ATOMIC LEVEL INTERACTIONS DISTINGUISHING THE ON AND OFF STATES OF SAM-III RIBOSWITCH

Transcription

1 ATOMIC LEVEL INTERACTIONS DISTINGUISHING THE ON AND OFF STATES OF SAM-III RIBOSWITCH Thesis submitted in partial fulfillment of the requirements for the degree of Master of Science (by Research) In Computational Natural Sciences By Harini Srinivasan Under the guidance of U. Deva Priyakumar Submitted to Center for Computational Natural Sciences and Bioinformatics (CCNSB) INTERNATIONAL INSTITUTE OF INFORMATION TECHNOLOGY Hyderabad , India

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4 ACKNOWLEDGEMENTS I take great pleasure in expressing my gratitude to all the people who have helped me in completing my thesis. First and foremost, I would like to thank my advisor, Dr. U. Deva Priyakumar, for his continuous guidance and support during the course of my research work, without which this thesis would not have been possible. His constant attention to perfection and hard work served as a benchmark for me and constantly reminds me of how one should lead by example. He served as role model by relentlessly supporting and inspiring me. His motivation, enthusiasm, and guidance have helped me complete my tenure of research. I would like to thank the other faculty members of the center Dr. Abhijit Mitra, Dr. Nita Parekh, Dr. Harjinder Singh, Dr. B. Prabhakar, Dr. Marimuthu Krishnan, Dr. S.V. Ramanan, Dr. Tapan Kumar Sau, Dr. Rameshwar (Formerly Professor, ANGRAU) and Dr. Gopalakrishnan Bulusu (Life Sciences R&D, Innovation Labs, TCS Hyderabad) for their constant support, encouragement and invaluable feedback. I would like to thank my group mates Prathyusha, Suresh Gorle, Chinmayee, Siladithya, Ramakrishna and Bikkina Swetha for their support and help during the course of my research. I would also like to thank Koushik and Navneet for always being open for discussions, giving suggestions and creating a competitive atmosphere for pursuing my research. I would like to thank Anubhooti and Pragya Saxena for being there for me at all times and making my stay at IIIT-H memorable. I would like to thank my other batch mates, juniors and other members of the department of CCNSB for providing a friendly atmosphere during my time at this institute. I would like to thank Mr. B. Bal Santosh and Mr. Girish Byrappa, CCNSB administrative assistants, for providing assistance and helping in lab-related issues. I also thank my institute, International Institute of Information Technology Hyderabad and, in particular, the Center for Computational Natural Sciences and Bioinformatics, CCNSB, for providing me with the necessary infrastructure and facilities that has ensured an ideal environment for pursuing the coursework and research. iii

5 I would also like to extend my sincere thanks to my dear friends C. Prashanth and R. Narendran for being by my side always and supporting me. Lastly, I dedicate this thesis to my dear parents, Mr. S. R. Srinivasan and Mrs. Usha Srinivasan and my loving sister Ranjani Srinivasan and thank them for being the most understanding family; without whose moral support, encouragement, push and timely advice, the successful completion of this research work would not have been possible. iv

6 CONTENTS Declaration of authorship Certificate Acknowledgements i ii iii Abstract 1 CHAPTER 1: INTRODUCTION Riboswitches Overview of Riboswitch Structures Mechanism employed for Gene Regulation by Riboswitches Nomenclature of Riboswitches Advantages of Gene Regulation by Riboswitches Riboswitches and the RNA World Riboswitches as Drug Targets Computational studies on Riboswitches Literature Review on SAM-III riboswitches Objective of this Study 28 CHAPTER 2: METHODOLOGY MD Simulation System chosen for this study Simulation Protocol 33 v

7 2.4. Analysis Details 34 CHAPTER 3: RESULTS AND DISCUSSION Structural Properties Riboswitch Level Structural Properties Residue Level Variation in Ligand Binding Region Variation in Shine-Dalgarno and anti Shine-Dalgarno sequence region Formation of Pseudo Duplex Verification of the Translational switch off mechanism 60 Conclusions 62 References 64 vi

8 LIST OF ABBREVIATIONS: A - Adenine AdoCbl - Adenosylcobalamin ASD - Anti Shine-Dalgarno BPTI Bovine Pancreatic Trypsin inhibitor C - Cytosine CHARMM - Chemistry at Harvard Macromolecular Mechanics DNA - Deoxyribonucleic acid FMN - Flavin mononucleotide G - Guanine GlcN6P - Glucosamine-6-phosphate glms - Glucosamine-6-phosphate synthetase GMP - Guanosine monophosphate GTP - Guanosine triphosphate MD - Molecular Dynamics mrna - Messenger RNA NAMD Nanoscale Molecular Dynamics NMR - Nuclear Magnetic Resonance PDB - Protein Data Bank RBS - Ribosome binding site REMD Replica exchange molecular dynamics RFN - Riboflavin mononucleotide RMSD - Root mean square deviation RMSF - Root mean square fluctuation RNA - Ribonucleic acid vii

9 SAH - S-adenosylhomocysteine SAM - S-adenosylmethionine SD - Shine-Dalgarno Se-SAM - Selenium derivitized SAM - [(3S)-3amino-4-hydroxy-4-oxo-butyl]-[[(2S,3S,4R,5R)- 5-(6-aminopurin-9-yl)-3,4-dihydroxy-oxolan-2-yl]methyl]-methyl-selenium SHAPE - Selective 2 -hydroxyl acylation analyzed by primer extension SMD - Steered Molecular Dynamics T - Thymine THF - Tetrahydrofolate TPP - Thiamin pyrophosphate U - Uracil UTR - Untranslated region vdw van der Waals VMD - Visual Molecular Dynamics viii

10 LIST OF FIGURES Figure Number Description Page Number 1 Depiction of the central dogma of molecular biology 3 2 Typical structure and placement of a riboswitch in a mrna 5 3 Mechanisms of gene regulation by riboswitches 6 4 (a) Structure of S-adenosylmethionine (SAM). (b) 8 Structure of S-adenosyl-L-homocysteine (SAH) 5 (a) Secondary structure of SAM-I riboswitch. (b) The 9 three-dimensional structural representation of the SAM-I riboswitch (3IQN) 6 (a) Secondary structure of SAM-II riboswitch. (b) The 10 three-dimensional structural representation of the SAM-II riboswitch (2QWY) 7 Secondary structure of SAM-III riboswitch 11 8 (a) Secondary structure of SAM-III riboswitch used in 12 this study. (b) The three-dimensional structural representation of the SAM-III riboswitch [PDB ID 3E5C] 9 Secondary structure adopted by SAM-III riboswitch (a) in the 13 absence of SAM and (b) in the presence of SAM. 10 Secondary structure of SAM-IV riboswitch Secondary structure of SAM-V riboswitch RMSDs (Å) of the SAM-III riboswitch in the presence and 37 absence of the ligand, SAM 13 RMSD (Å) of binding site residues of SAM-III riboswitch in 38 the presence and absence of the ligand, SAM 14 Probability distribution plot of the radius of gyration calculated for both the apo and holo form 42 ix

11 15 Probability distribution plots of basepair distances calculated between the N1 of purine and N3 of pyrimidine, throughout the simulation, of all the basepairs in the S MK box 16 RMSF (Å) of the apo and holo forms of the S MK box through the simulation 17 Basepair interaction energy (kcal/mol) of the basepairs near the ligand binding pocket in the apo and holo form represented as bar graphs 18 Position of basepairs (25C, 90G) and (27A, 71G) in the riboswitch and its loosening in apo form 19 Position of basepairs (75C, 89G) and (76C, 88G) in the riboswitch and its loosening in apo form 20 Basepair interaction energy (kcal/mol) of the basepairs around J3/2 junction region in the apo and holo form represented as bar graph 21 Position of basepairs (25C, 90G) and (75C, 89G) in the riboswitch and depicting its opening in apo form 22 The pseudo-duplex formation in the holo form which shows the five steps x

12 LIST OF TABLES Table Number Description Page Number 1 Details about adenosylcobalamin riboswitch 17 2 Details about cyclic di-gmp riboswitch 17 3 Details about FMN riboswitch 18 4 Details about glms riboswitch 18 5 Details about glutamine riboswitch 18 6 Details about glycine riboswitch 19 7 Details about lysine riboswitch 19 8 Details about preq riboswitch 20 9 Details about purine riboswitch Details about tetrahydrofolate riboswitch Details about TPP riboswitch Details about SAH riboswitch Details about SAM-SAH riboswitch RMSDs (Å) of the SAM-III riboswitch and its select regions 39 in the presence and absence of the ligand, SAM 15 The radius of gyration (Å) of the S MK box averaged through 41 the simulation 16 Basepair interaction energies (kcal/mol) of all the basepairs in 51 SAM-III riboswitch 17 The stacking interaction energy, only van der Waal s (vdw) 60 contributions, (kcal/mol) between consecutive steps of the pseudo duplex 18 Stacking interaction energies (kcal/mol) of the residues forming the SD and ASD sequence 61 xi

