How Tertiary Interactions Between the L2 and L3 Loops Affect the Dynamics of the Distant Ligand Binding Site in the Guanine Sensing Riboswitch

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1 How Tertiary Interactions Between the L2 and L3 Loops Affect the Dynamics of the Distant Ligand Binding Site in the Guanine Sensing Riboswitch Christian A. Hanke and Holger Gohlke Institute for Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University, Düsseldorf, Germany {christian.hanke, In order to investigate how tertiary interactions in the L2/L3 loop region of the guanine sensing riboswitch aptamer domain (Gsw) affect the domains ability to bind ligands, molecular dynamics simulations of wildtype Gsw and a G37A/C61U mutant of in total 9 µs length are performed. The simulations reveal a dynamic coupling between the loop region and the distant ligand binding site suggesting that there exists a complex pathway for the transmission of stability information through the aptamer domain. This finding may have important implications for understanding how Gsw functions at a molecular level. 1 Introduction Riboswitches are cis-acting mrna regulatory elements that modulate gene expression through their ability of binding small molecules with high specificity. They are mostly located in the 5 -untranslated region of bacterial mrna 1 3 and usually consist of two domains: the aptamer domain, which binds the ligand molecule, and the expression platform, which undergoes conformational changes upon binding of the ligand and, thereby, determines the expression of the genes under its control. Riboswitches in bacteria can act either on the translational or transcriptional level. In the latter case, the expression platform is involved in the formation of an intrinsic terminator or antiterminator, leading to the termination or activation of transcription, respectively. In order for such a riboswitch to be an effective regulator of gene expression, a decision must be made at a branchpoint during transcription in favor of one of the two folding pathways 4. This requires the unbound state of the riboswitch to maintain ligand-binding competence but at the same time to be able to follow the default pathway in the absence of the ligand. Another level of complexity arises from the observation that riboswitches involved in transcription regulation function through primarily a kinetically controlled mechanism 5, that is, the aptamer does not reach equilibrium between the unbound and ligand-bound state before the genetic decision. For understanding how the regulatory decision made at the branchpoint leads to one of the two structural states and the role of kinetic discrimination in this, the nature of the unbound riboswitch state and the folding pathways must be known in atomic detail. The guanine sensing riboswitch is one of the smallest riboswitches known, involved in transcription regulation, and experimentally well studied 6. Crystal structures of the guanine sensing riboswitch aptamer domain (Gsw) from the xpt-pbux operon of B. subtilis in published in Proceedings of the NIC Symposium 2014, Binder, K., Münster, G., Kremer, M. (eds.), Jülich

2 the ligand-bound state 7 revealed that the Gsw is built from three paired regions (P1, P2 and P3), two loops (L2 and L3) capping the P2 and P3 region and forming tertiary interactions, and three joining regions (J1/2, J2/3 and J3/1) connecting the paired regions and forming the ligand binding site (Figure 1A), in which the ligand is deeply buried 7. The stable tertiary interactions between the L2 and the L3 loop are important for the structural stability of the Gsw as well as for its ligand binding ability: Mutations that replaced L2 and L3 with stable UUCG tetraloops, that way eliminating the tertiary interactions, abolished the ligand binding ability of the Gsw 7. Introducing a destabilizing G37A/C61U double mutation in the loop region resulted in a structure displaying a Mg 2+ concentration dependence of the formation of the loop-loop interactions as well as