solution structure and thermodynamics of a divalent metal ion … · 2015-12-29 · berkeley and...

Article No. jmbi.1999.2841 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 289, 1267±1282

Solution Structure and Thermodynamics of a DivalentMetal Ion Binding Site in an RNA Pseudoknot

Ruben L. Gonzalez Jr and Ignacio Tinoco Jr*

Department of ChemistryUniversity of CaliforniaBerkeley and Structural BiologyDepartment, PhysicalBiosciences DivisionLawrence Berkeley NationalLaboratory, Berkeley, CA94720-1460 USA

E-mail address of the [email protected]

Abbreviations used: MMTV, mouvirus; NOE, nuclear Overhauser effHP, hairpin; SS, single-strand; NOEspectroscopy; HSQC, heteronuclearcoherence; DQF-COSY, double quacorrelated spectroscopy; ICP, inducplasma.

0022-2836/99/251267±16 $30.00/0

Identi®cation and characterization of a metal ion binding site in an RNApseudoknot was accomplished using cobalt (III) hexammine, Co(NH3)6

3�,as a probe for magnesium (II) hexahydrate, Mg(H2O)6

2�, in nuclear mag-netic resonance (NMR) structural studies. The pseudoknot causes ef®cientÿ1 ribosomal frameshifting in mouse mammary tumor virus. Divalentmetal ions, such as Mg2�, are critical for RNA structure and function;Mg2� preferentially stabilizes the pseudoknot relative to its constituenthairpins. The use of Co(NH3)6

3� as a substitute for Mg2� was investigatedby ultraviolet absorbance melting curves, NMR titrations of the iminoprotons, and analysis of NMR spectra in the presence of Mg2� or Co(NH3)6

3�. The structure of the pseudoknot-Co(NH3)63� complex reveals an

ion-binding pocket formed by a short, two-nucleotide loop and the majorgroove of a stem. Co(NH3)6

3� stabilizes the sharp loop-to-stem turn andreduces the electrostatic repulsion of the phosphates in three proximalstrands. Hydrogen bonds are identi®ed between the Co(NH3)6

3� protonsand non-bridging phosphate oxygen atoms, 20 hydroxyl groups, andnitrogen and oxygen acceptors on the bases. The binding site is signi®-cantly different from that previously characterized in the major groovesurface of tandem G �U base-pairs, but is similar to those observed incrystal structures of a fragment of the 5 S rRNA and the P5c helix of theTetrahymena thermophila group I intron. Changes in chemical shiftsoccurred at the same pseudoknot protons on addition of Mg2� as onaddition of Co(NH3)6

3�, indicating that both ions bind at the same site.Ion binding dissociation constants of approximately 0.6 mM and 5 mM(in 200 mM Na� and a temperature of 15 �C) were obtained forCo(NH3)6

3� and Mg2�, respectively, from the change in chemical shift as afunction of metal ion concentration. An extensive array of non-sequence-speci®c hydrogen bond acceptors coupled with conserved structuralelements within the binding pocket suggest a general mode of divalentmetal ion stabilization of this type of frameshifter pseudoknot. Theseresults provide new thermodynamic and structural insights into the roledivalent metal ions play in stabilizing RNA tertiary structural motifssuch as pseudoknots.

# 1999 Academic Press

Keywords: RNA structure and thermodynamics; NMR; metal binding;pseudoknot; frameshifting
*Corresponding author
ing author:

se mammary tumorect; PK, pseudoknot;SY, NOEsingle quantum

ntum ®lteredtively coupled

Introduction

The capacity of RNA to perform structural andfunctional roles in biological processes dependsupon its ability to fold into complex and compacttertiary structures. The close packing of helices andloops that is required for the formation of thesestructures is opposed by the presence of the nega-tively charged phosphodiester backbone. Positiveions non-speci®cally stabilize RNA structure(Anderson & Record, 1995) by screening the nega-

# 1999 Academic Press

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1268 Divalent Metal Binding in an RNA Pseudoknot

tively charged backbone. Additionally, speci®cmetal ion binding sites have been identi®ed thatare important in RNA structure and function (Stein& Crothers, 1976; Draper, 1985; Pan et al., 1993;Laggerbauer et al., 1994; Doudna & Doherty, 1997;Feig & Uhlenbeck, 1999). Until recently, detailedinvestigations of the interactions of metal ions withRNA was limited to crystallographic studies oftRNAs (Holbrook et al., 1977; Jack et al., 1977).Currently, crystal structures of hammerhead ribo-zymes (Scott et al., 1995; Pley et al., 1994), the P4-P6domain of the Tetrahymena thermophila self-splicinggroup I intron (Cate et al., 1996; Cate & Doudna,1996), and a 5 S ribosomal RNA (rRNA) domain(Correll et al., 1997) have revealed details of theinteractions between divalent metal ions andspeci®c binding sites in RNA. Some of these sitesoccur within elements of the secondary structureand others mediate tertiary contacts. The possi-bility of studying these types of interactions in sol-ution using cobalt (III) hexammine (Co(NH3)6

