Crystal Structure of the Lysine Riboswitch Regulatory mRNA Element

Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8Å resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding. Riboswitches are metabolite-sensitive elements found in mRNAs that control gene expression through a regulatory secondary structural switch. Along with regulation of lysine biosynthetic genes, mutations within the lysine-responsive riboswitch (L-box) play a role in the acquisition of resistance to antimicrobial lysine analogs. To understand the structural basis for lysine binding, we have determined the 2.8Å resolution crystal structure of lysine bound to the Thermotoga maritima asd lysine riboswitch ligand-binding domain. The structure reveals a complex architecture scaffolding a binding pocket completely enveloping lysine. Mutations conferring antimicrobial resistance cluster around this site as well as highly conserved long range interactions, indicating that they disrupt lysine binding or proper folding of the RNA. Comparison of the free and bound forms by x-ray crystallography, small angle x-ray scattering, and chemical probing reveals almost identical structures, indicating that lysine induces only limited and local conformational changes upon binding. Small non-protein-coding RNAs and mRNA sequences play a central role in cellular regulatory processes and are involved in virtually every aspect of the maintenance and transmission of genetic information. One prevalent form of riboregulation in bacteria is the riboswitch; at least 4% of all genes in Bacillus subtilis and related species are controlled in this fashion (1Irnov Kertsburg A. Winkler W.C. Cold Spring Harbor Symp. Quant. Biol. 2006; 71: 239-249Crossref PubMed Scopus (24) Google Scholar). This non-protein-coding element exerts genetic control in a cis-fashion by interacting with a cellular metabolite, thereby directing formation of one of two mutually exclusive mRNA secondary structures (reviewed in Ref. 2Winkler W.C. Breaker R.R. Annu. Rev. Microbiol. 2005; 59: 487-517Crossref PubMed Scopus (566) Google Scholar). Depending upon their placement within the mRNA, riboswitches control transcription or translation in bacteria (3Barrick J.E. Breaker R.R. Genome Biol. 2007; 8: R239Crossref PubMed Scopus (354) Google Scholar) and alternative splicing or mRNA stability in eukarya (4Cheah M.T. Wachter A. Sudarsan N. Breaker R.R. Nature. 2007; 447: 497-500Crossref PubMed Scopus (326) Google Scholar, 5Wachter A. Tunc-Ozdemir M. Grove B.C. Green P.J. Shintani D.K. Breaker R.R. Plant Cell. 2007; 19: 3437-3450Crossref PubMed Scopus (239) Google Scholar). Currently, there at least 20 distinct families of riboswitches that recognize a diverse set of metabolites including nucleo-bases, sugars, vitamin cofactors, amino acids, and metal ions (2Winkler W.C. Breaker R.R. Annu. Rev. Microbiol. 2005; 59: 487-517Crossref PubMed Scopus (566) Google Scholar). The lysine-binding riboswitch is of particular interest for several reasons. Although in vitro selection methods are capable of generating aptamers to an equally diverse set of compounds (6Wilson D.S. Szostak J.W. Annu. Rev. Biochem. 1999; 68: 611-647Crossref PubMed Scopus (975) Google Scholar), one of the few molecules for which an aptamer has failed to be raised is lysine (7Famulok M. J. Am. Chem. Soc. 1994; 116: 1698-1706Crossref Scopus (247) Google Scholar). This suggests that RNA may require a complex architecture for recognition of this otherwise simple amino acid. Second, the lysine riboswitch has been the focus of studies involving the potential of riboswitches as targets of antimicrobial agents (8Blount K.F. Wang J.X. Lim J. Sudarsan N. Breaker R.R. Nat. Chem. Biol. 2007; 3: 44-49Crossref PubMed Scopus (186) Google Scholar, 9Blount K.F. Breaker R.R. Nat. Biotechnol. 2006; 24: 1558-1564Crossref PubMed Scopus (354) Google Scholar, 10Ataide S.F. Wilson S.N. Dang S. Rogers T.E. Roy B. Banerjee R. Henkin T.M. Ibba M. ACS Chem. Biol. 2007; 2: 819-827Crossref PubMed Scopus (34) Google Scholar) because resistance to lysine analogs such as S-(2-aminoethyl)-l-cysteine (AEC 2The abbreviations used are:AECS-(2-aminoethyl)-l-cysteineSAXSsmall angle x-ray scatteringLysRSlysyl tRNA-synthetase. 