of the ligand binding ability Information in atomic detail how formation and stability of the loop-loop interactions affect the ligand binding ability in the Gsw has remained elusive, however. To this end, we performed explicit solvent molecular dynamics (MD) simulations of in total 9 µs length of the wild type (Gsw apt ) and the G37A/C61U mutant (Gsw loop ) of the guanine sensing riboswitch aptamer domain in the ligand-unbound state at different Mg 2+ concentrations. These simulations reveal a dynamic coupling between the loop region and the distant ligand binding site, suggesting that there exists a pathway transferring stability information through the Gsw. 2 Methods The starting structure for the MD simulations of Gsw apt was taken from the X-ray structure of the B. subtilis guanine sensing riboswitch aptamer domain bound to hypoxanthine (PDB code 4FE5 7 ). The starting structure for the MD simulations of Gsw loop was taken from the X-ray structure of this mutant bound to thioguanine (PDB code 3RKF 10 ). In both cases, the ligands were removed as were all ions and water molecules found in the structures. For both structures, three simulation systems with different Mg 2+ concentrations (0, 12 and 20 Mg 2+ ions per Gsw) were set up according to experimental findings on the Mg 2+ dependence of Gsw loop properties In order to allow for a sufficient equilibration of the Mg 2+ ions, which may be hampered by the slow exchange times of first shell ligands of Mg 2+11, the Mg 2+ ions were initially placed as hexahydrated complexes. These systems were then solvated using TIP3P water molecules, resulting in system sizes of 50,000 atoms. The parm99 force field 12 was used for the Gsw. For each of the six systems, three independent MD simulations at 300 K of 500 ns length each were performed with the GPU version of pmemd 13 of the Amber package 14 summing up to a total simulation time of 9 µs. 3 Results and Discussion Mg 2+ ions initially show a high mobility during MD simulations and occupy sites in very good agreement with those found in X-ray structures In order to investigate the Mg 2+ dependence of the formation of the loop-loop interactions and the ligand binding ability in Gsw apt and Gsw loop, we performed MD simulations with three different Mg 2+ /Gsw ratios (0, 12, 20 Mg 2+ ions per Gsw). Our careful initial placement of the Mg 2+ ions (see Methods section) resulted in all ions showing a pronounced 100

3 mobility in the first 100 ns of the simulations (Figure 1B), with several ions remaining mobile even after 500 ns (data not shown). This indicates that the ions can sufficiently equilibrate prior to making direct contacts with the Gsw, which is important considering the slow exchange kinetics of first shell ligands of Mg2+ on the order of µs11. In addition, we occasionally observed Mg2+ ions swapping their positions (data not shown). As a result, preferred occupation sites of the ions identified in the simulations are in very good agreement with those found in X-ray structures7, 10 (Figure 1C). These findings show that our setup of the Mg2+ ions results in simulation systems that should be well suited for investigating the influence of the concentration of Mg2+ ions on the structure and dynamics of the Gsw. A C B M L Figure 1. A: Structure of the Gsw bound to hypoxanthine (magenta spheres); the Gsw structure is colored according to secondary structure elements, which were assigned according to ref. 7: grey: P1; green: P2; orange: P3; red: L2; blue: L3; yellow: J1/2; cyan: J2/3; brown: J3/1. M marks the area of the G37A/C61U mutation; L marks the ligand binding site. B: Positions of 12 Mg2+ ions from an MD simulation over a simulation time of 100 ns; colors correspond to different Mg2+ ions; RNA in grey. C: Comparison of preferred sites of occupancy of Mg2+ ions during 100 ns of MD simulation (red) to experimentally determined Mg2+ binding sites (green/magenta: binding sites of cobalt hexammine ions in X-ray structures with PDB ID: 4FE57 /3RKF10 ); grey: RNA. The G37A/C61U mutation