3�) asan analog of fully hydrated magnesium (II) hexa-hydrate (Mg(H2O)6

2�) was suggested by Cowan(1993), based on similar ionic radii and covalentgeometries for the two metal ion complexes, aswell as evidence that Co(NH3)6

3� can activate someMg2�-dependent enzymes (Jou & Cowan, 1991). Itsuse in solution nuclear magnetic resonance (NMR)structural studies has been demonstrated byRobinson & Wang (1995) in DNA decamers and byKieft & Tinoco (1997) in the P5b stem-loop fromthe T. thermophila group I intron. These structuralstudies of Co(NH3)6

3� binding to nucleic acid sec-ondary structural elements prompted this study ofthe metal ion complex in the stabilization of RNAtertiary interactions in solution.

RNA pseudoknots are a classic family of tertiarystructures formed when nucleotides in the loop ofa hairpin pair with nucleotides from an adjacentsingle-stranded region, forming a second stem andloop (Pleij et al., 1985). Pseudoknots have manydifferent functional roles in biological systems.They occur at the 30 termini of several plant virusRNA genomes (Haenni et al., 1982; Rietveld et al.,1982; Joshi, et al., 1983; Guerrier-Takada et al., 1988;Kolk et al., 1998), in ribonuclease P RNA (Jameset al., 1988), within 16 S rRNA (Moazed & Noller,1987), and most recently a pseudoknot structurehas been identi®ed as a key component of a hepa-titis delta virus ribozyme (FerreÂ-D'AmareÂ et al.,1998). Pseudoknots are often involved in trans-lational control (Tang & Draper, 1989; Schimmel,1989), and cause programmed ribosomal frame-shifting in many retroviruses (Chamorro et al.,1992; ten Dam et al., 1994, 1995; Brierley et al.,1991; Chen et al., 1995; Brierley, 1995). Severalstudies have investigated the thermodynamics ofpseudoknot formation and stabilization (Wyattet al., 1990; Qui et al., 1996; Theimer et al., 1998;Nixon & Giedroc, 1998).

The VPK pseudoknot studied here (Shen &Tinoco, 1995) causes frameshifting in mousemammary tumor virus (MMTV). It differs from the

wild-type MMTV pseudoknot in that four of theG �C base-pairs were ¯ipped to C �G base-pairs todisrupt G-rich tracks that led to multiple confor-mations in the native sequence (Figure 1(a) and(b)). The modi®ed VPK sequence has equal frame-shifting ef®ciency as the wild-type MMTVsequence in two different constructs of ¯ankingsequences (Chen et al., 1995). The VPK pseudoknotforms below 25 �C in the presence of 200 mM Na�,but small concentrations of Mg2� (10 mM) or Co(NH3)6

3� (5 mM) greatly stabilize it. The highercharge of Mg2� or Co(NH3)6

3� compared to Na�

undoubtedly contributes to the increased stabilityof the pseudoknot, but in addition there could be aspeci®c metal ion binding site in the VPK pseudo-knot. This speci®city could be driven by the ionicradii and the octahedral coordination geometry ofMg2� and Co(NH3)6

3�, compared to the smallerionic radius and tetrahedral coordination geometryof Na�.

Here, we present the solution structure and athermodynamic study of a metal ion-RNA tertiaryinteraction: an RNA pseudoknot, stabilized by aspeci®cally bound Co(III) ion complex. NuclearOverhauser effect (NOE) crosspeaks between theprotons of Co(NH3)6

3� and 15 RNA protonsallowed identi®cation and characterization of thebinding site. Co(NH3)6

3� and Mg2� stabilize thepseudoknot as shown by ultraviolet absorbancemelting curves. NMR titration experiments demon-strated that Co(NH3)6

3� and Mg2� ions interactwith the pseudoknot structure at the same site,and provided estimates of their binding constants.We expect similar electrostatic and hydrogen-bonding interactions of Co(NH3)6

3� and Mg(H2O)62�

with the RNA, although the stability of binding isdifferent.

