Ligand-Induced Stabilization of a Duplex-like Architecture Is Crucial

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Ligand Induced Stabilization of a Duplex-Like Architecture is Crucial for the Switching Mechanism of SAM-III Riboswitch Gorle Suresh, Harini Srinivasan, Shivani Nanda, and U. Deva Priyakumar Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b00973 • Publication Date (Web): 01 Jun 2016 Downloaded from http://pubs.acs.org on June 6, 2016

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Biochemistry

Ligand Induced Stabilization of a Duplex-Like Architecture is Crucial for the Switching Mechanism of SAM-III Riboswitch

Gorle Suresh, Harini Srinivasan, Shivani Nanda and U. Deva Priyakumar*

Center for Computational Natural Sciences and Bioinformatics, International Institute of Information Technology, Hyderabad 500 032, India

*Corresponding author Email: [email protected] Phone: +91 40 6653 1161 FAX: +91 40 6653 1413

Running title: Switching mechanism of SAM-III riboswitch

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Abstract Riboswitches are structured RNA motifs that control gene expression by sensing the concentrations of specific metabolites and are a promising new class of antibiotic targets. Sadenosylmethionine (SAM)-III riboswitch, mainly found in lactic acid bacteria, is involved in regulating methionine and SAM biosynthetic pathways. SAM-III riboswitch regulates the gene expression by switching the translation process on and off with respect to the absence and presence of the SAM ligand respectively. In the present study, an attempt is made to understand the key conformational transitions involved in ligand binding using atomistic molecular dynamics (MD) simulations performed in explicit solvent environment. G26 is found to recognize the SAM ligand by forming hydrogen bonds, whereas the absence of the ligand leads to opening of the binding pocket. Consistent with experimental results, absence of the SAM ligand weakens the base pairing interactions between the nucleobases that are part of the Shine-Dalgarno (SD) and anti-ShineDalgarno (aSD) sequences, which in turn facilitates recognition of the SD sequence by ribosomes. Detailed analysis reveals that a duplex-like structure formed by nucleotides from different parts of the RNA and the adenine base of the ligand is crucial for the stability of the completely folded state in presence of the ligand. Previous experimental studies have showed that the SAM-III riboswitch exists in equilibrium between the unfolded and partially folded states in the absence of the ligand, which completely folds upon binding of the ligand. Comparison of the current results to the available experimental data indicates the structures obtained using the MD simulations to resemble the partially folded state. Thus, the current study provides detailed understanding of the fully and partially folded structures of the SAM-III riboswitch in the presence and absence of the ligand respectively. The present study hypothesizes a dual role for SAM ligand, which facilitates conformational switching between partially and fully folded states by forming a stable duplex-like structure and strengthening the interactions between SD and aSD nucleotides.

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Keywords: Non-coding RNAs, SAM biosynthetic pathways, molecular dynamics simulations, ONOFF switching, Shine-Dalgarno and anti-Shine-Dalgarno sequences.

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Introduction Riboswitches are non-coding, self-regulatory translational mRNA elements whose structure responds to the concentration of specific ligands. They are located in the 5'-untranslated regions of certain mRNA elements (5’-UTR) and undergo large conformational changes with respect to binding of specific ligands/metabolites.1-5 Regulation of gene expression through such RNAmediated mechanisms offers several advantages over the mechanism controlled by proteins. Firstly, they exhibit high selectivity towards the substrate, and secondly, binding is required only for a short period of time for the signal to be sensed for controlling gene expression. Lastly, there is no involvement of other gene products, thereby reducing the effect of mutations.6,7 Typically, a riboswitch has two integral parts in its structure: an aptamer domain which recognizes and binds metabolites and the other is an expression platform that controls the gene expression at the level of translation initiation or transcription termination. It has been shown that some riboswitches found in eukaryotes can also be functional at the level of splicing.8-11 The riboswitches identified so far have been classified into different classes based on the sequence, structure conservation and the metabolite involved.12 They also have been shown to serve as potential therapeutic targets as they control gene expression and hence can help in up- or down-regulating the formation of a particular gene product.13,14 Since antibiotic resistance is a major health problem at the global level, the need for new antibiotics has been increasing.15-17 One of the most primitive molecular mechanisms for regulating gene expression is the recently discovered riboswitch mediated mechanism.18-21 Due to the strong nature of discrimination of cognate ligands from their closely related analogs, riboswitches have been proposed as potential anti-microbial drug targets. Therefore, understanding the atomic details of the physical basis for this specific discrimination and underlying interactions provide a molecular level picture of the functions of riboswitches is crucial. S-adenosylmethionine (SAM) riboswitches function by responding to the metabolite SAM to regulate the biosynthesis of methionine and other sulfur-containing metabolites such as cysteine, 4