destabilizes the native hydrogen bond network in the loop region In MD simulations of Gswapt, we observe that the hydrogen bond network connecting the L2 and L3 loops is stable (data not shown). In contrast, in MD simulations of Gswloop, hydrogen bonds involved in the loop-loop interactions repeatedly break and then partially reform during the simulation time (data not shown). Thus, our MD simulations confirm the local destabilizing effect of the G37A/C61U mutation on the hydrogen bond network connecting the L2 and L3 loops8, which allows us to use Gswloop as a model system to investigate the influence of the stability of the loop-loop interactions on the overall structure and dynamics of the Gsw. 101

4 The G37A/C61U mutation leads to overall structural destabilization and increases the dynamics of Gsw loop, which is counteracted by the presence of Mg 2+ ions In order to investigate how the destabilization of the native hydrogen bond network in the loop region by the G37A/C61U mutation affects the overall structure and dynamics of the Gsw in a Mg 2+ -dependent manner 9, we simulated Gsw apt and Gsw loop in the presence of 0, 12 and 20 Mg 2+ ions per Gsw molecule. For reasons of space limitations, we only report results for 0 and 20 Mg 2+ per Gsw here. In the presence of Mg 2+ ions, the mean radius of gyration of both Gsw apt and Gsw loop is smaller by 1 Å than in the absence of Mg 2+ ions (Figure 2A), demonstrating a higher compactness of the Gsw structures in the former case. This arises from the Mg 2+ ions preferentially occupying the space between the RNA backbones, which decreases the electrostatic repulsion between the phosphate groups. Notably, the mean radii of gyration obtained from the MD simulations in the presence of Mg 2+ ions differ by < 0.1 Å with respect to the radii of gyration computed for the respective X-ray structures, which also contained Mg 2+ or [Co(NH 3 ) 6 ] 3+ ions (data not shown). Regarding the dynamics of the systems, Gsw loop shows a higher mobility than Gsw apt as demonstrated by mean root mean square fluctuations (RMSF) that are larger by 0.5 Å in the former case (Figure 2B). In the presence of Mg 2+ ions, mean RMSF are observed that are lower by 0.5 Å (0.7 Å) for Gsw apt (Gsw loop ) than in the absence of Mg 2+ (Figure 2B). This results in the mean RMSF of Gsw loop only being larger by 0.3 Å than that of Gsw apt in the presence of Mg 2+ ions then. In summary, these findings indicate that the mutation in Gsw loop does not influence the gross structure of the RNA but rather destabilizes the RNA compared to Gsw apt, which results in an increased dynamics in the case of Gsw loop. This destabilizing effect can be counteracted by the presence of Mg 2+ ions, with a stronger influence of Mg 2+ observed in the case of Gsw loop. This result is in agreement with experimental findings according to which the ability to bind a ligand is restored for Gsw loop if Mg 2+ ions are present 9. The destabilizing effect of the G37A/C61U mutation is most pronounced in the distant ligand binding site In order to gain insights which parts of the Gsw structures are most influenced in terms of the dynamics by the G37A/C61U mutation and the presence of Mg 2+ ions, we compared differences in RMSF values on a per-residue level (Figure 3). Regarding Gsw loop and Gsw apt (Figure 3A), the most pronounced difference is found for residue 74, which is part of the joining region J3/1, more than 25 Å away from the loop region, and has been found crucial for RNA-ligand interactions 15 : this residue s RMSF values are 1.5 Å higher in simulations for Gsw loop than for Gsw apt in the absence of Mg 2+ ions. The second largest differences are found for the joining region J2/3 (Figure 3A), which opposes residue 74 and has been implicated to act as an entrance gate to the ligand binding pocket 15. This region is also the most flexible one on an absolute scale in all simulations (except for the P1 region, which contains the termini of the RNA strands; Figure 2B), which is in agreement with experiments 16, 17. The third largest difference is found for the joining region J1/2, which is also part of the ligand binding site 7. Except for residue 66, which is part of L3 in the loop region, all other differences in the RMSF values between Gsw loop and Gsw apt are marginal (< 1 Å). 102