Results

Thermodynamics

Ultraviolet absorbance melting curves

Ultraviolet (UV) absorbance melting curves forVPK RNA dialyzed into 10 mM sodium phosphate,200 mM NaCl, 100 mM EDTA (pH 6.4) are shown inFigure 2. The UV melting curve in the absence ofMg2� or Co(NH3)6

3� (Figure 2(a)) displays twomajor melting transitions. The ®rst transition has amelting temperature (tm) of approximately 27 �Cand is assigned to the melting of the pseudoknotinteraction. The second transition has a tm ofapproximately 63 �C and is attributed to the meltingof the remaining hairpin. These transitions wereassigned based on the UV melting curves of each ofthe individual hairpins (Figure 1(c) and (d)) whichdisplay one transition each with a tm of approxi-mately 83 �C and no transitions at lower tempera-tures. Overall, the pseudoknot melting transitionsare unimolecular, as no increase in tm is observedwith increasing VPK concentration over a range of3 to 300 mM. Analysis of the derivative absorbance

Figure 1. Sequence comparisonof (a) wild-type MMTV pseudoknotand (b) the modi®ed VPK pseudo-knot. Boxed nucleotides indicatethe changes made to facilitateNMR studies. The two pseudo-knots have equal frameshifting ef®-ciencies. (c) The ST1 hairpin wasused as a model for stem 1 and (d)the ST2 hairpin was used as amodel for stem 2 in UV absorbancemelting experiments.

Divalent Metal Binding in an RNA Pseudoknot 1269

melting curve in the absence of multivalent ionsyielded estimates of the standard enthalpy changes(�Ho) for the pseudoknot (PK) to hairpin (HP) tran-sition and the hairpin (HP) to single-strand (SS)transition. As expected, due to the similarities ofthe two stems, the two �Ho values are within 10 %of each other (�Ho

PK ! HP � 37 kcal molÿ1 and�Ho

HP ! SS � 41 kcal molÿ1). Titration of VPK RNAwith Co(NH3)6

3� (Figure 2(b)) or Mg2� (Figure 2(c))shifts the tm of the two transitions to higher tem-peratures with increasing concentrations of metalion until both transitions overlap. Completely over-lapped transitions are observed at Co(NH3)6

3� con-centrations of 10 mM, or Mg2� concentrationsslightly higher than 50 mM. Shifts in tm of approxi-mately 40 deg. C for the ®rst melting transition and25 deg. C for the second melting transition areobserved. Because the �Ho of the pseudoknot tohairpin transition is within 10 % of the �Ho of thehairpin to single-strand transition, the 15 deg. Cdifference between the two tm shifts demonstratesthat the multivalent ions preferentially bind to thepseudoknot relative to the hairpin or single-strand.Titrations with Na� (Figure 2(d)) also shift the tm ofthe two transitions to higher temperatures withincreasing concentrations of metal ion, albeit atmuch higher concentrations of Na�. Even doublingthe Na� concentration from 200 mM to 400 mMwas not suf®cient to produce overlappingtransitions. The VPK pseudoknot requires approxi-mately ®vefold higher concentrations of Mg2� thanCo(NH3)6

3� to achieve a similar degree of stabiliz-

ation. Even with Na� concentrations at least 40,000-fold higher than Co(NH3)6

3� concentrations, thesame degree of pseudoknot stabilization is notattained. In the case of Na�, tm shifts of 30 deg. Cand 25 deg. C are observed for the ®rst and secondmelting transitions, respectively. The pseudoknotinteraction is somewhat preferentially stabilizedover the hairpin interaction, with only a 5 deg. Cdifference between the two shifts. These results areconsistent with previous ®ndings that very highconcentrations of Na� are necessary in order forNa� to replace the stabilizing role of divalent ionsin pseudoknots (Wyatt et al., 1990).

Equilibrium binding constants

Imino proton resonance assignments were basedon those made previously for VPK RNA in 5 mMMgCl2 (Shen & Tinoco, 1995). VPK RNA wastitrated with either Co(NH3)6

3� or Mg2� and thechemical shifts of the imino proton resonanceswere monitored as a function of concentration(Figure 3(a) and (b)). Chemical shift changes due toCo(NH3)6

3� binding are very similar to thoseinduced by Mg2� binding. This indicates similarbinding modes for both metal ions. In both casesthe imino protons of G15, G17, and G4 exhibit thelargest down®eld shifts, whereas the imino protonsof G9 and G11 undergo the largest up®eld shifts.The imino protons of G1 and G2 are shifted to agreater degree by Mg2�. This is presumably due todirect coordination of Mg2� ions to the 50-terminal