13 ABSTRACT SAM-III riboswitch, found at the 5' untranslated region (UTR) of the MetK gene that translates into SAM synthetase in lactic acid bacteria, is involved in regulating methionine and S- adenosylmethionine (SAM) biosyntheses in these organisms. The riboswitch acts as an on-off switch at the translational level regulated by SAM, wherein the binding of SAM switches off the translation as it blocks the Shine-Dalgarno sequence, thereby preventing the ribosome from initiating translation. The riboswitch distinguishes between SAM and its close analog, S- adenosyl-l-homocysteine (SAH), and binds more tightly with SAM. Experimental studies have reported two mutually exclusive RNA conformations in the presence and absence of ligand and have proposed intermediary state of the riboswitch. The Shine-Dalgarno sequence region is reported to be crucial for both the ligand binding and also acts as the regulatory domain. In this study, we have used molecular dynamics (MD) simulations to understand the structural details of the on-off transition of the riboswitch. The riboswitch is seen to sample larger conformational space in the absence of the ligand compared to the presence of the ligand. The binding pocket and the Shine-Dalgarno - anti Shine-Dalgarno region undergo major structural changes consistent with the experimental data. The off- state of the riboswitch is seen to be stabilized energetically by the formation of a pseudo-duplex like structure comprising the adenine part of the ligand. The phenomenon of translation switch off is verified by using the interaction energies which clearly show that the Shine-Dalgarno sequence region is more stable in the bound form and hence is very difficult to disrupt for the ribosome. 1

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15 INTRODUCTION The central dogma of molecular biology was first illustrated by Francis Crick in which detailed the irreversible sequential information transfer from DNA to RNA and then to protein (Fig 1). RNA is positioned as an intermediate in this transfer, while proteins, being complex and the final product, were believed to be regulators of gene expression and most of the intracellular interactions. But research in the recent years have shown that in spite of being short lived, the RNA molecules are capable of performing regulatory roles within the cells. Further investigation has shown a novel mechanism of gene regulation without the involvement of proteins mediated by RNA molecules. This mechanism is speculated as a remnant of the RNA world 2. In the absence of proteins, the RNA world organisms could have harnessed the structural and functional potential of aptamers, oligonucleotides or peptides that bind to specific target molecules, to create sophisticated regulatory networks. Figure 1: Depiction of the central dogma of molecular biology. 3

16 RNA molecules that are capable of regulating gene expression by responding to physiological signal, including binding of small molecules (vitamin cofactors, amino acids, nucleotides, or metal ions), without the need of proteins are called riboswitches. They are mrna segments that are metabolite-receptive, which control gene expression by interplaying between two alternative conformations in the leading region of the mrna. In order to respond to correct physiological signals each class of riboswitch specifically differentiates its cognate ligand from closely related ones. 1.1 RIBOSWITCHES: Riboswitches were initially discovered in bacteria and most of them known were from bacteria and a few from archaea 3. But later on the most common of the riboswitches, thiamine pyrophosphate (TPP) sensing riboswitches was found in fungal and plant genes, giving evidence of the existence of riboswitches in eukaryotic genome as well 4. Riboswitches are usually found in the 5 untranslated region (5 UTR) of a gene whose translation product takes part in the metabolism or transport of metabolite that controls the riboswitch. Physiological function of the gene regulated by the riboswitch is related to the stringency of the regulation 5. Riboswitches have also been reported to work in tandem in order to confer more finely tuned regulation 6, 7. Riboswitches being highly conserved RNA structures, there has been a pace up in their discovery recently due to the contribution of Bioinformatics in terms of the automated comparative genomics strategy. 1.2 OVERVIEW OF RIBOSWITCH STRUCTURES: Riboswitches, in general, have enough structural complexity to account for two of its important functions, which include (i) identification/recognition of the metabolite and (ii) control of gene expression by conformational switching. Riboswitches usually comprise of two parts, the aptamer part or the sensory domain, which recognizes the metabolite by differentiating it from other similar ones and the expression platform or the regulatory domain, which adopts two exclusive conformations that control the gene expression. Through interplay of two alternative 4

17 conformations, which enables the gene expression to be turned on or off, the metabolite-driven regulation of gene expression is achieved by riboswitches 8. Figure 2: Typical structure and placement of a riboswitch in mrna. The regions of the mrna are labeled and the regions colored cyan and pink denote the sensory and regulatory domain of the riboswitch respectively. 1.3 MECHANISM EMPLOYED FOR GENE REGULATION BY RIBOSWITCHES: Riboswitches can confer regulation at the level of transcription 9, translation 9, and also at the level of splicing 10 in eukaryotes and RNA cleavage Transcription termination: One of the most common ways of gene regulation by riboswitches is through transcription termination, 12 wherein the riboswitch stalls the process of transcription by forming a strong stem followed by a run of uridine residues forming the intrinsic transcription terminator (Fig 3A) 13, 14. The formation of the intrinsic transcription terminator is controlled by the ligand binding to the aptamer, leading to the formation of either an anti-terminator or a competing secondary structure. Bacillus subtilis pbue adenine riboswitch controls gene expression at the transcriptional level where, in the presence of the ligand, adenine, an antiterminator is formed, and hence, transcription of the downstream region occurs. The absence of the ligand, in this riboswitch, promotes the formation of a terminator region, thus leading to the formation of a transcription terminator 15. 5

18 Figure 3: Mechanisms of gene regulation by riboswitches. Image taken from Breaker et al., Translation initiation: The riboswitch can regulate gene expression at the level of translation by controlling the ribosome's access to the ribosome binding site. In this case also binding of ligand regulates the accessibility of the ribosome binding site to the ribosome by either being unpaired or pairing with the anti Shine-Dalgarno (ASD) sequence (Fig 3B). This mechanism is used to regulate gene expression from full length mrnas but it has been proposed that the transcription terminator protein Rho 16 might recognize the mrna not being translated and might cause transcription termination as well. Vibrio vulnificus add riboswitch, another adenine-sensing 6

19 riboswitch, controls gene expression at the translational level. The binding of ligand, adenine, makes the SD and start codon more accessible to the ribosome, while in the absence of ligand due to inaccessibility of SD, translation is blocked Splicing: The coenzyme, TPP riboswitch has been reported to be quite common in plants and fungi 4, regulating the gene expression by controlling the splicing (Fig 3C) 10, In this case, the binding of the ligand controls the splicing by either making the 5' splice site available for splicing or by pairing it up with the anti-splice site. TPP responsive riboswitch in C. reinhardtii present in THIC intron contains two potential splicing acceptor sites. In the presence of TPP, splicing occurs at both these sites and a long transcript with the additional exon (in between the two splice sites) is produced. In the absence of TPP, the first splice site is sequestered, leaving only the downstream acceptor site for splicing, thus completely removing the intron and producing a short transcript that is functional 18. Apart from the above three discussed common mechanisms, riboswitches also use some other rare mechanisms of gene regulation; these include transcription interference or basepairing between sense and antisense transcripts, 21 where the riboswitch is transcribed in the opposite direction of the genes it controls (Fig 3D); dual transcription/translation control, where the riboswitch aptamer lies upstream of the transcription terminator stem that is formed by the SD sequence (Fig 3E); and self cleaving, 11 where ligand binding and ribozyme activities are integrated for gene control (Fig 3F). 1.4 NOMENCLATURE OF RIBOSWITCHES: Riboswitches are classified into different classes based on the sequence, structure conservation and also the metabolite they respond to SAM riboswitch: S-adenosylmethionine (SAM) riboswitches function by responding to the metabolite S- adenosylmethionine (Fig 4(a)) to regulate the biosynthesis of methionine and SAM 22. Five types 7

20 of SAM riboswitches have been identified so far: SAM-I 23, 24, SAM-II 25, SAM-III 26, SAM-IV 27, and SAM-V 28 which respond to the intracellular SAM concentration and control the gene expression. SAM riboswitches are specific to SAM and can differentiate between SAM and its close analog SAH (Fig 4(b)). Figure 4: (a) Structure of S-adenosylmethionine (SAM). (b) Structure of S-adenosyl-L-homocysteine (SAH). SAM-I riboswitches, also called the S-box, were initially found upstream of the genes involved in biosynthesis of methionine, cysteine, SAM and other sulphur containing metabolites in many Gram-positive bacteria with low G+C content 22, and have a complex four-wayjunction architecture consisting of 4 stems, forming a compact U-shape conformation (Fig 5) 32. It controls the gene expression at the level of transcription, repressing the downstream gene expression by a premature transcription termination. The presence of SAM leads to repression of the S-box gene expression by forming a transcription terminator, promoting the termination of transcription 23, 24, 31. Also, S-box motifs have been found in a few Gram-negative bacteria and high G+C content Gram-positive bacteria to function by sequestering the translation initiation region and thereby regulating at the level of translational initiation 33, 34. The S-box is rare in members of the Lactobacillales branch of the Gram-positive bacteria where the genes involved in methionine metabolism are regulated by the S MK box 34, 35. SAM binds to this riboswitch in a compact conformation. This riboswitch distinguishes between SAM and SAH by attracting the 8