2The abbreviations used are:AECS-(2-aminoethyl)-l-cysteineSAXSsmall angle x-ray scatteringLysRSlysyl tRNA-synthetase., see Fig. 1A) in Escherichia coli and B. subtilis is the result of mutations within the lysine riboswitch that regulates the lysC gene (see Fig. 1B, highlighted in blue) (11Lu Y. Shevtchenko T.N. Paulus H. FEMS Microbiol. Lett. 1992; 71: 23-27Crossref PubMed Google Scholar, 12Patte J.C. Akrim M. Mejean V. FEMS Microbiol. Lett. 1998; 169: 165-170Crossref PubMed Google Scholar). S-(2-aminoethyl)-l-cysteine small angle x-ray scattering lysyl tRNA-synthetase. S-(2-aminoethyl)-l-cysteine small angle x-ray scattering lysyl tRNA-synthetase. A recent study of AEC resistance in E. coli uncovered a mechanism that implicates lysyl tRNA-synthetase (LysRS) as the primary target of this compound (10Ataide S.F. Wilson S.N. Dang S. Rogers T.E. Roy B. Banerjee R. Henkin T.M. Ibba M. ACS Chem. Biol. 2007; 2: 819-827Crossref PubMed Scopus (34) Google Scholar). The toxic effects of AEC are a result of its incorporation into proteins in the place of lysine due to the inability of LysRS to discriminate between the two compounds. Mutations in the lysine riboswitch confer resistance to AEC because they result in a loss of lysine-dependent regulation of key lysine biosynthetic enzymes, increasing the intracellular lysine concentration. This allows lysine to effectively outcompete AEC for binding to LysRS, alleviating its toxic effects (10Ataide S.F. Wilson S.N. Dang S. Rogers T.E. Roy B. Banerjee R. Henkin T.M. Ibba M. ACS Chem. Biol. 2007; 2: 819-827Crossref PubMed Scopus (34) Google Scholar). Thus, development of new effective lysine analog antimicrobials will require targeting both LysRS and the lysine riboswitch (10Ataide S.F. Wilson S.N. Dang S. Rogers T.E. Roy B. Banerjee R. Henkin T.M. Ibba M. ACS Chem. Biol. 2007; 2: 819-827Crossref PubMed Scopus (34) Google Scholar). In the current study, we have solved the crystal structure of the lysine riboswitch in complex with lysine, revealing the basis for recognition of both the cognate ligand and the antimicrobial analogs, and provided insights into how mutations in the riboswitch might confer AEC resistance through two different means. RNA Preparation and Crystallization—A 161-nucleotide double-stranded DNA coding for the Thermotoga maritima lysine riboswitch aptamer domain controlling the asd gene was constructed by PCR using overlapping oligonucleotides. The RNA was transcribed and purified using previously published techniques (13Montange R.K. Batey R.T. Nature. 2006; 441: 1172-1175Crossref PubMed Scopus (322) Google Scholar). The refolded RNA was then exchanged into 10 mm Na-HEPES, pH 7.0, 5 mm MgCl2, and 2 mm lysine before storage at 4 °C. For the free state, RNA was refolded in the lysine supplemented buffer, exchanged three times into 10 mm Na-HEPES, pH 7.0, 5 mm MgCl2 followed by overnight dialysis into 1 liter of lysine-free buffer. The final concentration was determined by absorbance at 260 nm (ϵ = 1,570,000 m–1 cm–1, molecular weight = 52,433 g mol–1). RNA was stored at 4 °C until use. The riboswitch was crystallized by the hanging drop vapor diffusion method in the presence of 1 mm lysine or in the absence of lysine for the free state crystals. Drops were set up by mixing 1 μl of RNA with 1μl of a mother liquor solution consisting of 2 m Li2SO4, 5 mm MgCl2, and 10 mm Na-HEPES, pH 7.0, and 60 mm iridium hexammine to obtain the heavy atom derivative crystals. Identical conditions were used to grow the free state crystals except that no iridium hexammine was used in the mother liquor. Crystals were obtained within 24 h and required no additional cryoprotection agent; they were looped with 0.2–0.3-mm loops and flash-frozen in liquid nitrogen before data collection. Data Collection—Data for the bound state iridium hexammine derivative crystal were collected on beamline X29A at the Brookhaven National Synchrotron Light Source X-rays at the iridium absorption peak. These data were integrated and scaled using HKL2000 (14Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar). All data used in phasing and refining came from a single crystal. Data for the unliganded structure were collected using CuKα wavelength (1.5418 Å) radiation on an R-AXIS IV++ home source (Riguaku MSC), and the data were indexed and scaled using D*TREK (15Pflugrath J.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1718-1725Crossref PubMed Scopus (1417) Google Scholar). Phasing and Structure Determination—Phases were determined by single wavelength anomalous diffraction using data extending to 2.8 Å. SHELXD (16Schneider T.R. Sheldrick G.