Figure 2. UV melting curves of the VPK pseudoknot. The VPK concentration in all melts is 4 mM. All samples weredialyzed against a buffer solution of 10 mM sodium phosphate, 200 mM NaCl, 100 mM EDTA (pH 6.4). Meltingcurves were acquired in the presence of (a) no additional salt, (b) 0-10 mM Co(NH3)6

3�, (c) 0-50 mM Mg2�, and (d) 200and 400 mM Na�. The absorbances in (b), (c), and (d) are normalized with respect to the absorbance of (a).


triphosphate which does not occur with Co(NH3)63�

ions. Proton chemical shift changes can be causedby metal ion binding in the local environment ofthe proton, or by structural changes brought aboutby metal ion binding not necessarily near theproton. A H2O NOE spectroscopy (NOESY) exper-iment in the presence of Co(NH3)6

3� identi®ed theimino protons which are directly near the bindingsite. NOE crosspeaks from Co(NH3)6

3� protons tothe imino protons of G10, G11, and G29 were

observed, identifying these imino protons as directprobes of the metal ion binding site (Figure 4).

Binding curves can be constructed by plotting thechemical shift of individual imino protons as a func-tion of metal ion concentration. Two such curvesare plotted in Figure 3(b) for the imino proton ofG11 versus Co(NH3)6

3� and Mg2� concentrations,respectively. For a system in fast exchange, thebinding data for a 1:1 stoichiometry can be ®t to thefollowing binding isotherm:

dobs � df � �db ÿ df ��ion�t � �RNA�t � Kd� ÿ

��ion�t � �RNA�t � Kd�2 ÿ 4�RNA�t � �ion�t

q2�RNA�t

24 35

Figure 3. Imino proton titrations of VPK with Co(NH3)63� and Mg2�. The sample conditions are 400 mM VPK,

10 mM sodium phosphate, 200 mM NaCl, 100 mM EDTA (pH 6.4) at a temperature of 15 �C. (a) Plot of chemical shiftversus Co(NH3)6

3� and Mg2� for all observed imino protons. The pattern of chemical shift changes are very similar forboth metal ion complexes. (b) Binding curves for the G11 imino binding to Co(NH3)6

3� and Mg2�. Equilibrium dis-sociation constants (Kd) are calculated by ®tting the data to a one binding site model.


in which dobs is the observed RNA chemical shift, db

is the chemical shift of the fully bound proton, df isthe chemical shift of the unbound proton, [RNA]t isthe total RNA concentration, [ion]t is the total ionconcentration, and Kd is the equilibrium bindingdissociation constant for the RNA-metal ion com-plex (Roberts, 1993). This binding isotherm appliesto one ion binding site per RNA molecule; it can be®t to obtain the fully bound chemical shift and theequilibrium binding dissociation constant Kd. Bind-ing constants calculated for imino protons withsigni®cant chemical shift dependence on metal ionconcentration agree within three- to fourfold ofthose calculated for the imino proton of G11(Figure 3(b)), consistent with a one binding sitemodel. This method was used to obtain equilibriumdissociation constants of Kd(Co(NH3)6

3�) � 0.6(�-0.4) mM and Kd(Mg2�) � 5(�3) mM, for binding toVPK in 10 mM sodium phosphate, 200 mM Na�,100 mM EDTA (pH 6.4), at a temperature of 15 �C.

There is an approximately eightfold increase in af®-nity for the Co(NH3)6

3� metal ion versus Mg2� metalion, presumably due to the greater positive chargeof Co(NH3)6

3�. Although it would be advantageousto measure binding constants using a VPK non-exchangeable proton, no non-exchangeable protonresonance was suf®ciently well resolved in the 1Dspectra.

In addition to the imino protons at the Co(NH3)6

3� binding site, imino protons from otherregions of the pseudoknot display chemical shiftchanges. Imino protons from G17 and G4 in stem 1exhibit down®eld chemical shifts on binding of thecobalt ion complex. The G15 imino proton, adja-cent to the junction region of the pseudoknot,demonstrates the largest chemical shift change inboth the Co(NH3)6

3� and Mg2� titrations. It is clearthat during these titrations considerable structuralchanges are occurring within the pseudoknot, par-ticularly the junction region, which cause these

Figure 4. (a) Imino to Co(NH3)63� and (b) imino to

imino region of the 300 ms mixing time H2O NOESY ofthe VPK pseudoknot in the presence of Co(NH3)6

3�.Lines show connectivities for the intermolecular NOEcrosspeaks of the Co(NH3)6

3� ammine protons with theimino proton diagonal peaks. Sample conditions are2 mM VPK, 2 mM Co(NH3)6