21 positive charge on sulphur through a partial negative-charged surface created because of the carbonyl oxygen atoms (O2) of two uracil residues 32. Figure 5: (a) Secondary structure of SAM-I riboswitch. Figure is an adaptation of the original image from Rfam 10.1 database 36. (b) The three-dimensional structural representation of the SAM-I riboswitch (3IQN) showing the ligand, SAM, in CPK and surface representation. SAM-II riboswitches are found mainly in proteobacteria 37 and have a H-type pseudoknot consisting of two loops and two stems (Fig 6) 38. This riboswitch was discovered by comparative sequence analysis of 116 complete bacterial genomes 25. They are predominantly found in α- proteobacteria and some other Gram-negative species but are absent in Gram-positive bacteria. Some SAM-II riboswitches have putative terminator sequences while some have the ability to sequester the SD region. The mechanism of gene regulation in response to SAM level, however, is still not well characterized in this riboswitch 25, 37. SAM binds to this riboswitch in an extended conformation. This riboswitch distinguishes between SAM and SAH by recognizing SAM in part by the positive charge on sulphur bound to carbonyl oxygen atoms (O4) of uridine 37. 9

22 Figure 6: (a) Secondary structure of SAM-II riboswitch. Figure is an adaptation of the original image from Rfam 10.1 database 36. (b) The three-dimensional structural representation of the SAM-II riboswitch (2QWY) showing the ligand, SAM, in CPK and surface representation. SAM-III riboswitches, also called the S MK box, are found in lactic acid bacteria and are involved in regulating methionine and SAM biosynthesis in these organisms 26. It was found in the 5 UTR of the SAM synthetase (metk) gene which is responsible for the synthesis of SAM from methionine and ATP. The binding of SAM ligand to this riboswitch blocks the SD sequence, thereby blocking the translation. The S MK box, found in Lactobacillales species, exhibits no similarity to SAM-I or SAM-II at the primary sequence, secondary structure or tertiary structure level. Previously determined crystal structure of the Enterococcus faecalis S MK box reveals an inverted Y-shaped arrangement, a three-way junction consisting of 4 stems, with a 10

23 set of conserved nucleotides around the SAM binding region (Fig 7) 39. In some cases, there are large insertions up to 200 nucleotides within its structure. Figure 7: Secondary structure of SAM-III riboswitch. Figure is an adaptation of the original image from Rfam 10.1 database 36. Three structures of SAM III riboswitch have been deposited in PDB so far. Each of them consists of a 53nt RNA crystallized with a different ligand: SAM (3E5C) 39, SAH (3E5E) 39 and Se-SAM (3E5F) 39. The structure of SAM-III riboswitch used in this study [PDB ID - 3E5C] has 4 stems: P1 (SD-ASD helix), P2 (middle helix), P3 (top helix) and P4 (linker helix); and 2 junctions: J2/4 and J3/2 (Fig 8) 26, 38. The stems P2 and P3 stack on top of each other to give rise to a long arm. The SD sequence consists of five Gs which span the P1 and P4 stems. 11

24 Figure 8: (a) Secondary structure of SAM-III riboswitch used in this study [PDB ID 3E5C]. The structure consists of 4 stems [P1 (blue), P2 (red), P3 (grey) and P4 (green)] and two junctions [J2/4 (magenta) and J3/2(orange)] colored differently and the Shine-Dalgarno (SD) sequence [G88-G92] and anti Shine-Dalgarno (ASD) sequence [C23-C25 andc75-c76] are highlighted. (b) The three-dimensional structural representation of the SAM-III riboswitch used in this study colored based on the different secondary structural feature [stems and junctions]. The SAM ligand is shown in CPK and surface representation. The three stems around the junction which binds SAM are P1, P2 and P4 stems with junctions J2/4 and J3/2 also forming a part of the binding pocket. The double-strand reversal in the J3/2 bulge results in a conformation that partially encloses the SAM binding site, leading to further stabilization of the three-way junction through base-triple interactions enlarging the major groove of P2 39. The base-triple interaction (A73.G90-C25), where the N1 of A73 accepts a 12

25 hydrogen bond with N2 of G90, forms the floor of the SAM binding pocket. The base triplet ties J2/4 to P1 stem and also orients N6 of A73 for SAM recognition 39. Mutational studies have shown that the SD, ASD regions and the pairing between these regions, P2 stem, and a minimal P3 stem are essential for SAM binding 26. While the overall size of P3 stem and P4 stems were shown not to be critical for SAM binding, they might help fine tuning the affinity of the RNA for SAM 26. The arrangement discussed above has been reported in the presence of SAM, while in the absence of SAM it was reported that the stems P1, P2 and P4 rearrange and form another stem P0 (Fig 9) 26, 40. The region that pairs with a part of P2 stem and J2/4, in the absence of ligand, to form the P0 stem is called the leader sequence. On formation of the P0 stem, the SD and ASD regions become single stranded, allowing the SD to be accessible for translational initiation. In the S MK box, 23 residues are conserved and 10 of these form a part of the pocket formed in the three-way junction, where the adenosine moiety of SAM intercalates 39. Figure 9: Secondary structure adopted by SAM-III riboswitch (a) in the absence of SAM and (b) in the presence of SAM. The leader sequence is denoted in grey, the different secondary structural regions, the Shine-Dalgarno (SD) sequence and anti Shine-Dalgarno (ASD) sequence are labeled to the side. Image taken from Wilson et al.,

26 The SAM-III aptamer has a distinct tertiary structure and binding pocket compared to the other SAM riboswitches previously described due to its distinctive sequence and secondary structure 65. The Enterococcus faecalis S MK box has been reported to bind to SAM with a K d of 0.85 μm, showing greater than 100-fold preference for SAM over its close analog SAH 26, 39, 42. The binding of SAM to this riboswitch causes a structural rearrangement in the riboswitch causing the SD sequence region to be paired up with the anti Shine-Dalgarno (ASD) sequence, thereby making the ribosome binding site (RBS) inaccessible for the ribosome, which in turn blocks the translation initiation. Unlike many other metabolite riboswitches, in the case of SAM- III riboswitch both the aptamer and the regulatory domain coincide. The paring of the SD-ASD sequence has been reported to be required for the binding of SAM 26 and this region also acts as the regulatory domain. Previous genetic and enzymatic probing analyses have shown that the SD sequence of this riboswitch is essential for the binding-site formation and also specific SAM recognition 26, 42. This work deals with understanding the structural changes that accompany the on-off transition of the SAM-III riboswitch. SAM-IV riboswitches adopts a quite distinct scaffold (Fig 10) but the conserved features and experiments on this riboswitch suggest that they share a similar SAM-binding site as the SAM-I riboswitches 126. They regulate the gene expression at the translation initiation level, wherein the terminator stem is replaced by a region that sequesters the RBS 28. These riboswitches were identified by using Bioinformatics techniques 43 and are confined to the Actinomycetales. These were identified using CMfinder comparative genomic pipeline 44 by systematic comparative sequence analysis of intergenic regions of gene families of many bacterial groups. 14

27 Figure 10: Secondary structure of SAM-IV riboswitch. Figure is an adaptation of the original image from Rfam 10.1 database 36. SAM-V riboswitches are found in Pelagibacter ubique and related marine bacteria and carry a putative ligand binding core identical to SAM-II riboswitches 28 (Fig 11). These two classes are suggested to have undergone convergent evolution 28. This riboswitch has been reported to work as a tandem riboswitch which is capable of regulating both at the transcriptional and translational level. When the levels of SAM are high, transcription of gene regulated is terminated, while in low concentration of SAM, translation of the downstream genes is initiated as the RBS is available for the ribosome

28 Figure 11: Secondary structure of SAM-V riboswitch. Figure is an adaptation of the original image from Rfam 10.1 database 36. Each type of SAM riboswitch is seen to exhibit a distinct overall tertiary fold, form a very different binding pocket and interact differently with its cognate ligand, SAM. The hydrogen bonding pattern for recognition of SAM is also different in each one of these types. In case of SAM-I and SAM-II riboswitches,, all the functional groups of SAM are recognized except the reactive methyl group of SAM, while in SAM-III riboswitches, in addition to the reactive methyl group even the methionine moiety (beyond the S atom) is not recognized specifically 40. Some common features include the electrostatic interaction with the positively charged sulphonium moiety, which is the basis of discrimination between SAM and SAH by these riboswitches and stacking interaction of the adenine moiety of SAM with the RNA bases

29 1.4.2 Other riboswitches: Breaker reports that there are a total of 17 riboswitch classes with at least some experimental evidence/validation and a lot more that remain to be discovered 8. Some of the important riboswitches, apart from the different types of SAM riboswiches described above, that respond to different metabolites by regulating the gene expression at the transcriptional or translational level are described in the tables (Table 1 to Table 13) that follow: Table 1: Details about Adenosylcobalamin (or) Cobalamin riboswitch: Riboswitch Name Adenosylcobalamin or Cobalamin riboswitch Metabolite Adenosylcobalamin Organism / Species Bacteria and Eukaryota (2823 species) Genes that it regulates Genes involved in vitamin B 12 synthesis and transport of other metals. Regulation level Both at the transcriptional activation or deactivation level and translational activation level. Secondary Structure adopted Structure is very complex consisting of a set of conserved stems closed by a single base stem. PDB structures -nil- References 36, 46, 47 Table 2: Details about Cyclic di-gmp riboswitch (I & II): Riboswitch Name Cyclic di-gmp ribowitch (I & II) Metabolite Organism / Species Genes that it regulates Regulation level Secondary Structure adopted PDB structures Cyclic di-gmp, a second messenger Bacteria, Eukaryota and Viruses (615, 111 species) Its role is not primarily in regulating metabolism but instead plays a part in signaling by regulating a variety of genes controlled by this second messenger, cyclic GMP. Both at the transcriptional and translational level. Pseudoknotted structure with at least two stems. 19, 2 structures References 36,