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1772-1779Crossref PubMed Scopus (1576) Google Scholar) was used to find three iridium heavy atom sites within the asymmetric unit that had reasonably high occupancy. These heavy atom sites were used to calculate phases in SHELXE (17Sheldrick G.M. Z. Kristallographie. 2002; 217: 644-650Crossref Scopus (360) Google Scholar). The resulting experimental density map, following density modification (0.5 solvent fraction), displayed clear features corresponding to RNA backbone and base pairing (supplemental Fig. S1). This map was used for initial building of the model. The model was built in Coot (18Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23226) Google Scholar) and refined in PHENIX (19Adams P.D. Grosse-Kunstleve R.W. Hung L.W. Ioerger T.R. McCoy A.J. Moriarty N.W. Read R.J. Sacchettini J.C. Sauter N.K. Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1948-1954Crossref PubMed Scopus (3624) Google Scholar) using iterative rounds of building and refinement. The RNA nucleotides were initially built along with four iridium hexammine molecules. This model was brought through multiple rounds of simulated annealing and atomic displacement factor refinement before building lysine into the model. At this point, the density for the entire ligand was clearly visible and was validated by inspection using a simulated annealing omit map in which the ligand and a few surrounding nucleotides were omitted from the model (supplemental Fig. S2). One round of water picking was carried out by the PHENIX-ordered solvent protocol; waters were chosen based on peak size in an Fo – Fc map. Rfree was monitored in each round to ensure that it was dropping. Figures were prepared using PyMOL (20DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA2002Google Scholar). The unliganded RNA model was built using the bound form as a molecular replacement solution using only the RNA and re-refined using iterative rounds of simulated annealing and atomic displacement factor refinement. The final refinement statistics are shown in supplemental Table S1, and the structure factors and models have been deposited in the Protein Data Bank (accession codes 3D0U and 3D0X). Chemical Probing Using Selective 2′-Hydroxyl Acylation Analyzed by Primer Extension Chemistry—RNA sequences were constructed to correspond to the T. maritima RNA that was crystallized as well as the riboswitch controlling the B. subtilis lysC gene (21Sudarsan N. Wickiser J.K. Nakamura S. Ebert M.S. Breaker R.R. Genes Dev. 2003; 17: 2688-2697Crossref PubMed Scopus (280) Google Scholar). The B. subtilis sequence was truncated in the P5 region to match the length of the T. maritima sequence to ensure that the RNAs are comparable, and the 5′- and 3′-structure cassettes were appended to these sequences as described previously (22Wilkinson K.A. Merino E.J. Weeks K.M. Nat. Protoc. 2006; 1: 1610-1616Crossref PubMed Scopus (554) Google Scholar). RNA was generated by run-off transcription and purified according to the same protocol used to generate RNA for the crystallographic studies. RNA was prepared for modification by placing 1 μl of 2 μm RNA (2 pmol) into 11 μl of 0.5× Tris EDTA buffer. This sample was heated/cooled to allow the RNA to refold and then supplemented with 6 μl of buffer consisting of 333 mm K-HEPES, pH 8.0, and 333 mm NaCl. This buffer was supplemented with 2 mm lysine for the plus ligand reactions (final concentration of 667 μm), and MgCl2 was included in the folding buffer at concentrations ranging from 6.8 mm to 425 μm in 2-fold dilutions to yield the concentrations shown in the magnesium titration All reactions were supplemented with 1 μl of mm to ensure proper by the the transcription were carried out using mm for the at 20 transcription and were as described previously (22Wilkinson K.A. Merino E.J. Weeks K.M. Nat. Protoc. 