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SAM and S-adenosyl-L-homocysteine (SAH) across a wide range of bacterial species.22 SAM, being a principal methyl donor, is of fundamental importance for the metabolism of polyamines, nucleic acids and the precursor of aminopropyl groups, glutathione.23,24 Five types of SAM riboswitches have been identified so far: SAM-I,25,26 SAM-II,27 SAM-III,28 SAM-IV,29 and SAMV,30 all of which respond to the intracellular SAM concentration and control gene expression. SAM riboswitches have been shown to exhibit preferential binding (~100 fold) to SAM in comparison to closely related SAH.22 SAM and SAH are analogous ligands and differ by just a methyl group and the positive charge on the sulfur atom. SAM-III riboswitch, also called the SMK box, is found in lactic acid bacteria (Figure 1) and is involved in regulating methionine and SAM biosynthesis in these organisms.28 This riboswitch is found in the 5’-UTR of the MetK gene, and the binding of SAM ligand to this riboswitch blocks the Shine-Dalgarno (SD) sequence thereby blocking translation. Previously determined crystal structure of the Enterococcus faecalis SMK box revealed an inverted Y-shaped arrangement with a set of conserved nucleotides around the SAM binding region.31 Earlier experimental studies have suggested that SAM-III riboswitch binds to SAM more tightly than SAH by exploiting the positive charge on sulfur.31 Previously, we have used MD simulations on SAM and SAH bound SAM-III riboswitch to identify the factors that enable the riboswitch to differentiate the closely related ligands.32

Molecular dynamics simulations have been used to investigate the structures and dynamics of riboswitches in the presence and absence of the cognate ligand.33-47 Computational studies on the SAM-I riboswitch have helped in the understanding the route of entry of SAM ligand and the intermolecular interactions.33 Furthermore, mutational studies have revealed that certain modifications in the SAM-binding region locked the riboswitch in a conformation similar to the bound form, and hence resulted in the loss of SAM binding capability.28 Simulations have been carried out on SAM-I riboswitch which showed that the folding of the nonlocal helix is the ratelimiting step in the formation of aptamer domain where SAM assists in its formation by lowering 5


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the associated free energy barrier.34 Studies have also revealed that the aptamer follows a two-step hierarchical folding that is selectively induced by the metal (Mg2+) ions and ligand binding.35 Computational studies have shown modification of the curvature and base-pairing of the expression platform upon binding of the ligand to the SAM-II riboswitch which affects its interaction with the ribosome.36 Previous studies on adenine riboswitch have helped to characterize the ligand interactions with the riboswitch, and how the ligand binding stabilizes the riboswitch.37,38 Studies on guanine riboswitch have shown the structural changes in the riboswitch on binding to guanine, adenine, and in the absence of ligand.39 Computational studies suggested that long-range interactions play an important role in the induced-fit binding of the ligand in guanine sensing riboswitches.40 A recent study has helped to understand the crucial interactions and structural basis for the regulation of gene expression by glycine riboswitch41 and preQ1 riboswitch.42,43 Despite several attempts, the mechanism of riboswitch folding and discrimination mechanism is not well understood primarily due to the timescale at which the ligand assisted folding takes place. The present study attempts to identify the role of ligand in stabilizing the OFF state of the riboswitch, and on the key conformational transitions that are crucial in the translational termination process. This is examined by analyzing the differences between the structures and dynamics of the riboswitch in the presence and absence of the ligand (referred to as holo and apo forms respectively in the rest of the manuscript) obtained using all atom MD simulations.