30 Table 3: Details about FMN riboswitch: Riboswitch Name FMN riboswitch or RFN element Metabolite FMN Organism / Species Archaea, Bacteria and Eukaryota (3143 species) Genes that it regulates Genes that encode for riboflavin biosynthesis and transport protein. Regulation level At transcriptional level as a terminator and translational level as an initiator. Secondary Structure adopted Four hairpins and a stem joined by a five way junction. PDB structures 16 structures References 36, Table 4: Details about glms riboswitch: Riboswitch Name Glucosamine-6-phosphate activated ribozyme Metabolite Glucosamine-6-phosphate Organism / Species Bacteria (726 species) Genes that it regulates glms gene Regulation level Shows ribozyme activity, glucosamine-6-phosphate initiates the cleavage of glms ribozyme, thereby lowering the production of glms enzyme, encoded by the glms gene. Secondary Structure adopted Double psuedoknotted structure PDB structures 37 structures References 11, 36, 55 Table 5: Details about Glutamine riboswitch: Riboswitch Name Glutamine riboswitch or glna RNA motif Metabolite Glutamine Organism / Species Bacteria and Viruses (52 species) Genes that it regulates Genes involved in glutamine and nitrogen metabolism Regulation level Both at the transcriptional and translational level. Secondary Structure adopted Three-stem junction also called an E-loop structure or bulged-g module. PDB structures -nil- References 36, 56, 57 18

31 Table 6: Details about Glycine riboswitch: Riboswitch Name Glycine riboswitch Metabolite Glycine Organism / Species Bacteria and Eukaryota (2059 species) Genes that it regulates Genes encoded by the gcvt operon that allow glycine to be used as an energy source. Regulation level It has two metabolite-binding domains with similar functions in tandem. Excess glycine level causes binding of glycine to both the aptamer domains and activates genes that facilitated glycine degradation. Secondary Structure adopted Three way junction with a stem, a hairpin and two stems stacked one over the other. PDB structures 21 structures References 6, 36 Table 7: Details about Lysine riboswitch: Riboswitch Name Lysine riboswitch or L box Metabolite Lysine Organism / Species Bacteria and Eukaryota (1354 species) Genes that it regulates Genes involved in lysine metabolism including lysc. Regulation level In gram-positive organisms as a transcription terminator and in gramnegative organisms as a translational initiator. Secondary Structure adopted Three helical and two helical bundles connected by a five-way junction PDB structures 14 structures References 36, 58, 59 19

32 Table 8: Details about PreQ riboswitch (I & II): Riboswitch Name PreQ1 and PreQ1-II riboswitch Metabolite Pre-queosine 1 (7-aminomethyl-7-deazaguanine) Organism / Species Bacteria (647, 423 species) Genes that it regulates Genes involved in the biosynthesis of nucleoside queuosine from GTP. Regulation level At the level of transcription as a transcription terminator. Secondary Structure adopted H-type pseudoknot structure, predicted to have four stems PDB structures 5, 0 structures References 36, Table 9: Details about Purine riboswitch: Riboswitch Name Purine riboswitch Metabolite Adenine and Guanine Organism / Species Bacteria and Eukaryota (1123 species) Genes that it regulates Genes involved in purine transport and purine nucleotide synthesis Regulation level Both at the transcriptional activation or deactivation level and translational activation level. Secondary Structure adopted Three way junction, with paired regions (P1, P2 and P3), joining regions (J1/2, J2/3, J3/1) and loop regions (L2 and L3). PDB structures 26 structures References 36,

33 Table 10: Details about Tetrahydrofolate riboswitch: Riboswitch Name Tetrahydrofolate riboswitch or THF riboswitch Metabolite Tetrahydrofolate (THF) Organism / Species Bacteria and Eukaryota (508 species) Genes that it regulates Genes that encode either folate transporters or enzymes involved in folate metabolism Regulation level Both at transcriptional or translational level. Secondary Structure adopted Inverted three-way junction with long-range tertiary pseudoknot interaction. Comprises of four helical segments joined by an internal loop and a three-way junction. PDB structures 7 structures References 36, 66, 67 Table 11: Details about TPP riboswitch: Riboswitch Name TPP riboswitch or THI element Metabolite Thiamine pyrophosphate (TPP), an active form of Vitamin B 1 Organism / Species Archaea, Bacteria, and Eukaryota (4095 species) Genes that it regulates Regulates genes involved in thiamine biosysnthesis and transport. Regulation level Both at the transcriptional activation or deactivation level and translational activation level. Secondary Structure adopted Hairpin structure formed by two stems, one interior loop and a hairpin loop. PDB structures 18 structures References 4, 10, 19, 36, 68, 69 21

34 Table 12: Details about SAH riboswitch: Riboswitch Name SAH riboswitch Metabolite S-adenosylhomocysteine (SAH) Organism / Species Bacteria and Eukaryota (313 species) Genes that it regulates Up regulate genes involved in recycling SAH to create more SAM Regulation level Both transcriptional and translational level. Secondary Structure adopted Pseudoknot architecture formed at the interface between the three stems. PDB structures -nil- References 36, 70, 71 Table 13: Details about SAM-SAH riboswitch: Riboswitch Name SAM-SAH riboswitch Metabolite S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) Organism / Species Bacteria (58 species) Genes that it regulates Regulate SAM synthesis Regulation level At the translational level as a initiator Secondary Structure adopted Consists of a stem-loop structure and SD sequence forms a pseudoknot architecture with the terminal loop within the main stemloop region. PDB structures -nil- References 36, ADVANTAGES OF GENE REGULATION BY RIBOSWITCHES: Proteins are generally considered to be more versatile than RNAs as they are made up of 20 amino acid building blocks while RNAs consist of a combination of just four nucleotides. But studies on RNA sensors have shown that despite simpler composition they resemble proteins in many ways 9. Like proteins, RNA sensors, especially riboswitches interact with small molecules with high affinity and specificity. RNA sensors, like riboswitches, have an advantage over their protein counterparts as direct sensing of metabolites by the mrna eliminates the need for regulatory proteins and so saves energy and resources 9. Also, as RNA are short-lived molecules, for the control of gene expression, binding of the metabolite to the riboswitch is required only for 22

35 a short period of time, within which, the binding is sensed and signals are sent. There is no involvement of any other gene product, thus reducing the effect of mutations on control of gene expression 11, 73. Some riboswitches have been reported to be kinetically driven based on the concentration of the ligand to trigger the genetic control rather than being thermodynamically driven 74, 75. This can be advantageous as they can be tuned to change the rate constant for ligand association by mutating the aptamer residues and thus respond differently with change in concentrations of metabolite 8, RIBOSWITCHES AND THE RNA WORLD: Riboswitches represent one of the oldest regulatory systems 77 and have for long been speculated as descendants of the RNA-based sensors used for regulation of gene expression in the RNA world organisms. However, all riboswitches are not necessarily direct descendants from homologous riboswitches from the RNA world. Some riboswitch classes like the TPP, AdoCbl, and FMN are very widespread 12 and have complex aptamer structures which lead to speculations of them having an ancient origin. This is because such complex and intricate structures are unlikely to emerge frequently during evolution. While in contrast, some riboswitches have narrower distribution in the phylogeny and less complex aptamer structures which could be recent emergences in the evolutionary tree 8. It is also speculated that widespread riboswitches with less complex structures could simply have been reinvented many times during evolution rather than direct descent from the ancient ancestor. Another interesting hypothesis is that some riboswitch aptamers may be descendents from the ancient ribozymes that synthesized the particular metabolite RIBOSWITCHES AS DRUG TARGETS: For a very long time, RNA was considered as just an intermediate between DNA and proteins. But the discovery that RNA molecules can control gene expression by sensing small molecules has changed that perception. RNA molecules are now prime target for most antibiotics 78. Discovery of riboswitches that regulate gene expression based on metabolite 23

36 binding increased the hope on the chances of targeting RNA successfully. Riboswitches act like a switch and over 20 families of riboswitches from the three domains of life (bacteria, archaea, and eukaryotes) have been reported so far 79. And also it has been reported that over 4% of genes in some bacteria are regulated by riboswitches 80. Hence riboswitches are a new gateway to targeting RNA as they can serve as druggable molecular switches and alternatives for the traditional protein targets. Some natural antimicrobials were reported to target riboswitches which include pyrithiamine pyrophosphate which binds to a bacterial TPP riboswitch 81, roseoflavin that targets bacterial FMN riboswitch 82 causing gene repression. Also, it has been reported that in some bacterial pathogens, riboswitches regulate genes essential for their survival and virulence 83. Hence, riboswitches could serve as novel targets to the new generation of drugs, by targeting the ligand (metabolite) binding pocket. Development of metabolite analogs can help control the gene expression externally/artificially that can prove lethal to the invading cell and advantageous to the host cell. This can be exploited to develop a new generation of antibacterials and antifungals targeting the riboswitches to help relieve the current resistance crisis. 1.8 COMPUTATIONAL STUDIES ON RIBOSWITCHES: Previous studies on different riboswitches helped understand the structural features, their interaction with the ligand leading to stabilization and mechanism of action. Studies on SAM-I riboswitch have assisted in understanding the route of entry of SAM ligand into the riboswitch and how it interacts to stabilize the riboswitch in that conformation 84. Also, mutational studies have revealed that certain modifications in the SAM-binding region locked this riboswitch in a form similar to the bound form and hence resulted in the loss of SAM binding capability 84. In another study, simulations on SAM-I riboswitch observed that the folding of the nonlocal stem (P1) is rate-limiting in the aptamer domain formation which is assisted by SAM, by lowering the associated free energy barrier 85. Studies have also indicated that the aptamer of this riboswitch follows a two-step hierarchical folding that is selectively induced by metal ions and ligand binding and helped characterize the ligand-controlled riboswitch folding pathway extensively 86. Computational studies showed the change in RNA dynamics on addition of Mg 2+ to SAM-I riboswitch through ten 2μs MD simulations 87. Computational studies have shown modification of the curvature and base-pairing of the expression platform on binding of the ligand to SAM-II 24