5 ARadiusSofSgyrationS[SÅS] Å 16.2 Å 17.5 Å 16.4 Å TimeS[SnsS] TimeS[SnsS] B RMSFS[SÅS] P1 J1/2 P2 L2 P2 J2/3 P3 L3 P3 J3/1 P1 P1 J1/2 P2 L2 P Å 1.19 Å 2.22 Å 1.49 Å J2/3 P3 L3 P3 J3/1 P Residues Residues Figure 2. Comparison of the radius of gyration and RMSF for simulations of Gsw apt and Gsw loop in the absence (red) and presence of 20 Mg 2+ ions per Gsw (blue) A: Radius of gyration of the Gsw without the P1 region for simulations of Gsw apt (left) and Gsw loop (right); insets contain the mean radii of gyration, the SEM is 0.002; B: RMSF per residue for simulations of Gsw apt (left) and Gsw loop (right); residues belonging to the P1 region, which show large fraying motions, were omitted for the calculation of the RMSF; insets contain the mean RMSF, the SEM is < Secondary structure elements were assigned according to ref. 7. Regarding the influence of the presence of 20 Mg 2+ ions per Gsw versus the absence of Mg 2+, only small (< 1 Å) differences in the per-residue RMSF values are found for Gsw apt, particularly in regions J1/2, J2/3, and the loop region (Figure 3B). In contrast, pronounced differences occur for Gsw loop, with the largest influence of Mg 2+ exerted on the regions J3/1 (residue 74 shows the overall largest difference of 2.3 Å), J2/3, J1/2, and part of L3 (Figure 3C), i.e. those regions that become most destabilized due to the G37A/C61U mutation. The increased mobility of residues involved in ligand binding in Gsw loop versus Gsw apt provides an explanation why Gsw loop is not able to bind the ligand productively in the absence of Mg 2+ ions; at the same time, these residues show the largest decrease in the mobility upon addition of Mg 2+, which explains why the binding ability is restored in the presence of these ions 9. 4 Conclusion We performed explicit solvent molecular dynamics simulations of the wildtype Gsw and its G37A/C61U mutant in the ligand-unbound state at three different Mg 2+ ion concentra- 103

6 A B C >= Difference in RMSF [ Å ] <= Figure 3. Difference in RMSF values determined for each residue projected on the RNA. A: Gsw loop - Gsw apt in the absence of Mg 2+ ions; residue numbers are denoted; B: Gsw apt with 0 Mg 2+ - Gsw apt with 20 Mg 2+ ions; C: Gsw loop with 0 Mg 2+ - Gsw loop with 20 Mg 2+ ions. tions in order to investigate how tertiary interactions in the L2/L3 loop region affect Gsw s ability to bind ligands. Initially, we validated our simulation setup by monitoring Mg 2+ ion mobility, sites in the Gsw occupied by Mg 2+ ions, and the local hydrogen bond network in the loop region. On a global scale, we observed that the G37A/C61U mutation leads to overall structural destabilization and increases the dynamics of Gsw loop, which is counteracted by the presence of Mg 2+ ions. In contrast, structural differences between Gsw apt and Gsw loop are small. On a local scale, the destabilizing effect of the G37A/C61U mutation is most pronounced in the distant ligand binding site, and the presence of the Mg 2+ ions restores the stability of this site almost to the level of Gsw apt. These findings yield a possible explanation on an atomic level as to why Gsw loop is not able to bind the ligand productively in the absence of Mg 2+ ions but can do so in the presence of Mg 2+, as observed experimentally 9. Our findings furthermore reveal a long-range transmission of stability information through the Gsw from the loop region to the ligand binding site, which is 25 Å away. This suggests that both sites are dynamically coupled. This may have important implications for understanding how Gsw functions at a molecular level. Thus, we will next set out to characterize the pathway of information flow through the Gsw applying Constraint Network Analysis 18, which was successfully used for predicting signal transmission pathways in the ribosomal exit tunnel already 19. Acknowledgments We gratefully acknowledge the computing time granted by the John von Neumann Institute for Computing (NIC) and provided on the supercomputer JUROPA at Jülich Supercomputing Centre (JSC) (NIC project 4722). Additional computational support was 104

