 duplex adopts a highly ordered conformation, with kex~ 83 s−1, similar to the global motion of the molecule, excluding the loops and binding pocket, at 84 s−1. The µs–ms motions in both the apo and bound states are consistent with RT B-factors. Reduced spatial atomic fluctuation, ~ 50%, in P1 upon ligand binding coincides with significantly attenuated temporal dynamic exchanges. The binding pocket is structured in the absence or presence of ligand, as evidenced by relatively low and similar RT B-factors. Therefore, despite the dramatic rearrangement of the binding pocket, those residues exhibit similar spatial thermal fluctuation before and after binding.
KeywordsAdenine riboswitch Aptamer CPMG Conformational exchange B-factor
The 71-nucleotide aptamer domain of add adenine (A) riboswitch
X-ray free electron laser
Heteronuclear single quantum coherence
Riboswitches are structured RNAs that selectively bind metabolites for controlled gene expression. A riboswitch consists of two domains: an “aptamer” and an “expression platform” (Mandal and Breaker 2004; Winkler and Breaker 2003). Binding of a metabolite to the aptamer domain leads to an allosteric conformational change that is transmitted to the expression platform via a structurally regulated “switching” sequence (Batey 2012), enabling control over gene expression (Peselis et al. 2015; Wickiser et al. 2005). Purine riboswitches, the largest known class of riboswitches, include transcriptional regulators of pbuE (on-switch) and xpt-pbuX (off-switch), and the translational regulator of add (on-switch). Although the mode of gene regulation by riboswitches is diverse, the ligand recognition by their aptamer domains is relatively conserved across classes (Mandal and Breaker 2004; Winkler and Breaker 2003). The structural dynamics of the add (adenosine deaminase) adenine (A) riboswitch has been studied (Warhaut et al. 2017; Lee et al. 2010; Reining et al. 2013). Previous studies of the adenine-sensing aptamer domain revealed that the ligand-free state is conformationally heterogeneous, but in the presence of Mg+2 is pre-organized for adenine binding by forming kissing-loop interactions (Lee et al. 2010; Noeske et al. 2007; Nozinovic et al. 2014; Reining et al. 2013). In other studies, however, the ligand-binding pocket was suggested to be locally disordered in the absence of ligand (Batey 2012; Gilbert et al. 2006), and that rearrangement and stabilization of the pocket occur upon ligand binding (Gilbert et al. 2006; Di Palma et al. 2013). Our recent real-time, ligand-triggered crystallography study using XFEL determined structures of two apo states (apo1/apo2), a transient intermediate ligand-bound state (IB·ade), and the final bound state formed in crystallo (B·ade) (Stagno et al. 2017). The presence of four conformational states agreed with the four-state kinetic model in solution, thus revealing the structural basis for the switching action (Stagno et al. 2017). The crystallographic data, i.e. B-factor, which was recorded at room temperature, may provide a unique insight into a spatial aspect of molecular motion and complement the information from the µs-ms conformational dynamics to present a complete view of the RNA motion. Here we present the conformational flexibility of the aptamer domain in both the ligand-free and ligand-bound states using solution NMR spectroscopy and RT B-factor analysis derived from the crystallographic experiments using XFEL. We used spin relaxation and relaxation compensated Car-Purcell-Meiboom-Gill (rc-CPMG) dispersion experiments to observe the signals in hetero-nuclear single quantum coherence (HSQC) spectra of imino groups. Combined spatial and temporal information provides atomic-level details and timescales associated with the binding and switching process, thereby furthering our understanding of the regulatory mechanism of purine-riboswitches.