37 riboswitch which affects its interaction with the ribosome 88. Studies carried out by Doshi et al., identified the molecular basis for ligand recognition and specificity of SAM-II riboswitch 89. It was reported that SAM or SAH bind to SAM-II riboswitch by following a combination of conformational selection and induced-fit mechanism 89. Previous studies on adenine riboswitch characterized the ligand interaction with the riboswitch, and how the ligand binding stabilizes the riboswitch 90, 91. Lin et al., studied the unfolding and refolding of add riboswitch aptamer and thereby characterized the energy landscape 92. Multiple folding states were identified and it was reported that binding of adenine stabilizes the folded structure, thereby significantly decreasing the unfolding rate 92. MD simulations gave insights into structural organization of add A-riboswitch in the ligand-bound, free and mutated states 93. Studies on guanine riboswitch have shown the structural changes in the riboswitch on binding to guanine (its cognate ligand), adenine and in the absence of ligand. The study also characterized the interactions of the riboswitch with the ligands and the associated conformational changes in the riboswitch 94. Computational studies showed that long-range interactions play an important role in the induced-fit binding of the ligand in guanine sensing riboswitches 95. Nonequilibrium MD simulations of the temperature-induced energy flow in guanine riboswitch indicate anisotropic transfer of energy from the ligand, guanine, to its neighboring residues 95. Long-range energy transfer between the ligand and the distant kissing loop has been identified and special correlations up to distance of 4nm were identified 95. MD studies suggest a possible two-step ligand recognition process in guanine sensing riboswitches, where the ligand binds first and then subsequently selection of the cognate ligand occurs 94. Nonequilibrium simulations deciphered the initial steps of ligand unbinding in the guanine sensing riboswitches 94. The atomistic details of the interaction of amino-purine, aminopyrimidines and imidazole derivative with a mutant guanine riboswitch were studied using MD simulations 96. This identified the range of compounds that riboswitches can specifically bind and thus can help when targeting them for antibiotic therapeutics 96. Phylogenetic analysis and comparative genomics have been used to map purine riboswitches across all bacterial groups by identifying the class of genes/operons regulated by them and also recognize their origin during evolution

38 Recent study reported the crucial interactions and structural basis for the regulation of gene expression by glycine riboswitch 98. Computational modeling approaches in combination with experimental procedures identified models of the ligand-free state of the TPP riboswitch 99. Molecular dynamics simulations and free energy calculations identified the role of GlcN6P in the self-cleavage of glms and found that the amine group of GlcN6P is responsible for the phdependent binding to glms 100. MD simulations revealed the protonation states of the key active site residues in glms riboswitch and thereby helped propose a mechanism for self-cleavage 100. Dynamical hinge points, virtual torsion angles, were identified in the conformational switch transition from the inactive to active state of the L1 ligase, which can contribute to understanding the conformational switch mechanism of the L1 ligase 102. Computational studies along with experimental procedures suggested that the apo form of preq1 riboswitch is structured but does not possess a ligand-binding pocket and characterized the conformational changes in the aptamer upon ligand binding 103. Two different preq1 aptamers were studied and it was identified that they have similar folding or unfolding pathways and ligand binding processes, though they have different bound/unbound states and regulatory functions 104. NMR studies and REMD simulations characterized the 12 nucleotide single stranded RNA tail derived from prequeuosine riboswitch which adopts an A-form-like conformation, with the level of order decreasing towards the terminal ends LITERATURE REVIEW ON SAM-III RIBOSWITCHES: The S MK box was identified by examining the metk genes in lactic acid bacteria, including Enterococcus, Streptococcus and Lactococcus species 26. This examination was driven by the fact that a similar conserved region was found in the Gram-positive bacteria, which controlled the methionine and cysteine biosynthesis genes, named as S-box 27. It has been seen that mutations that disrupt the pairing of the SD sequence with its complementary sequence blocked the binding of SAM, while compensatory mutations that restored the pairing, reinstated the binding of SAM 26. Translational repression of the lacz gene (used as a reporter) was observed when fused with the S MK box and grown under conditions of elevated SAM pools and loss of repression was observed when mutations resulting in loss of SAM binding were incorporated. These confirm the functioning of the SAM-III riboswitch in vivo 26. Further studies showed that the SD-ASD pairing 26

39 required for SAM binding helped the riboswitch to function in translational repression by blocking the access of the ribosome to the RBS 42. Experimental studies have shown that SAM- III riboswitch binds to SAM more tightly than SAH by exploiting the positive charge on sulphur 39. Fluorescence binding assays were used to demonstrate the rapid switching of the metk leader between the SAM-bound and SAM-free conformations in response to fluctuating levels of SAM. This demonstrated that the riboswitch can function as a reversible switch taking multiple SAM-dependent regulatory decisions during the lifetime of the transcript to modulate the expression of the SAM synthetase gene, in vivo 40. Differentially populated, distinct mutually exclusive RNA conformations of the S MK box were reported in the presence and absence of SAM by NMR spectroscopic studies 41. This study also reported the thermodynamic relationship between the bound, unbound and their proposed intermediary state of the S MK box 41. Mutational study based on single-nucleotide substitutions in a hypervariable region outside SAM binding region helped to explore the folding landscape of the S MK box and revealed a two step folding/ unfolding process followed by this riboswitch 106. We have recently reported the molecular recognition mechanism of the SAM-III riboswitch using molecular dynamics simulation 107. Differentiation of SAM over SAH was attributed primarily to the nonspecific interaction of the sulphonium ion with the electronegative atoms (O and N) in the vicinity of the binding pocket. SAM acts as a coenzyme for the methylation reactions, which lead to the formation of SAH that is toxic to the cells 108, 109. The SAH formed needs to be selectively recycled to regenerate SAM. SAM, being a principal methyl donor, is of fundamental importance for the metabolism of polyamines, nucleic acids and the precursor of amiopropyl groups and glutathione 108, 110. SAM is also found to regulate the activities of various other enzymes. It is involved in transmethylation, transsulfuration and aminopropylation. It has been shown that SAH is a potent inhibitor for the SAM-dependent methyltransferase reactions 111. It has also been seen in mice models that elevated SAH pools alone or in combination with reduced SAM pools is associated with DNA hypomethylation 112. Furthermore, decrease in SAM:SAH ratio is seen to reduce methylation capacity only when associated with high SAH pools, while that associated with SAM pool depletion alone was not sufficient to influence the DNA methylation 112. Consequently, SAM-III riboswitch which regulates the synthesis of SAM in lactic acid bacteria can serve as a good target in case of SAM-aided disease modifications as targeting it can control the levels of SAM 27

40 synthesized as required. Also, targeting SAM and SAH riboswitches simultaneously can help to control the toxicity levels in the cell OBJECTIVE OF THIS STUDY: The purpose of this study is to understand how the SAM-III riboswitch functions by switching conformations and thereby controls the gene expression. The study focuses on the various structural changes and the differences between the two conformations. We have used MD simulations to look into and understand the differences in the riboswitch in the presence and absence of SAM, the structural features that uniquely identify each of them, and the possible series of steps in the transition from one state to another. 28

41 CHAPTER 2 29

42 METHODOLOGY Alder and Wainwright were the first to introduce molecular dynamics method in the late 1950 s to study interactions of hard spheres 113, 114. Their studies helped uncover many important insights regarding the behavior of simple liquids. In 1964, for the first time realistic potentials were used to simulate a system of liquid argon by Rahman 115. The first molecular dynamics simulation on a realistic system of liquid water was done in 1974 by Rahman and Stillinger 116. Later in 1977, first protein simulation was done on bovine pancreatic trypsin inhibitor (BPTI) 117. Today, simulations are performed on solvated systems of proteins, protein-ligand, DNA, RNA, lipids and complexes of these as well to study various aspects including thermodynamics of ligand binding, folding of protein, and behavior of biomolecules in different environments. 2.1 MD SIMULATION: In molecular dynamics, simulation is performed by integrating Newton s equations of motion over a small time step. In such a simulation, the initial positions of all the atoms are assigned from crystal structure, wherever available. Velocities are assigned randomly from Maxwell- Boltzmann distribution at the desired temperature. Forces on atoms are computed using an appropriately selected force field. Different integration algorithms such as Verlet, Velocity Verlet, Leap-frog, Beeman s use the current positions, velocities and forces to compute the position and velocity of the atoms, thereby advancing the simulation by one time step. And the cycle repeats to find the position of the atoms at every time step, thus generating a trajectory, a time dependent location for each atom, over a period of time. MD simulations generate information at microscopic level, which include atomic positions and velocities that can be converted to macroscopic observables, like energy, heat capacity using statistical mechanics. The general form of the force field equation is: E tot = E bonded + E nonbonded where E bonded = E bond + E angle + E dihedral and E nonbonded = E electrostatic + E van der Waals 30