Materials and Methods Simulation setup: The initial structures of riboswitches used for running simulations of both the bound and unbound form were obtained from the experimental X-ray crystallographic data with PDB ID 3E5C.31 The secondary and tertiary structure of SAM-III riboswitch is shown in Figure 1 which has four stems: P1, P2, P3 and P4; and two junctions: J24 and J32. The three stems around the junction which binds SAM are P1, P2 and P4 stems with junctions J24 and J32 also forming a 6


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part of the binding pocket. The key nucleotides around SAM mentioned in the experimental structure were considered as the binding pocket (BP). All the MD simulations in this study were performed using the NAMD program48 with the CHARMM27 all atom nucleic acid force field49,50 for both SAM-III riboswitch and SAM ligand. This force field has been shown to be adequate to study conformational changes in RNA molecules despite having few limitations.32,37,38,42 The initial system preparation and equilibration were done using the CHARMM program.51 The X-ray crystal structure (PDB ID: 3E5C) corresponding to the SAM ligand bound to the SAM-III riboswitch was used as the initial structure for bound form riboswitch.31 Previously, we have reported MD simulations on different conformations of SAM and SAHC bound to the riboswitch.32 Two independent 100 ns MD simulations were performed on the original crystal structure for further understanding of the holo structure. The final configuration of the trajectory corresponding to the original crystal structure reported in previous study was subjected to three independent 100 ns MD simulations in absence of the SAM ligand (termed as apo). The ligand was removed from the holo structure and was immersed in a water box built by using modified TIP3P water model52 and the size of the water box was chosen by extending 9 Å padding beyond system dimensions. The interacting water molecules within 2.2 Å of the non-hydrogen atoms of the riboswitch were deleted while retaining the crystallographic water molecules. All the crystallographic Sr2+ ions were replaced by Mg2+ ions, and the system was neutralized by adding Mg2+ ions. After a 500-step minimization and a short 100 ps MD simulations in the NVT ensemble with a harmonic mass weighted restraint of 10 kcal mol-1Å-2 on the non-hydrogen atoms. SHAKE algorithm53 was used to constrain the covalent bonds involving hydrogens. An integration time step of 2 fs was used while employing the Leapfrog algorithm. Periodic boundary conditions were employed in all the MD simulations.54 Particle mesh Ewald summation method55,56 was used to treat the long range electrostatic interactions and a real space particle smooth function from 10 Å to 12 Å was used for van der Waals interactions by truncating the Lennard-Jones (LJ) potential at 12 Å.57 Nose-Hoover thermostat58 and Langevin piston algorithm59 were used to maintain constant temperature and 7


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pressure respectively. Multiple independent 100 ns MD simulations started with different sets of initial velocities were performed in the NPT ensemble at 300 K. All the simulations were performed at room temperature (298 K). The main objective of this study is not characterizing the ON state of the riboswitch that would require practically improbable long time scale simulation along with employing advanced sampling methods given the size of the system and the extent of conformational changes that are deemed to be responsible for the OFF to ON state transition. To identify the structural features that enable the ligand to stabilize the folded state, and to identify key conformational differences in the riboswitch in the absence of the ligand, the three 100 ns long MD simulations are deemed adequate. One of the MD simulations corresponding to apo system was extended to 0.5µs to examine the transitions between folded and intermediate states.

Analysis of the trajectories: All the results presented here are based on the trajectories excluding the first 20 ns of the simulations of the apo and holo systems. The multiple and independent simulations yielded qualitatively similar results, and cumulative averages and probabilities over different MD simulations performed here are presented. The interaction energies presented here were calculated using the INTER command in CHARMM program that require two selections of interacting sites, and gives interaction energies that include both the electrostatic and van der Waals contributions, using infinite non-bond cutoffs. The images of structures depicted in this manuscript were rendered using the VMD program.60 All the stacking interactions corresponding to nucleobases have been calculated in a similar way as mentioned elsewhere.61-63 Principal component analysis (PCA) has been performed on the combined trajectory of all the trajectories corresponding to holo and apo systems by considering all the atoms of the nucleotides. The covariance matrix of the x, y and z coordinates of all the atoms were obtained from each snapshot of the combined trajectory after aligning the non-hydrogen atoms of SAM-III structure. Then eigen vectors and corresponding eigen values were obtained after diagonalization of these covariance

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matrices. The first three eigen vectors that contribute most of the atomic fluctuations were used to project the conformational space along two dimensions.

Results and Discussion This section discusses the structural differences between the holo (riboswitch in the presence of ligand) and apo (riboswitch in the absence of ligand) forms of the riboswitch first. This is followed by a comparison of the available experimental information on the structural intermediate and the closed state with results from the current study. Finally, a hypothesis is proposed for the switching of ON state to OFF state upon ligand binding to the SMK box resulting in termination of the translation process.