Materials and methods
RNA sample preparation
The two strands of the DNA template, for transcription of the 71-nucleotide (nt) aptamer domain of the add A-riboswitch (rA71), were synthesized by Integrated DNA Technologies (IDT). The sequence of the template strand is as follows:
5′-TCT GAT TCA GCT AGT CCA TAA TAC GAC TCA CTA TAG GGA ACA TAT AAT CCT AAT GAT ATG GTT TGG GAG TTT CTA CCA AGA GCC TTA AAC TCT TGA TTA TGT TCC C-3′
The template strand contains an 18-nt spacer sequence prior to the T7 promoter (shown as underlined). This spacer gives flexibility to the template strand and helps to reduce molecular crowding when binding to the agarose beads. It also reduces steric hindrance for binding of the T7 RNA polymerase and increases the binding efficiency. First, the two complementary DNA strands were dissolved in water and annealed at room temperature. The double-stranded DNA was then used as template for touch-down PCR (TD-PCR) (Liu et al. 2015).
Primer sequences used for TD-PCR are as follows:
Forward primer: 5′-/Bio/TCT GAT TCA GCT AGT CCA TAA TAC GAC TCA CTA TAG G-3′
Reverse primer: 5′-mGmGG AAC ATA ATC AAG AGT TT-3′
The forward primer is 5′ biotinylated and the reverse primer contains two 2′-O-methyl guanosines (mG) at the 5′ end, which reduce the non-templated nucleotide addition to the transcription product. Both primer sequences were purchased from IDT.
The TD-PCR reaction mixture contained the following:
0.001 μM double stranded DNA template
0.001 mM primers
0.2 mM NTPs
Taq DNA polymerase reaction buffer (10 mM Tris-HCl, 50 mM KCl, 2 mM MgCl2, pH 8.0).
A TD-PCR was performed as reported previously (Liu et al. 2015) with some modifications. The first step: 95 °C for 2 min, followed by the touch-down phase and PCR phase. The touch-down phase starts with 95 °C for 30 s, annealing for 45 s followed by elongation at 72 °C for 40 s. The annealing step of the touch-down phase has a temperature ramp from 75 to 45 °C in 20 cycles (1.5 °C per cycle). The PCR phase has 45 thermal cycles, and each cycle has melting at 95 °C for 30 s, annealing at 50 °C for 45 s, and elongation at 72 °C for 40 s.
Template attachment and transcription
The template preparation and transcription were performed according to the method described previously (Liu et al. 2015). Briefly, commercially purchased neutravidin (Thermo Fisher Scientific) coated agarose beads (30–165 μm diameter) were used as solid-phase support. First, the neutravidin beads were washed with water, then 3 times with buffer A (10 mM Tris-HCl, 50 mM KCl, pH 8.0). The PCR product (10 ml) was incubated with neutravidin beads at room temperature overnight to immobilize the DNA template. The next day, the beads were added to a Pierce Centrifuge column (~ 30 μm pore size) and centrifuged at 500 rpm, 4 °C for 1 min. The beads were then washed 3 times with buffer A. Approximately 80% of the template could be attached to the neutravidin beads. The bead-attached templates were stable for weeks and reusable for multiple RNA preparations. In vitro transcription was used to prepare all RNA samples, which includes the following steps for a 10 ml transcription reaction. DNA templates attached neutravidin agarose beads were incubated with 80 mM HEPES-KOH (pH 7.5), 28 mM MgCl2, 2 mM Spermidine, 40 mM DTT, 6 mM rNTPs, T7, SUPERase In RNase Inhibitor (Invitrogen) and deionized H2O to a final volume of 10 ml for 3 to 5 h. The RNA product was purified by urea-denaturing polyacrylamide gel electrophoresis, eluted from gel by RNA elution buffer (0.3 M sodium acetate, 2 mM EDTA, pH 5.3) at 4 °C overnight and finally buffer changed to NMR buffer (10 mM potassium phosphate, 30 mM KCl, 2 mM MgCl2, pH 6.8).
NMR sample preparation
15N uniformly labeled rA71 samples were made by the above in vitro T7 transcription protocol using 15N-labeled rNTPs. The final RNA concentration used for NMR measurements was 0.6 mM. For bound rA71, adenine was added to a final concentration of 5 mM.