2006; 1: 1610-1616Crossref PubMed Scopus (554) Google Scholar). Small Data and B. subtilis lysC riboswitch was prepared for using an liquid with a 6 an and a distinct sample buffer conditions were used and are to as and 2 mm lysine and 5 mm MgCl2 or 2 mm All sample buffer conditions 20 mm Na-HEPES, pH and mm to of the the was with the buffer. RNAs were refolded as described at 5 μm and to μl to a final concentration of 5 μl of and a corresponding to the peak was for data were collected at the beamline of the Light Source All scattering data were collected at using a sample of A scattering was as two at 6 and 60 for the sample and buffer. The x-ray scattering due to the riboswitch RNA was determined by the x-ray scattering of the buffer from that the RNA and buffer. Data were and with to the final x-ray scattering J. Crystallogr. 1992; Scopus Google Scholar). Data were collected a range of RNA concentrations changes were in the scattering The of which the of a around its of was determined by using the A. Small of Scholar) within the range of or by the in J. Crystallogr. 1992; Scopus Google Scholar, M. Rev. 2007; PubMed Scopus Google Scholar). All final were prepared with and understand the basis for lysine recognition and AEC we have solved the structure of a riboswitch that the expression of the T. maritima gene This which is a conserved all of the nucleotides is conserved 1B, (3Barrick J.E. Breaker R.R. Genome Biol. 2007; 8: R239Crossref PubMed Scopus (354) Google Scholar). iridium hexammine derivative high data from which an experimental density map be (supplemental Fig. S1). Data and refinement statistics for both and unliganded structures that all nucleotides are in supplemental Table the bound final = Rfree = The 2.8 resolution structure of the complex well with and of the RNA (21Sudarsan N. Wickiser J.K. Nakamura S. Ebert M.S. Breaker R.R. Genes Dev. 2003; 17: 2688-2697Crossref PubMed Scopus (280) Google Scholar, Henkin T.M. S. A. 2003; PubMed Scopus Google Scholar, M.S. 2003; PubMed Scopus Google Scholar). The architecture of the RNA three of and to one a of in RNAs A. A. E. RNA Spring 2007; Scholar). At the of this is the the of the nucleotides with in which a single lysine is between and the architecture of the RNA is by formation of a structure of the and by their A is between the loops of and that was as for the of the B. subtilis lysC riboswitch to transcription S. RNA Spring 2007; Scholar). interactions, there is a between and which are to the These two form with the of the central four base of the This additional may an for at were in the selection of mutations that new or are for to T.R. RNA Spring 2006; Scholar). The of the two loops to is by a at using a In the of lysine this is by the T.M. J. PubMed Scopus Google Scholar). Thus, the of the aptamer domain is highly elements of the region of the lysine riboswitch have to the of a the of RNA structure in A. E. Biol. 2006; PubMed Scopus Google Scholar). The element the is an between the of and an to the between and A. Z. S. A. 1998; PubMed Scopus Google Scholar). The of forms a structure to a by out a with using the of the three as for P. N. S. A. PubMed Scopus Google Scholar), the with the of using their forms the central base of a base that the ligand-binding pocket is within the of the between the and and is by the base of the and The of lysine forms a set of with the amino of the the and the of to the and of are by the of lysine. The of lysine is by a of and within a pocket that it to the of along with the atom of the The small size of the pocket recognition by and (8Blount K.F. Wang J.X. Lim J. Sudarsan N. Breaker R.R. Nat. Chem. Biol. 2007; 3: 44-49Crossref PubMed Scopus (186) Google Scholar, N. Wickiser J.K. Nakamura S. Ebert M.S. Breaker R.R. Genes Dev. 2003; 17: 2688-2697Crossref PubMed Scopus (280) Google Scholar). This is the basis for between the related metabolites lysine and between lysine and related compounds is through recognition of the of the The lysine is bound in an that allows it to the two sites of of the with the of a lysine analog that a between the and to this riboswitch (8Blount K.F. Wang J.X. Lim J. Sudarsan N. Breaker R.R. Nat. Chem. Biol. 2007; 3: 44-49Crossref PubMed Scopus (186) Google Scholar). a compounds or and are bound because their is of the length to allow the proper between all of the of lysine and the RNA. The are through with and the a of proper the of lysine are the RNA. The around lysine the of antimicrobial lysine analogs at the such as and to reasonably well to the riboswitch (8Blount K.F. Wang J.X. Lim J. Sudarsan N. Breaker R.R. Nat. Chem. Biol. 2007; 3: 44-49Crossref PubMed Scopus (186) Google Scholar, N. Wickiser J.K. Nakamura S. Ebert M.S. Breaker R.R. Genes Dev. 2003; 17: 2688-2697Crossref PubMed Scopus (280) Google Scholar). the riboswitch only the central of A. A. Breaker R.R. Nature. 