Structural differences between the holo and the apo forms of the SMK box: Mean root mean square deviations (rmsd) of the holo and apo forms with respect to the Xray crystallographic structure were calculated and are depicted in Figure 2. The apo form exhibits large deviation (6.37 Å) compared to the holo form (3.10 Å) indicating significant structural distortions in the former. Plots corresponding to the rmsd time series of the holo and apo forms of the RNA are given in Figure S1 in the Supporting Information, and the average values are presented in Table S1. The riboswitch in the absence of the ligand undergoes major structural changes within few ns of the simulation to reach an equilibrated state and samples a larger conformational space, which is significantly different from the bound state. In order to understand which part of the RNA undergoes major changes, rmsd values were calculated for individual regions, including the binding pocket (BP), Shine-Dalgarno and anti-Shine-Dalgarno nucleotides, individual stems (P1, P2, P3 and P4), and the junctions (J32 and J24). Most of the regions of the RNA, exhibit larger rmsd values in the apo form compared to the holo form with respect to the experimental structure. However, experimental studies have reported that the P2 and P3 stems remain largely unaltered when the 9


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riboswitch transitions from the ON to the OFF state.64 Even though the difference in RMSD values corresponding to P2 and P3 stem regions are higher, the two base pairs present in these two stems are largely intact during the simulations consistent with the experimental results. This is further supported by the structural changes observed in the longer 0.5 µs MD simulation of the apo1 system (see later).

Differences in the stem dynamics of SMK box in the presence and absence of ligand: NMR spectroscopic study on a 51-nt construct of the SMK box,65 which is similar to the 53nt RNA used in the present study, had revealed 13 exchangeable imino proton resonances in the presence of SAM. These signals are observed only if the imino protons are protected from the solvent and hence are direct indicators of the presence or absence of base pairing interactions. Out of the possible 21 imino protons participating in base pairing, only 13 signals were observed and the rest are proposed not to yield observable signals due to local breathing motions in the RNA. In the absence of SAM, it has been observed that at 4˚C all the signals are detected with slight shift in the peaks, which indicates change in local chemical environment including conformational changes. However, at 25˚C, few signals corresponding to imino protons in P1 and P4 were undetectable while those in P2 and P3 remained largely intact in the absence of SAM. All 18 base pair interactions shown in the secondary structure in Figure 1, which includes the 13 base pairs discussed above, have been monitored throughout the MD simulations in both the holo and apo forms. Additionally, probability distributions of the N1-N3 distances between the base pairs have also been calculated (Figure S2-S5). Consistent with experiments, it is observed that the base pair interactions within the P2 and P3 stem are reasonably stable in both the holo and apo forms. One exception is the destabilization of A27:G71 base pairing of the P2 stem in the apo form, which is present in the vicinity of the binding pocket observed from the multiple MD simulations of apo systems. Loss of π-stacking interactions between the adenine base of the SAM ligand and A27 in the apo form leads to such instability (see later). The stability of the base pairs is well supported by 10


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the base pair interaction energies and small rmsd values discussed above. Other minor differences are marginal differences in the interaction energies of the 31-67, 32-63 and 33-62 base pairs. This indicates that P2 and P3 stems of the SMK box are pre-organized with reasonably well-defined structure before the ligand binds. The NMR study also showed that out of the four signals from P1 and P4 regions, the signal from G89 is undetectable in the absence of ligand.65 Figure 3 shows that the interaction between C75 and G89 is completely lost in the absence of the ligand, which leads to the exposure of the imino proton on G89 to the solvent environment. Additionally, the interaction between C25 and G90 is significantly reduced in the absence of the ligand leading to exposure of the SD nucleotides. The weakening of the base pair interactions near the terminal of the holo form of the RNA are due to fraying effects.32 Consistent with the NMR experiments, the apo form was found to have weak base pair interactions in the P1 and P4 compared to the holo form, and no significant distortions in the P2 and P3 stems.