NMR relaxation experiments
NMR relaxation experiments were performed on Bruker Avance spectrometers operating at proton frequencies of 850 MHz, 700 MHz or 600 MHz. 2D 1H-15N TROSY-HSQC and 3D 15N NOESY-HSQC spectra were recorded on the 850 MHz spectrometer. All spectrometers are equipped with proton-cooled cryogenic 1H/13C/15N triple resonance probes. Sample temperature was calibrated with a 100% methanol sample prior to each experiment.
R 1, R 2 and [1H]-15N NOE experiments
15N CPMG relaxation dispersion experiments
Small angle X-ray scattering
The detailed procedure for SAXS data collection, processing and analysis are previously described (Wang et al. 2009) using an in-house program package NCI-SAXS or a program package by Svergun et al. (http://www.embl-hamburg.de/biosaxs/). The experimental radius of gyration (Rg) was calculated from the data at low q values in the range of qRg < 1.3, using the Guinier approximation of lnI(q) ≈ ln(I(0)) − R g 2 q2/3.
The detailed description of serial femtosecond crystallography experiments using an XFEL, including the preparation of nano/microcrystals of rA71, data collection, data processing, and structure determination, were reported previously (Stagno et al. 2017). Briefly, using a “mix-and-inject” approach, adenine ligand (20 mM) was diffused into the flowing slurry of crystals, upstream of the X-ray interaction region. The delay time between ligand mixing and X-ray exposure was controlled by the path-length of HPLC tubing. Data were recorded in real-time at room temperature, and processed with Cheetah (Barty et al. 2014) and CrystFEL (White et al. 2012). The binning of data at a 10 s delay interval post-mixing revealed an intermediate bound (IB·ade) conformation. For B-factor analysis, the average B-factors per residue (Fig. 6) were extracted from the PDB coordinate files 5E54 (apo1/apo2) and 5SWE (B·ade) using Baverage from CCP4 suite (Winn et al. 2011). The spatial conformational flexibility of the four states was calculated based on the B-factors using PHENIX (Adams et al. 2002).
Results and discussion
NMR assignments for rA71 in absence or presence of ligand
To obtain more information for the junction and loop regions where no imino signals were detected, we used long-range (N1, N3, N7 & N9) 1H-12C-15N two-bond coupling HSQC experiments. The ligand-induced chemical shift changes for these spins are shown in Fig. 2b. Besides the residues already detected by imino HSQC experiments, additional residues in P1 (A16, A17, A19 and A76), the binding pocket (A23, A24, G46, A52 and A73), P2 (G44 and A45), and L3 (A65 and A66), showed large chemical shift changes.
Pico- to nano-second timescale conformational flexibility of rA71
The P1 stem, which harbors the “switching sequence” critical for add regulation, is dynamic in the apo state, indicated by both the increased R2/R1 and decreased [1H]-15N NOE values (Fig. 4). The dynamic behaviors among these residues have a poor correlation to one another, reflected by the random fluctuation in R2/R1 values. Such high flexibility might be caused by the opening of the P1 helix, as revealed by molecular dynamics simulations (Di Palma et al. 2013). In contrast, the fluctuation in the P1 stem is significantly reduced upon ligand binding, as suggested by the R2/R1 values closer to the average, consistent with the formation of a stable P1 duplex upon ligand binding. Stabilization of the P1 duplex upon ligand binding that results in regulation of gene expression downstream is a common feature among this class of riboswitches (Montange and Batey 2006; Huang et al. 2011; Haller et al. 2013; Suresh et al. 2016). With respect to the binding pocket, residues U49 and U51, which were not detected in the apo state, were stabilized upon binding, as reflected by their small R2/R1 values. Furthermore, the high [1H]-15N NOE values (~ 0.9) for the residues in this region, indicate that the imino groups of the local structure are ordered after adenine binding.
Micro- to milli-second timescale conformational dynamics of rA71
Our previous kinetics study showed that the conformational exchanges among the four states are on subsecond-second timescales (Stagno et al. 2017). Nevertheless, as mentioned in the previous section, several residues in rA71 have elevated R2/R1 values in both apo and bound states, indicating possible micro- to-milli-second timescale conformational dynamics before and after binding. We then examined the relaxation dispersion data of rA71 in the absence or presence of ligand.