2006; 441: PubMed Scopus (326) Google Scholar, S. M. N. 2006; PubMed Scopus Google Scholar). In each through which are the are to yield antimicrobial agents T.E. Structure 2006; PubMed Scopus Google Scholar). in the is completely within the indicating that there is form of folding with binding. To the that lysine-dependent conformational changes within the we crystallized the RNA in the absence of lysine and determined its The RNA crystallized the same conditions and in the same The resulting structure is identical to the form (supplemental Fig. with only between the two structures in the of the of the (supplemental Fig. This reveals that the architecture be in the absence of of the binding pocket that of of the nucleotides is by the of base the same The be an of the crystal induces a that in To the potential between the bound and lysine riboswitch in we backbone using by to local changes in the RNA (22Wilkinson K.A. Merino E.J. Weeks K.M. Nat. Protoc. 2006; 1: 1610-1616Crossref PubMed Scopus (554) Google Scholar) and to To ensure that the is a of the we both the T. maritima asd and the B. subtilis lysC RNAs at magnesium concentrations in the absence and presence of 667 μm lysine and supplemental and In we to these RNAs concentrations mm mm as to the high conditions of the crystals (2 m Li2SO4, 5 mm MgCl2, 60 mm iridium These two in the of the to magnesium and both RNAs clear changes in upon the of These changes are on and involving the loops of and For the conserved in in both RNAs at 2 mm magnesium its to the solvent in the Second, both RNAs limited lysine-dependent changes to the the base of and of the is that at high magnesium concentrations lysine to the structure in the B. subtilis lysine-dependent effects are magnesium concentrations These data are in with both probing of the B. subtilis lysC riboswitch (8Blount K.F. Wang J.X. Lim J. Sudarsan N. Breaker R.R. Nat. Chem. Biol. 2007; 3: 44-49Crossref PubMed Scopus (186) Google Scholar, N. Wickiser J.K. Nakamura S. Ebert M.S. Breaker R.R. Genes Dev. 2003; 17: 2688-2697Crossref PubMed Scopus (280) Google Scholar) and an of a riboswitch at the to in the J. 2007; PubMed Scopus Google Scholar). these data that the structure of the be by magnesium and in the absence of lysine. To the chemical probing we the B. subtilis lysC riboswitch by This solution method is to the of a and a of the of and of the J. S. Annu. Rev. 2007; PubMed Scopus Google Scholar). The of 5 mm magnesium in the absence of lysine induces a of the RNA supplemental Fig. This form is by the of 2 mm lysine, as the both the bound and the forms of the riboswitch in 5 mm magnesium are The of the RNA in the presence of of lysine supplemental Table is with the crystal structure into the In the absence of the riboswitch is as by the of the In the presence of the at small scattering a well R. L.W. B. V. S. S. A. 2002; PubMed Scopus Google Scholar). with the chemical probing these that this riboswitch a form lysine in solution with the of Thus, lysine to the binding pocket through in the RNA within Mutations in the to the antimicrobial lysine analog AEC is in both E. coli and B. subtilis mutations within the lysine riboswitch (8Blount K.F. Wang J.X. Lim J. Sudarsan N. Breaker R.R. Nat. Chem. Biol. 2007; 3: 44-49Crossref PubMed Scopus (186) Google Scholar, Y. Shevtchenko T.N. Paulus H. FEMS Microbiol. Lett. 1992; 71: 23-27Crossref PubMed Google Scholar, 12Patte J.C. Akrim M. Mejean V. FEMS Microbiol. Lett. 1998; 169: 165-170Crossref PubMed Google Scholar). of these mutations map and around the binding with lysine. are in the of the and and formation of key of these lysine with the same as the B. subtilis lysC RNA (8Blount K.F. Wang J.X. Lim J. Sudarsan N. Breaker R.R. Nat. Chem. Biol. 2007; 3: 44-49Crossref PubMed Scopus (186) Google Scholar), that AEC resistance is by the at which the RNA into a regulation has a in which to the secondary structural J.K. Winkler W.C. Breaker R.R. Cell. 2005; PubMed Scopus (354) Google Scholar), a folding to loss of regulatory In this regulation is by the at which elements are to between the of each structure in the switch. studies will be to the role of the ligand in the folding of the riboswitch and its to genetic for with model building and and the Batey for and of this with