SAM binding stabilizes SD-aSD base pairing that is crucial for blocking the translation initiation: Shine-Dalgarno (SD) sequence or Shine-Dalgarno box is the binding site for the ribosome in the mRNA64 which is located upstream of the start codon. It is the site that is recognized by the ribosome, and that helps the ribosome bind to the mRNA and initiate translation by aligning it to the start codon. SMK box is a translational control riboswitch and has SD sequence that spans over five guanine bases: G88 to G92 (Figure 4). It is proposed that in the OFF state (in presence of the ligand) the SD and aSD sequences are base paired to each other, and hence the translation cannot proceed. On the other hand, in the ON state (in the absence of the ligand) the SD sequence opens for the ribosome, allowing translation to take place. The effect of the presence and absence of the ligand on the base pairing interactions between the nucleotides that are part of the SD and aSD sequences are investigated based on the hydrogen bond distances, and hydrogen bond/π-stacking interaction energies. Probability distributions of distances corresponding to the hydrogen bonds are 11


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presented in Figure 4 along with the structure of the SD-aSD region. The base pairs adjacent to the ligand binding region (C75-G89 and C25-G90) are completely distorted by opening up in the absence of the ligand (apo state). In case of the holo state, the base pairs are reasonably stable with minor sampling of the partially open state by the C75-G89 base pair. Probability distributions have also indicated that the other three base pairs C76-G88, C23-G92 and C24-G91 are almost intact in both the cases even though slight shift towards ideal hydrogen bond distance is observed in case of C76-G88 base pair. Previous experimental studies have suggested that the SD-aSD pairing occurs to some extent even in the absence of the ligand, SAM, and that the binding of SAM shifts the equilibrium selectively to a perfectly paired SD-aSD.64 This is also reflected in the base pair interactions energies corresponding to these five base pairs that form the SD-aSD sequence. Since the ligand binding increases the stability of the SD-aSD base pairs, initiation of translation process is not expected to occur. This is because the ribosome has to recognize and bind to this region in order for translation to initiate. This is quite difficult in holo SMK box as compared to apo because it would require higher energy to disrupt the base pairs, which are held by strong interactions of base pairing in the region in the presence of SAM ligand. This is consistent with the experimental results which showed that the SD-aSD interaction is stabilized in the presence of SAM, resulting in SAMdependent inhibition of ribosome binding by blocking the access of ribosome to the ribosome binding site (RBS).64

Opening up of the J32 junction in the apo form: The conformational differences between the holo and apo states that possibly play a role in the OFF to ON transition of the SAM-III riboswitch were analyzed in detail. The J32 junction (A64 to G66), which interacts with the ligand-binding region in the holo state was found to lose such interactions and open up in the apo state observed in all three MD simulations of apo system. The structural alignment of RNA conformations in holo and apo states disposition of the J32 junction in the absence of the ligand is given in Figure 5A. Snapshots obtained from 0.5µs long simulations of 12


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apo system showing complete opening of the J32 junction are depicted in Figure S6. Contact maps generated based on all the pairwise distances between the center of masses of the nucleobases of the riboswitch are given in Figure 5B, 5C and S10. Loss of contacts between the P1 stem and J32 junction of RNA was observed for SAM-III in its apo system. The loss of contacts between the P1 stem and the J32 junction further support the importance of the role of ligand in correct positioning of the nucleotides of J32 in the binding region. Additionally, quantitative comparison of the movement of J32 from the binding pocket is illustrated by the probability distribution of the distance between the center of masses of nucleobases G66 and G90 (Figure 5D). The two different conformations of the J32 junction were further examined by calculating the phosphodiester dihedral angles corresponding to the three nucleotides. The distributions where significant differences in the sampling of these dihedral angles were observed are depicted in Figure 6. Major differences were observed in the sampling of the χ, ζ and ε dihedral angles. While changes in the ζ and ε dihedral angles lead to opening up of nucleobase, change in χ is related to the orientation of the base with respect to the ribose moiety to which it is connected.66,67 Deviations from the characteristic backbone angles in absence of favorable interactions enable the disposition of J32 when SAM is not bound to the SMK box.

Destabilization of a duplex-like structure in the apo form: Careful examination of the holo-structure reveals a duplex-like structure formed by two sets of nucleobases that form single strand like architecture: (1) C24, C25 and G26 of P1 near the 5’end, and A64 and G66 of J32; (2) G71 and U72 of P2, adenine base of the ligand, and G90 and G91 of P1 near the 5’-end of the RNA (see Figure 7). SAM ligand, which is strategically positioned in the middle of such a duplex-like form, seems to be essential for the stability. The stabilities of the duplex-like structure in the holo and apo states were elucidated by calculating the π-stacking interactions between adjacent base pair steps that form the duplex-like structure (Figure 7). The inplane interaction energies and hydrogen bond distance distributions of these interactions are 13