In the presence of ligand, a total of 17 resolved resonances show conformational exchange. Similar to the apo state, these sites are mainly located in helical regions: P1 (G14, U20, U75, U77, U80), P2 (U39, U40), and P3 (G57, U68, U70, U71, G72); few are sparsely distributed in loops and junctions: J1/2 (U22), J2/3 (G46) and L2 (G37, G38) (Fig. 5b). Individual fits yield kex ranging from 74 s−1 (G72) to 1194 s−1 (G59). The segmental fits for three duplexes, P1, P2, and P3, result in kex of 83, 94 and 89 s−1, respectively (Table S2), significantly lower than those in the absence of ligand (Table S2). It is interesting to note that kex for the binding pocket remains similar in the absence and presence of ligand (Table S1). But the lack of sufficient detectable signals from the residues in the binding pocket makes the fitting less reliable.
In general, the values of the RT B-factors are consistent with kex values from the global and segmental fittings (Table S2). The average RT B-factors for the two apo conformations (~ 77 Å2) (Fig. 6a; Table S2) is considerably higher than that observed for the bound conformation (~ 58 Å2), despite the fact that the apo crystal data are of better quality, and to higher resolution (2.3 Å for apo1/apo2 vs. 3.0 Å for B·ade). In particular, the P1 helix exhibits the greatest changes in B-factors (Fig. 6a), with average values of ~ 123, 131, and 69 Å2 for apo1, apo2 and B·ade, respectively. This reflects a high degree of atomic motion of residues in the P1 stem, most probably associated with duplex opening in the absence of ligand and stabilization of the duplex upon ligand binding. The dramatic changes in the RT B-factors of the P1 duplex upon ligand binding are clearly illustrated by plotting the differences of the RT B-factors between the apo and bound structures (Fig. 6b). We then performed ensemble calculation based on the B-factors of all four states using PHENIX (Adams et al. 2002) and results provide visualizations of comparison of spatial motions of the four states (Supplemental Video 1). The binding pocket, on the other hand, shows relatively small B-factor differences, despite large conformational changes (Fig. 3), which appears to be consistent with the μs-ms relaxation date (Table S2). This implies that the binding pocket, although temporally flexible, exhibits comparable spatial fluctuation before and after ligand binding.
Our NMR relaxation data, combined with the RT XFEL structural information, provide temporal and spatial evidence for the differential conformational dynamics of the add riboswitch aptamer domain. Ligand binding reduces overall motions in all duplex regions, in particular, the P1 duplex based on both the relaxation data and the RT B-factors, and result in the more compact structure as indicated by the radius of gyration.
This four-state model was further supported by RT SFX experiments that captured the structures of these states. The data analysis of the stopped-flow experiments provided detailed kinetic rate constants (Stagno et al. 2017): kop = 2.1 s−1, kcl = 0.53 s−1, kon = 0.37 µM−1 s−1, koff = 45 s−1, kf = 132 s−1, kr = 5.8 s−1, sc = 2.58 with the fitting error Err(k,sc) = 0.025, with kf being the fastest rate in the four-state model, and the rest being on the subsecond-second timescale. Those rates are slower than conformation exchange rates, kex, from relaxation measurements. Slower rates observed from the kinetic measurements may be attributed to an artifact caused by replacing U48 with 2-aminopurine (2AP) in the ligand-binding pocket. The fluorescent 2AP substation is used as a reporter to trace the trajectory of conformational changes triggered by ligand binding. Such a substitution in the binding pocket may perturb the kinetic landscape. Furthermore, the two methods might probe different types of motion on a different time scale. The much slower kinetic rates measured by the stopped-flow experiments might be relevant to such as domain-wise diffusive motion, whereas the CPMG measurements in this study probe micro- to milli-second timescale motion.
This work was supported by the NIH Intramural Research. We thank Dr. Jinfa Ying and Dr. Janusz Koscielniak for technical assistances.
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