7 provided by the Center for Information and Media Technology (ZIM) at the Heinrich- Heine-University of Düsseldorf (Germany). References 1. H. Schwalbe, J. Buck, B. Fürtig, J. Noeske, and J. Wöhnert, Structures of RNA switches: insight into molecular recognition and tertiary structure, Angewandte Chemie International Edition 46, , A. Roth, and R. R. Breaker, The structural and functional diversity of metabolitebinding riboswitches, Annual Review of Biochemistry 78, , R. K. Montange, and R. T. Batey, Riboswitches: emerging themes in RNA structure and function, Annual Review of Biophysics 37, , C. D. Stoddard, R. K. Montange, S. P. Hennelly, R. P. Rambo, K. Y. Sanbonmatsu, and R. T. Batey, Free state conformational sampling of the SAM-I riboswitch aptamer domain, Structure 18, , M. Sharma, G. Bulusu, and A. Mitra, MD simulations of ligand-bound and ligandfree aptamer: Molecular level insights into the binding and switching mechanism of the add A-riboswitch, RNA 15, , R. T. Batey, Structure and mechanism of purine-binding riboswitches, Quarterly reviews of biophysics 45, , R. T. Batey, S. D. Gilbert, and R. K. Montange, Structure of a natural guanineresponsive riboswitch complexed with the metabolite hypoxanthine, Nature 432, , J. Noeske, J. Buck, B. Fürtig, H. R. Nasiri, H. Schwalbe, and J. Wöhnert, Interplay of induced fit and preorganization in the ligand induced folding of the aptamer domain of the guanine binding riboswitch, Nucleic Acids Research 35, , J. Buck, J. Noeske, J. Wöhnert, and H. Schwalbe, Dissecting the influence of Mg 2+ on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain, Nucleic Acids Research 38, , J. Buck, A. Wacker, E. Warkentin, J. Wöhnert, J. Wirmer-Bartoschek, and H. Schwalbe, Influence of ground-state structure and Mg 2+ binding on folding kinetics of the guanine-sensing riboswitch aptamer domain, Nucleic Acids Research 39, , H. Ohtaki, and T. Radnai, Structure and dynamics of hydrated ions, Chemical Reviews 93, , V. Hornak, R. Abel, A. Okur, B. Strockbine, A. Roitberg, and C. Simmerling, Comparison of multiple Amber force fields and development of improved protein backbone parameters, Proteins: Structure, Function, and Bioinformatics 65, , A. W. Goetz, M. J. Williamson, D. Xu, D. Poole, S. Le Grand, and R. C. Walker, Routine Microsecond Molecular Dynamics Simulations with AMBER on GPUs. 1. Generalized Born, Journal of Chemical Theory and Computation 8, , D. A. Case, T. E. Cheatham 3rd, T. Darden, H. Gohlke, R. Luo, K. M. Merz Jr., A. Onufriev, C. Simmerling, B. Wang, and R. J. Woods, The Amber biomolecular simulation programs, Journal of Computational Chemistry 26, , S. D. Gilbert, C. D. Stoddard, S. J. Wise, and R. T. Batey, Thermodynamic and kinetic characterization of ligand binding to the purine riboswitch aptamer domain, Journal 105

8 of Molecular Biology 359, , C. D. Stoddard, S. D. Gilbert, and R. T. Batey, Ligand-dependent folding of the threeway junction in the purine riboswitch, RNA 14, , C. D. Stoddard, J. Widmann, J. J. Trausch, J. G. Marcano-Velazquez, R. Knight, and R. T. Batey, Nucleotides adjacent to the ligand-binding pocket are linked to activity tuning in the purine riboswitch, Journal of Molecular Biology 425, , C. Pfleger, P. C. Rathi, D. L. Klein, S. Radestock, H. Gohlke, Constraint Network Analysis (CNA): a Python software package for efficiently linking biomacromolecular structure, flexibility, (thermo-)stability, and function, Journal of Chemical Information and Modeling 53, , S. Fulle, H. Gohlke, Statics of the ribosomal exit tunnel: implications for cotranslational peptide folding, elongation regulation, and antibiotics binding, Journal of Molecular Biology 387, ,