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discussed above. The SAM ligand binds to the RNA by interacting with G26 nucleotide, which is the principal recognizing element, via hydrogen bonding interactions. In the holo state, the πstacking interactions between the successive base pair steps are highly favorable indicating stable duplex-like architecture. In the apo state, the absence of the ligand implies absence of a base pair partner for the G26 in the duplex-like arrangement. This is analogous to an abasic site, but without the backbone connecting the two nucleotides (U72 and G26). The missing hydrogen bonded interactions, and the stacking interactions in the middle of the duplex-like structure make it unstable. As the energies given in Figure 7 indicate, the π-stacking is significantly reduced in the apo state. Thus, difference in the stacking interactions show how the presence of the ligand, SAM, leads to the formation of a duplex-like structure, which is energetically, more stable than in the apo form. Furthermore, the absence of ligand disrupts base pairing in U72-A64 and G71-G66 and moves the bases G64 and G66 away. These two bases are part of the trinucleotide bulge J32 and leads to disposition of J32 junction, seen in the MD simulation corresponding to apo system. The formation of the duplex-like structure is proposed to play a crucial part in the stabilization of the RNA in the holo state.

Apo state observed here is the intermediate structure primed for SAM binding: Recent experimental studies using NMR spectroscopy by Wilson et al65 and chemical probing analysis by Lu et al68 suggested three different states during the SAM binding to the SAMIII riboswitch: (a) unbound ON-state in which the SD sequence is exposed; (b) unbound intermediate state resembling the SAM-bound state but with partially exposed SD sequence; and (c) fully folded bound OFF-state where the SD sequence is excluded from the solvent via base pairing to the aSD nucleotides (Figure 8). Experimental studies on the SAM-III riboswitch have shown that the absence of the leader sequence will stall the formation of P0 helix, which is usually formed in the absence of ligand.28 The conformation thus adopted by this riboswitch without its leader sequence in the absence of ligand may correspond to the intermediate state (READY state) in the 14


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transition from ON to OFF state. The RNA used in this study lacks the leader sequence and hence, in the absence of the ligand, SAM, resembles the intermediate state reported in the earlier studies. The experimental studies proposed that this intermediate state is favorable as it has a pre-organized SAM binding pocket, which is stabilized upon binding of the SAM ligand. Comparison of the structural and dynamic details of the apo state modeled here with the experimental data (see above) indicate that the equilibrated structure of the apo state obtained in this study is the intermediate state that is primed for SAM binding.65,68 Previous SHAPE probing analyses have suggested that the apo structure in the ON state converts to the OFF state in presence of the ligand via a conformational intermediate state referred to as the READY state (Figure 8).68 The READY state was proposed to be similar to the OFF state nevertheless with weak intramolecular interactions in the binding site region, P1 and P4 stems, and that the preorganization of the apo form into SAMbound like conformation (READY state) before the ligand binds. Long 0.5 µs apo system simulation further resulted a structure with weak interactions in binding site, P1 and P4 stems similar to READY conformation (Figure S6).The analyses presented above and comparison to the experimental data reveals that the apo structure obtained in the present study seems to be close to the READY state.

Opening of binding pocket in the absence of ligand: Previous NMR spectroscopic studies on the SMK box had suggested that the ligand binding induces major structural changes in the binding region.65 The experimental studies had also revealed that these changes were due to the extensive loss of imino proton signals in the ligand binding region reflecting the reduced stability of this region in the intermediate state formed in the absence of ligand. As mentioned earlier, the apo state used in the present study is comparable to this intermediate state. The binding pocket region includes C25-G90-A73 (the triplet base pair) which is ceiling by A64-U72 base pair and triplet A27-G71-G66 (Figure 9). The interaction between C25-G90 is crucial for SAM binding activity suggested by mutational studies.31 The base 15