43 The total energy contribution (E tot ) is divided into bonded term (E bonded ) and non-bonded term (E nonbonded ). The bonded part consists of contributions from bond stretch (E bond ), angle bends (E angle ), and variation in dihedral angle (E dihedral ). The non-bonded part consists of contribution from electrostatic interactions (E electrostatic ) and van der Waals interactions (E van derwaals ). Additional terms are included to account for the improper torsions, cross-terms, etc depending on the force field used. In the present study, CHARMM all atom nucleic acid force field has been used. The energy function in CHARMM is: U CHARMM = U bonded + U nonbonded where U bonded = U bond + U angle + U UB + U dihedral + U improper with U bond = K b (b - b 0 ) 2 U angle = K θ (θ - θ 0 ) 2 U UB = K UB (b b 1-3, 0 ) 2 U dihedral = K φ ((1 + cos(nφ - δ)) U improper = K ω (ω - ω 0 ) 2 and U nonbonded = U LJ + U elec with U LJ = ε ij [(r min ij /r ij ) 12 2(r min ij /r ij ) 6 ] U elec = [q i q j /εr ij ] The energy function is divided into two parts: the bonded (U bonded ) and the non-bonded (U nonbonded ) part. The first term in the bonded part accounts for the bond stretches (U bond ), the second for the bond angles (U angle ) and the fourth for the dihedrals (U dihedral ). The terms K b, K θ, K φ correspond to the bond, angle, and dihedral force constant respectively while b, θ, φ correspond to the bond length, bond angle between 3 bonded atoms and dihedral angle between 4 bonded atoms at a particular instant respectively. The terms b 0, θ 0 correspond to the equilibrium bond length and bond angle respectively. n in the dihedral term refers to the multiplicity while δ 31

44 is the phase shift. The third term in the bonded part, Urey-Bradley term (U UB ), is considered for 3 bonded atoms where K UB is the force constant for the Urey-Bradley term, b 1-3 is the distance between atoms 1 and 3 at a particular instant and b 1-3,0 is the equilibrium distance. The fifth term in the bonded part accounts for impropers or out of plane bending (U improper ) where K ω is the force constant and ω ω 0 is the out of plane bend angle from the equilibrium between the 4 atoms. The non-bonded part consists of the van der Waals contribution (U LJ ), which is calculated with a standard 12-6 Lennard-Jones potential and electrostatic contribution. Nonbonded interactions are calculated for all atom pairs except for the covalently bonded atom pairs (1,2 pairs) and atom pairs separated by two covalent bonds (1,3 pairs). The term ε is the relative dielectric constant, r ij is the distance between atom i and j, r min ij is the distance at which the LJ term has its minimum, and q is partial charge. The values of equilibrium terms (b 0, θ 0, etc), the various force constants (K b, K θ, etc) and partial charges (q i ) are taken from the force field parameters. In CHARMM 118, ε ij = (ε i ε j ) 0.5 and r min ij = (r min i + r min j )/2 The energy terms given above has one addition compared to the standard terms of molecular mechanics force fields, which is the Urey-Bradley (U UB ) term. Urey-Bradley term is important for in-plane deformations and for separating symmetric and asymmetric bond stretching modes in aliphatic molecules. MD simulation provides an insight into the time-dependent behavior of a molecular system, which helps understand the conformational changes and fluctuations in biomolecules. One of the biggest advantages of simulation is the level of detail that one can get from the simulation and often simulation can give insights to trends that are not experimentally measurable. Also, simulations can help save resources which include physical resources required to build the system of study and money, though they require computational power. One of the major limitations of simulations is the dependency on force fields i.e. a simulation results are only as good as the underlying force field used. Also simulations neglect electronic motions and quantum effects and consider only nuclear motions i.e., it cannot be used to study processes of bond breaking and formation. Descriptions of interatomic forces can be improved by incorporating features such as salt effects or ph to better mimic experimental conditions 99,

45 2.2 SYSTEM CHOSEN FOR THIS STUDY: SAM-III riboswitches were initially identified in lactic acid bacteria in It is considered to have an inverted Y-shaped arrangement 65. The structure of SAM-III riboswitch used in this study [PDB ID - 3E5C] has 4 stems: P1 (20-25 and 90-95), P2 (26-31 and 67-71), P3 (32-63) and P4 (75-89); and 2 junctions: J2/4 (72-74) and J3/2 (64-66) 53. The numbers in the parenthesis indicate the residue numbers. The PDB structure 3E5C contains the crystal structure of SAM-III riboswitch bound to the ligand SAM, which is used as the initial structure for bound form, holo simulation. The initial structure for the unbound form, referred as apo form, simulation is obtained by removing the ligand from the final structure after simulating the bound form for 50 ns. This structure is subjected to the preparatory steps, including energy minimization, to provide an environment similar to that in the unbound form. This is used as the starting structure to simulate the apo form of the riboswitch. 2.3 SIMULATION PROTOCOL: The MD simulations in this study were performed using CHARMM 118 and NAMD 119 biomolecular simulation programs using CHARMM 27 all atom force field. The parameters for SAM are available along with the CHARMM27 biomolecular force fields 120. Two simulations lasting for 50 ns each were carried out in this study, which include that of the riboswitch in the presence of ligand, referred to as holo, and that of the riboswitch in the absence of ligand, referred to as apo. Hydrogen atoms were added to the initial structure of the riboswitch obtained from PDB in standard orientations using Hbuild utility of CHARMM. The simulations were performed using modified TIP3P water model 121, as explicit solvent environment, and periodic boundary conditions. The systems were solvated in a truncated octahedron TIP3P water box. The solvent molecules within 2.2 Å of the non-hydrogen atoms of the riboswitch or ligand were removed in both the systems. A padding of 9 Å was taken on all sides and then the size of the water box was chosen for the systems. The crystallographic water molecules were retained and the hydrogens 33

46 were added to them using the Hbuild utility of CHARMM. The 15 crystallographic Sr 2+ ions were replaced by 15 Mg 2+ ions. The systems were neutralized by placing positively charged ions at random positions within the water box. 25 Mg 2+ ions and 1 Na + ion is added to the holo system, while 26 Mg 2+ ions is added to the apo system, to neutralize the systems. The solvated systems were then subjected to 500 steps of adopted basis Newton-Raphson (ABNR) minimization. MD simulations in the NVT ensemble were performed for 100 ps as equilibration. All the non-hydrogen atoms of the RNA and ligand were restrained using harmonic constraints of 10 kcal/mol/å 2 during both minimization and equilibration. The systems were then allowed to undergo another 500-step adopted basis Newton-Raphson minimization after the restraints were removed. And further the systems were allowed for a production simulation for 50 ns with a timestep of 2 fs. Steps up to the initial minimization and the analysis of the trajectories were performed using the CHARMM program, while the production simulations were performed in NAMD using NPT ensemble with Leapfrog integrator. Hoover thermostat is used for temperature control and Langevin piston method is used to maintain a pressure of 1 atm with a piston mass of 600 amu. Long range electrostatics interactions were calculated using particle mesh Ewald method 122, 123. All covalent bonds involving hydrogens were constrained using SHAKE algorithm 124, which allowed using an integration time of 2 fs during the simulation. 2.4 ANALYSIS DETAILS: The coordinates were saved after every 5 ps of the production simulation for the purpose of further analysis. Trajectories used for analysis include the RNA molecule and the ligand alone, if applicable, without the water molecules and ions. All the analyses presented here were performed on the entire trajectory of 50 ns. The entire 50 ns of the simulation time had 10,000 time frames and all the calculations done were averaged over each of the time frames, except the RMSD calculations which show the deviation as a function of time. The rotational and translational motions of the RNA in the trajectory were removed by superimposing all the nonhydrogen atoms of the riboswitch onto those of the X-ray crystal structure. The interaction energies presented here were calculated using the INTER command in CHARMM program, which include both the electrostatic and van der Waals contributions, using 34

47 infinite non-bond cutoffs. RMSDs were calculated following least-square fitting of all the nonhydrogen atoms in the structures being analyzed. The RMSD values signify the extent of deviation of the structures obtained through the MD simulations from the reference structure, which in this case is the X-ray crystal structure of the riboswitch obtained from PDB. RMSDs were also calculated for individual secondary structure elements or regions (SD, ASD, binding site, etc) by superposing all the non-hydrogen atoms in that region alone. The residues which have one or more atoms within a cutoff distance of 3Å from the ligand, SAM, in the original PDB structure [3E5C] were considered as the binding site residues. The RMSF values presented here give the fluctuation of each of the nucleotides in comparison to the average structure from the trajectories. The probability distribution of the hydrogen bond distances presented in this study are generated by using the command COOR MINDIST in CHARMM program to calculate the distance between the hydrogen bond donor and acceptor and then the probability distribution plots of these distances were generated. The radius of gyration is calculated by using the GYRA command in CHARMM. The structure presented in this study were created using VMD 125 and the graphs were plotted using the Grace plotting tool. 35

48 CHAPTER 3 36

49 RESULTS AND DISCUSSION The analysis of trajectories from the MD simulations of the aptamer, in the presence (holo form) and absence (apo form) of the ligand, primarily concentrate on differentiating the two and thereby trying to identify the unique structural features of each of the conformations. 3.1 STRUCTURAL PROPERTIES RIBOSWITCH LEVEL: The root mean square deviations (RMSDs) through the simulation as a comparison between the holo and apo forms, by aligning the entire riboswitch with the crystal structure used in this study, 3E5C, is illustrated in Figure 12. The figure shows that the two systems have stabilized after the initial 5ns and that the holo system seems to have a lesser deviation from the reference structure than the apo system. Figure 12: RMSDs (Å) of the SAM-III riboswitch in the presence (red) and absence (black) of the ligand, SAM, with respect to the crystallographic structure 3E5C. 37

50 To understand the differences between the structures in greater details, we looked into the contribution to the RMSD from the structurally important regions (binding site and SD-ASD) and each of the secondary structure regions individually. Figure 13 shows the RMSD of the binding site region throughout the simulation time, as a comparison between the holo and apo forms of the riboswitch by aligning the entire riboswitch. Figure 13: RMSD (Å) of binding site residues of SAM-III riboswitch in the presence (red) and absence (black) of the ligand, SAM, with respect to the crystallographic structure 3E5C. Residues containing one or more atoms within a cutoff distance of 3Å from the ligand, SAM, are considered as binding site residues. The graph clearly shows that the binding site regions show lesser deviations in the holo form, which is contributed to the presence of the ligand that interacts with the binding site residues. The tabulated values from Table 14 indicate that though the overall structure is similar in the holo and apo forms, there are variations in the ligand binding region which are discussed in the later sections. 38

51 Table 14 presents the RMSDs in the holo and apo forms by aligning the entire riboswitch structure and each of the individual secondary structure regions of the riboswitch as shown earlier. Also the residues lying in the binding site region and the bases forming the SD and ASD sequences were aligned separately and the RMSDs were calculated. The RMSD calculations are done taking the crystal structure, 3E5C, as reference structure for the calculation. The residues in the binding site region were defined by considering all the residues, which contain one or more atoms within a cutoff distance of 3Å from the ligand, SAM. Table 14: RMSDs (Å) of the SAM-III riboswitch and its select regions in the presence and absence of the ligand, SAM, with respect to the crystallographic structure 3E5C. Region holo a holo b apo a apo b Full Structure P P P P J2/ J3/ SD ASD Binding Site Residues a - The structures were aligned based on all the non-hydrogen atoms of the riboswitch. b - The structures were aligned based on the non-hydrogen atoms of the region for which the RMSDs were calculated. 39

52 These values clearly show that the holo form is more stable than the apo form. Greater deviations are seen in regions close to the ligand binding which include the P1, P2 and P4 stem, SD and ASD sequence and J3/2 junction. The small difference in the RMSD values of P3 stem for both the apo and holo form indicates that the P3 stem mostly retains its conformation without much disturbance. The RMSD value for the residues in the binding site region is suggestive of the fact that the presence of ligand stabilizes the binding site. RMSD of the holo form shows that the holo form is closer to the experimental structure than the apo form, which signifies that the apo form samples larger conformational space. In the holo form, the RMSD of all regions lie within acceptable range of values (< 3.5Å) except for the P1 stem, which is a consequence of the opening of the 2 terminal basepairs. In the holo form, we can see from the table that the RMSD values are low (< 2.5 Å) for each of the selected regions of the riboswitch when aligned with respect to itself, contrast to the global alignment. This suggests rigid body motions of the regions with respect to each other. In the apo form, though the RMSD values indicate similar rigid body motions, there are major deviations in the P4 stem and the SD- ASD region, in addition to the P1 stem. However, the P2 and P3 stem regions are largely intact in the case of apo. Experimental studies on the S MK box have shown that the absence of the leader sequence will stall the formation of P0 stem (Fig 9), which is usually formed in the absence of the ligand 41. The conformation thus adopted by this S MK box without its leader sequence in the absence of the ligand is proposed as an intermediate state in the transition from on- to off- state. This intermediate state is favorable for SAM binding as it has a pre-organized SAM binding pocket, though energetically it is less favorable than the on- state. SAM is then found to selectively stabilize this intermediate state through ligand-mediated interactions and drives the equilibrium towards the holo form 41, 106. The RNA used in this study lacks the leader sequence and hence, in the absence of the ligand, SAM, it resembles the intermediate state reported in the earlier studies. As a result of this we don t see any major conformational changes in the secondary structure of the riboswitch between the apo and holo forms. From the RMSD data presented above in Table 14, it can be seen that the difference in RMSD in the individual secondary structures, on aligning with themselves, is less than 1 Å. This indicates that there are no major conformational changes that have taken place in the secondary structural regions in the transition from apo to holo. 40

53 Previous experimental studies on the S MK box show that the holo and apo form used in this study, resembling the off and the intermediate state of the riboswitch, maintain the overall structure, but the variation is observed in the ligand binding region 41. These results are consistent with those from the present study indicated by the RMSD values. To elucidate the difference in compactness of the S MK box in the presence and absence of ligand, SAM, radius of gyration calculations were performed. The average radius of gyration values of the S MK box in the presence and absence of ligand, considering only the heavy atoms in the molecule, are presented in Table 15. Table 15: The radius of gyration (Å) of the S MK box averaged through the simulation System S MK box (All Atoms) Apo Holo The tabulated values, clearly, show that the S MK box is more compact in the presence of ligand. The radius of gyration values, calculated by averaging through the simulation, is comparable to that reported in previous study 41. The radius of gyration of the crystal structure of the S MK box containing 53-nt was reported as 18.5 Å in the presence of ligand, SAM 41. They also reported that in solution, both in the 51-nt and 59-nt construct of the S MK box, the holo form was more compact than the apo form consistent with our present result. 41

54 Figure 14: Probability distribution plot of the radius of gyration calculated for both the apo (black) and holo (red) form, considering only the heavy atoms in the S MK box, through the simulation. The green line marks the experimental value reported for the 53-nt X-ray crystal structure containing the ligand, SAM 41. The probability distribution plots of the radius of gyration are shown in Figure 14. The plot shows that the radius of gyration of the riboswitch is lesser in the presence of the ligand, i.e., in the holo form. The plot also shows the experimentally reported value for the radius of gyration of the riboswitch, in the presence of ligand, as a green line 41. From the figure, it can be seen that the experimentally determined radius of gyration value, in the presence of ligand, coincides with the peak of the frequency distribution of the holo form. This indicates that for the maximum amount of time in the simulation, the holo form has a radius of gyration which is almost equivalent to that determined experimentally. 3.2 STRUCTURAL PROPERTIES RESIDUE LEVEL: The basepairs in the riboswitch were studied by calculating the distance between the N1 of purine and N3 of pyrimidine of each of the basepairs. The basepair distances were monitored throughout the simulation. The probability distribution of the distances between the 19 basepairs 42

55 in the riboswitch is presented in Figure 15. The distance between the pairs of bases in the loops at the end of P3 stem (38G, 57A) and P4 stem (77G, 87A) were also monitored throughout the simulation and their probability distribution are presented in Figure

56 44

57 45

58 Figure 15: Probability distribution plots of basepair distances calculated between the N1 of purine and N3 of pyrimidine, throughout the simulation, of all the basepairs in the S MK box. The basepair (20G, 95U) is seen to predominantly sample greater distances and this is because of the opening up of the terminal residues (Fig 15 (a)). (21U and 94A), being the penultimate one, is seen to sample greater distances in the holo form as a result of the opening up of the terminal basepair (Fig 15 (b)). (22U, 93A) is seen to sample greater distances in the apo form (Fig 15 (c)). (23C, 92G) and (24C, 91G) have overlapping probability distribution curves with a peak at around 3Å indicating that there is strong basepairing between these basepairs (Fig 15 (d) (e)). (25C, 95G) is seen to have a peak around 3Å in the holo form while in the apo form it sample greater distances which indicates that in the latter it loses the basepairing (Fig 15 (f)). (27A, 71G) is seen to sample greater distances than the usual 3Å as it is non-watson Crick basepairing and is also seen to open up in the apo form (Fig 15 (g)). (28A, 70U), (29A, 69U) and 46