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pairing in this triplet increases interactions between J24 and SD-aSD helix in P1. Probability distributions corresponding to the hydrogen bonds of these base pairs are calculated and are shown in Figure 9. These distributions indicate that the base pairs sample the regions corresponding to the distance > 4.5 Å in the apo form which suggest that the base pairs are open in the apo form. This is further supported by the interaction energies calculated for individual base pairs (Figure 3), which is consistent with the NMR imino proton exchange studies. In the holo form, the base pairs sample the regions corresponding to the distances of hydrogen bonds between them. This subsequently leads to compactness of the binding region of the SMK box. When SAM binds to the SMK box, the duplex becomes stable by increasing the stability of the base pairs in binding pocket, which are otherwise open in the absence of ligand. The non-standard base pairs A27-G71-G66 joins the P2 stem with trinucleotide bulge J32 via extensive hydrogen bonds mediated by G71 (Figure 9). As seen in the probability distributions corresponding to the hydrogen bonds, the absence of ligand abolishes the hydrogen bond between these bases, which results in the disposition of J32 junction. This suggests that the opening of binding pocket occurs via disposition in J32 junction followed by opening of P1 stem. The longer time scale simulation of apo1 system indicate that the absence of SAM-III ligand results in the large displacement of J32 that weakens the interactions among the base pairs in the pseudo-duplex, binding pocket and P1 stem. Such opening of the J32 loop results is followed by the distortion of the SD-ASD base pairs thus further destabilizing the P4 stem (Figure S6, S9). A schematic depiction of these conformational changes occurring in apo system is shown in Figure 10. To further elucidate the compactness of the binding region and SMK box in holo and apo forms, radius of gyration calculations were performed (Figure 11). The radius of gyration of the crystal structure of the SMK box containing 53-nt was reported as 18.5 Å65 and, it was also observed that the holo form was more compact than the apo form for both the 51-nt and 59-nt constructs of the SMK box in solution. The probability distributions of radius of gyration indicate that the ligand, SAM, binding transforms the SMK box and binding pocket into more compact structure. Principal 16


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component analysis was performed to understand the differences in the conformational sampling space of the riboswitch in the presence and absence of the ligand. The projections of principal components indicate that the presence of ligand restrict the sampling of conformational space to a smaller region, but samples a large conformational space in the absence of the ligand (Figure S7). The overlap of conformational space of apo system with holo system indicate that the apo riboswitch had also visited the conformations that were sampled by the holo riboswitch (Figure S17, S18), transition between two conformations, thus by indicating the small differences between the holo and apo conformations (READY state) of SMK box. This further supports the earlier discussion that the presence of SAM-III stabilizes the holo conformation than the READY state.

Ligand plays dual role to anchor the ON to OFF switching: Opening of the P1 stem was observed in the absence of ligand SAM in all the apo simulations that are performed here. The binding of SAM leads to the stabilization of the P1 stem through interactions with the G26 base via hydrogen bonds. This direct interaction between cognate-ligand and G26 would add an equivalent base pair AG to P1 stem. This subsequently leads to organize the P1 stem and strengthens the SD-aSD base pairs via strong hydrogen bonding and stacking interactions. Furthermore, the SAM binding also assists in forming duplex-like structure comprising base pairs from P1, P2 and P4 stem regions which blocks the access of SD sequence to ribosome whose recognition is necessary in order to initiate the translation process. This results in termination of the translation process. Similar kind of preorganization in P1 stem during the recognition mechanism was also seen in SAM-I33,35 and adenine riboswitches.37,38 The obtained results suggest a two-fold modular role for the SAM ligand in the switching from ON to OFF state of SAM-III riboswitch. In one side, the ligand enhances the stability of the SD and aSD pairing and blocks the access of SD sequence to ribosome binding. On the other hand, the ligand binding assists in the formation of duplex-like structure, which subsequently blocks the opening of the P1 stem.

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Conclusions MD simulations have been performed on the SAM-III riboswitch (SMK box) in the presence and absence of SAM ligand to identify the key factors that are responsible for switching the conformation of SMK box from ON state to OFF state. While characterizing the ON state is difficult, comparison of the current results with the available experimental results indicate that the apo structures obtained here correspond to the READY state, an intermediate state that connects the ON and OFF states. The results suggest that the transition between the READY and OFF states is associated with structural changes in the J32 junction, binding pocket region, SD and aSD regions. Probability distributions corresponding to hydrogen bonds and base pair interaction energies suggest that the P2 and P3 stems are pre-organized before SAM binding. Multiple MD simulations of apo system indicate disposition of the J32 junction in the absence of SAM ligand, which facilitates the opening of binding pocket. Analysis of the 0.5 µs trajectory of the apo state reveals that while P2 and P3 stems are still preorganized, absence of the ligand leads to loss of the tertiary interactions involving J32 followed by the base pairing interactions of the P1 and P4 stems. This suggests that the binding of SAM increases interactions between bases in SD-aSD sequence favoring the OFF state. Furthermore, the SAM binding assists in the formation of a stable duplexlike structure crowned by J32, as observed in the probability distributions of hydrogen bond distances among base pairs and corresponding interaction energies. The formation of such a duplexlike structure blocks the access of SD sequence to ribosome binding which is a necessary step during the initiation of translation process, thus blocking the translation process. Further, SAM binding to RNA duplex increases the compactness of the duplex consistent with experimental results. The formation of stable duplex-like structure in the presence of SAM ligand and increase in the interactions between SD-aSD base pairs are likely to be the possible factors for this ON-OFF switching of the SAM-III riboswitch in response to the SAM level. The present study further 18