59 (30G, 68C) are seen to have overlapping probability distribution plots with peak at 3Å indicating that these basepairs are preserved in both the apo and holo forms (Fig 15 (h) (i) (j)). (31G, 67C), (32A, 63G) and (33U, 62A) are seen to sample greater distances in the holo form when compared to the apo form signifying that in the former, it loses the basepairing, while in the latter basepairing exists (Fig 15 (k) (l) (m)). (34G, 61C), (35G, 60C), (36C, 59G), (37G, 58C) are seen to sample similar distances with overlying probability distribution graphs indicating that the basepairs are maintained in both the apo and holo forms (Fig 15 (n) (o) (p) (q)). (38G, 57A) samples almost same distance in both the forms but is seen to sample distances greater than 3Å as it is a non-watson Crick basepair (Fig 15 (r)). (75C, 89G) is seen to sample greater distances in the apo form, which indicates that in the apo form the basepairing between these bases is lost while in the holo form it is maintained (Fig 15 (s)). (76C, 88G) is seen to have a slight shift in the probability distribution curve peak in the apo form but in both the cases the peak lies within 3.5Å and hence the basepairing is preserved (Fig 15 (t)). The shift in the peak in the apo form to a slightly larger distance can be attributed as an effect of the moving apart of (75C, 89G). (77G, 87A) is seen to sample almost same distance in the two forms while the peak lies around 10Å as a result of the non-watson Crick basepairing (Fig 15 (u)). NMR spectroscopic study on a 51-nt construct of the S MK box 41, which is similar to the 53-nt RNA used in this study, has revealed 13 exchangeable imino proton resonances in the presence of SAM. These signals are observed only if the imino proton is protected from the solvent and hence are direct indicators of the base-pairing interactions. Out of the possible 21 imino protons participating in base-pairing, only 11 signals were observed at 25 C and the rest are proposed to not yield observable signals due to local breathing motions in the RNA. These results were compared with the results from the present study. The eleven signals observed in the holo form at 25 C include those from 30G, 31G, 34G, 35G, 37G, 59G, 69U, 88G, 89G, 91G and 92G, while in the apo form, two of them are lost which include 69U and 89G. The basepairing of 8 out of the 9 signals reported are seen to be preserved while the basepairing of 31G is lost in the holo form according to the present study. This is due to the rearrangement that takes place in the P2 stem and J3/2, so as to make the latter a part of the ligand binding site. This is explained in detail in the following section where the variations in the ligand binding region are explained. The basepairing of 89G is seen to be lost in the apo form even in the present study, while that of 69U is seen to be preserved contrasting to that reported in the NMR study. 47

60 The root mean square fluctuation (RMSF) of each of the residues throughout the simulation is calculated in the apo and holo form and presented in Figure 16. The difference in the RMSF between the apo and holo form is also presented in the Figure 16. Figure 16: RMSF (Å) of the apo (black) and holo (red) forms of the S MK box through the simulation. The difference between the RMSF of apo and holo form is shown in green color. The orange line denotes zero mark signifying equal fluctuation in holo and apo forms. The five regions circled in orange denote the regions discussed in Lu et al., as protected regions on binding of the ligand, SAM, which include anti Shine-Dalgarno (ASD), SAM site (SS), J3/2 and P4 stem. The strip shown below the graph shows the secondary structure region that each of the residue falls in and is colored accordingly (P1 in blue, P2 in red, P3 in grey, P4 in green, J3/2 in orange, and J2/4 in violet). The residues in one strand of the P1 stem show greater fluctuation in apo form while those in the other strand show greater fluctuations in the holo form. The residues in P2 and P3 48

61 stem predominantly show greater fluctuations in holo form while those in J3/2 show greater fluctuations in apo form. The initial residue of J2/4, 72U, has almost similar fluctuations in both the forms but residues 73A and 74A have greater fluctuations in holo form. The residues of the P4 stem have predominantly greater fluctuation in the holo form with the exception of 78A, and 86A which have higher fluctuation in apo form. The junction J3/2, regions P1 and P4 stems are seen to mainly have higher fluctuation in the apo form while in the holo form they have lesser fluctuation due to their interaction with the ligand. Selective 2 -hydroxyl acylation analyzed by primer extension (SHAPE) experiments on the S MK box have provided significant insights into the conformational flexibility of the on and off states 106. The study included selective destabilization of the ON state (apo form) by introduction of mutations called as READY state (intermediates). The most stable READY state which includes introduction of a Poly A tail in place of the leader sequence and using a perfect linker in P4 adopts a conformation closest to the apo form used in our study. Thus, this mutant in the presence and absence of SAM is the one that closely mimics the conformation of holo and apo form used in this study. So the SHAPE experiment results on this mutant can be compared to the RMSF differences observed in our study. The results of the SHAPE experiment for this mutant that closely resemble the apo and holo form used in the present study, indicate that residues belonging to 4 sets: ASD sequence, SAM-site, J3/2 and P4 stem, denoted as orange circled regions in Figure 16, are protected in the presence of SAM due to SAM induced structural changes in the RNA. This signifies that the residues in these regions have greater fluctuation in the apo form when compared to the holo form and hence the difference in RMSF shown in Figure 16 is positive for them. According to the study 23C, 24C, 25C, the ASD sequence, have greater fluctuations in apo form, which is consistent with the results from the present study that shows that the difference is positive in the case of these 3 residues. The residues 26G, 27A, 72U, 73A and 74A form the SAM-site, wherein for residues 26G, 27A and 72U it is seen that the apo form has greater fluctuation consistent with the experimental results, while in the case of 73A and 74A the fluctuation is greater in the holo form. The residues 64A, 65U, 66G form a part of the J3/2 or the U-bulge and in these residues we find that 65U has greater fluctuation in apo form, while the other two have almost same fluctuation in both the holo and apo forms. The residues 78A and 86A in the P4 stem are seen to have higher fluctuation in apo form, while 76C and 89G have greater fluctuation in holo form. The other residues in P4 stem have almost equal 49

62 fluctuations in both the apo and holo form. This variation in the fluctuations in the P4 stem can be attributed to both the opening of basepairs in the apo form and also breathing motion in the presence of ligand in the holo form. 3.3 VARIATION IN LIGAND BINDING REGION: The major overall change that is seen during the comparison of the apo and holo form is the opening up of the basepairs in P1, P2 and P4 stems, next to the binding pocket. Generally, in riboswitches, a clear trend is observed on comparing the energy contributions from the holo and apo forms i.e., one form is clearly more stable than the other. But in the case of our present study, on the S MK box comparing the riboswitch in the presence and absence of SAM, we are unable to find uniform energy contribution from the entire structure towards the stabilization of the holo form. From experimental studies, we know that the holo form of the riboswitch is more stable than the apo form 106. But, the basepair interaction energies presented in Table 16 show that in the P1 stem and P4 stem, the holo form is more stable than the apo form due to lower interaction energies, except for the terminal basepair of P1 stem (21U-94A). In the case of P2 and P3 stems, some basepairs have lower interaction energy in apo form while others have in holo form. This implies that certain parts of the P2 and P3 stems are more stable in apo form while others are in holo form. Thus, there is no uniform pattern of energy contributions in the riboswitch overall. 50

63 Table 16: Basepair interaction energies (kcal/mol) of all the basepairs in SAM-III riboswitch Secondary Structure Basepair holo apo Element P1 stem 21U-94A P1 stem 22U-93A P1 stem 23C-92G P1 stem 24C-91G P1 stem 25C-90G Average P2 stem 27A-71G P2 stem 28A-70U P2 stem 29A-69U P2 stem 30G-68C P2 stem 31G-67C Average P3 stem 32A-63G P3 stem 33U-62A P3 stem 34G-61C P3 stem 35G-60C P3 stem 36C-59G P3 stem 37G-58C Average P4 stem 75C-89G P4 stem 76C-88G Average But on closely looking at the pattern of the energy contributions, it is seen that the major changes indicate a collective motion of the basepairs surrounding the ligand binding region (in P1, P2 and P4 stems) moving away from each other in the apo form and basepairs close to the J3/2 junction moving away from each other in the holo form. In the absence of ligand, it is seen 51

64 that the basepairs close to the ligand binding region loosen out. The interaction energy between the basepairs (25C, 90G), which is the terminal basepair of P1 stem, (27A, 71G), which is the initial basepair in the P2 stem, and (75C, 89G) & (76C, 88G), which are the initial basepairs in the P4 stem, are much lower in the holo form when compared to the apo form as shown in the bar graphs in Figure 17. Figure 17: Basepair interaction energy (kcal/mol) of the basepairs near the ligand binding pocket in the apo and holo form represented as bar graphs. This is indicative of the loosening of these basepairs in the apo form (Figure 18 and 19). The probability distribution of the basepair distances for these 4 basepairs as presented earlier in Figure 15 (f), (g), (s), and (t) also show that in the apo form these basepairs sample larger distances more than shorter ones, supporting the idea of loosening of these basepairs. 52

65 Figure 18: The position of basepairs (25C, 90G) and (27A, 71G) in the riboswitch, where the riboswitch is rendered in tube representation while the basepairs in apo and holo form are rendered in sticks with colors red and blue respectively. The inset image shows the exact positioning of the basepairs, with distances, depicting the loosening of the basepairs in the apo form. 53

66 Figure 19: The position of basepairs (75C, 89G) and (76C, 88G) in the riboswitch, where the riboswitch is rendered in tube representation while the basepairs in apo and holo form are rendered in sticks with colors red and blue respectively. The inset image shows the exact positioning of the basepairs, with distances, depicting the loosening of the basepairs in the apo form. The basepairs (31G, 67C), (32A, 63G) and (33U, 62A), surrounding the J3/2 junction, are seen to be more stable in the apo form than in the holo form, as in the latter they rearrange slightly in order to facilitate J3/2 to be a part of the ligand binding site. The basepair interaction energies of these basepairs are depicted as bar graphs in Figure