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suggests a dual role for SAM ligand in anchoring the switching process by forming stable duplexlike structure and providing better interactions between SD and aSD sequences.

Funding Information This work was supported by a grant to U.D.P. from Department of Atomic Energy (DAE)-Board of Research in Nuclear Sciences (BRNS), India [Grant: 37(2)/14/05/2015/BRNS/20046].

Supporting Information Available Figures of RMSD, hydrogen bond distances of base pairs in four stem regions, contact maps, hydrogen bond distances of base pairs in triplets, opening of SD-aSD region, principal components of SMK box, and Table of RMSD values of individual regions of SMK box. The same analyses from multiple MD simulations corresponding to apo system are also provided (Figures S8-S19 and Tables S2-S5).This material is available free of charge via the Internet at http://pubs.acs.org.

Abbreviations SAM, S-adenosylmethionine; MD, molecular dynamics; SD, Shine-Dalgarno; RMSD, root mean square deviations; PCA, Principal component analysis

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Figures

Figure 1: The secondary (A) and tertiary (B) structures of the SAM-III riboswitch. The stems and junctions are represented in different colors, and the numbering of the nucleotides is according to the previous studies.31 The SD sequence (G88GGGG92) is highlighted in the secondary structure. The binding pocket is highlighted by transparent surface representation, and the ligand, SAM, is given in stick representation (B).

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Figure 2: Mean rmsd (Å) values of the holo and apo forms of the riboswitch. These values were calculated with respect to the crystal structure (Same data with standard deviation values is given in Table S2).


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Figure 3: Base pair interaction energies (in kcal mol-1) of all the base pairs present in the stem regions (P1, P2, P3 and P4) in SAM-III riboswitch in holo and apo states (Same data with standard deviation values is given in Table S3).


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Figure 4: Probability distributions of hydrogen bond distance of SD and aSD sequences of riboswitch in holo (black) and apo (red) forms.


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Figure 5: Disposition of J32 junction. (A) Alignment of the three dimensional structures of SMK box in holo and apo states. Contact maps showing interactions among nucleobases of SMK box in (B) holo and (C) apo states (see Figure S10 also for other systems). (D) Probability distributions of distances between center masses of nucleotides G66 and G90 in holo and apo states.


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Figure 6: Probability distributions of glycosidic bond angles and phosphodiester backbone angles for nucleotides in J32 of SMK box in its holo (black) and apo (red) states.


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Figure 7: Three dimensional representation of the pseudo-duplex in SAM-III riboswitch. The stacking interaction energies (in kcal mol-1) of successive base pair steps corresponding to holo (blue) and apo (black) are also included.


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Figure 8: Proposed free energy landscape for binding process of SAM ligand to SMK box and proposed ON, READY, OFF and transition states during the binding process.


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Figure 9: Opening of binding pocket. (A) Representation of binding pocket of SMK box including triplets A73:G90:C25 (green), A27:G71:G66 (orange), base pair U72:A64 (pink) along with the SAM ligand. (B) The representation of hydrogen bonding among bases present in two triplets and (C) the probability distributions corresponding to hydrogen bonds among bases forming triplets of RNA in holo (dotted lines) and apo (straight lines) states.


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Figure 10: Conformational changes occurring in SMK box in the absence of the ligand derived from the 0.5µs long MD simulations. ‘a’ is the fully folded structure in the presence of the SAM-III ligand. Removal of the ligand leads to opening of J32 junction (b), then losing interactions at binding site (c), followed by losing interactions in P1 and P4 stems (d & e). The base pairs in P2 and P3 stem regions are largely intact during the course of the simulations.


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Figure 11: Probability distributions of radius of gyration values corresponding to SMK box in holo (black) and apo (red) states.


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Page 38 of 38

Ligand-Induced Stabilization of a Duplex-like Architecture Is Crucial

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