3�, 10 MM sodium phos-phate, 200 mM NaCl, 100 mM EDTA (pH 6.4), at a tem-perature of 20 �C.


chemical shift changes. Thus, unlike the P5b stemloop (Kieft & Tinoco, 1997), the pseudoknot struc-ture changes upon divalent metal ion binding.Even small changes in the relative position of theG15 imino with respect to A6, A14, and/or A27nucleotides at the junction may cause notablechemical shift changes due to the large ring cur-rents associated with the adenine bases. Spectraacquired in 200 mM Na� but in the absence ofMg2� or Co(NH3)6

3� exhibit very broad, low inten-sity or absent NOE crosspeaks in the junctionregion. This may be due to low pseudoknot stab-ility in the absence of Mg2� or Co(NH3)6

3� at highertemperatures, and/or broadening due to largerotational correlation times associated with lowertemperatures. These broad or absent NOE cross-peaks, coupled with signi®cant spectral overlap inthe one dimensional spectra made it dif®cult tomeasure chemical shift changes for non-exchange-able protons in the junction region.

Similar binding studies were done and equili-brium binding dissociation constants were obtainedfor the Co(NH3)6

3� binding site formed by tandemG �U base-pairs in the P5b stem loop from theT. thermophila group I intron (Kieft & Tinoco, 1997).The dissociation constant determined from iminoproton titrations in 10 mM sodium phosphate,

200 mM Na�, 100 mM EDTA (pH 6.4), and a tem-perature of 10 �C was Kd � 0.19(�0.01) mM (Kieft,1997). Subsequent equilibrium dialysis experimentswere done on P5b to validate the binding constantsusing imino proton chemical shift changes observedduring metal ion titrations. The results of the equili-brium dialysis experiments are consistent with onemetal ion binding at one strong bindingsite (Kd � 0.72(�0.06) mM) and approximatelytwo ions binding at weaker binding sites(Kd � 0.06(�0.04) mM2). The equilibrium dialysisKd of the one strong binding site is about fourfoldweaker binding than that calculated from the iminotitrations. The Kd obtained from the imino protontitrations is expected to be smaller (stronger bind-ing) than the value obtained from equilibriumdialysis due to assumptions about non-speci®cbinding used in the analysis of the imino protontitration data. Non-speci®c binding leads to adecrease in the concentration of free Co(NH3)6

3�; inthe equilibrium dialysis experiments, the free con-centration of Co(NH3)6

3� is determined directly, andthe effect of non-speci®c binding is included. Never-theless, using imino proton chemical shift changesduring metal ion titrations provides equilibriumbinding constants for strong binding sites that arein reasonable agreement with those measured fromequilibrium dialysis experiments.

Comparison of spectra in Mg2� andin Co(NH3)6

3�

NOESY spectra were acquired in H2O and 2H2Ofor VPK in the presence of Mg2� and in the pre-sence of Co(NH3)6

3�. In both cases the same NOEconnectivities and relative intensities are observed.The most notable changes occur in the chemicalshifts of the RNA protons. It was observed that forVPK in the presence of Mg2� the optimum tem-perature for experiments involving exchangeableprotons was 15 �C, and for experiments involvingnon-exchangeable protons it was 30 �C. At thesetemperatures the VPK-Co(NH3)6

3� complex dis-played very broad lines and a number of NOEcrosspeaks were not observed due to the linebroadening. The VPK-Co(NH3)6

3� complex dis-played optimal spectral qualities at temperaturesof 20 �C for exchangeable protons, and 35 �C fornon-exchangeable protons. This is probably due togreater stabilization of VPK by 2 mM Co(NH3)6

3�

as compared to 5 mM Mg2�. As indicated by theUV absorbance melting curves, about ®vefoldhigher concentrations of Mg2� are necessary toprovide the same amount of stabilization as 2 mMCo(NH3)6

3�. Unfortunately, such large concen-trations of Mg2� generally tend to broaden spectrallines. The greater stabilization by Co(NH3)6

3�

allows for work at higher temperatures and pro-vides sharper lines. Aggregation due to Mg2� ionsinteracting with 50 triphosphate groups on multipleVPK molecules may also cause broader lines in theMg2� spectra. This type of aggregation would notbe present in the Co(NH3)6

3� spectra.


Analysis of exchangeable proton resonances

Spectra recorded in H2O displayed the samenumber of imino proton resonances for VPK in thepresence of Mg2� as in the presence of Co(NH3)6

3�.The same imino to imino NOE crosspeaks wereobserved in both complexes. There were nochanges in the base-pairing scheme upon additionof Co(NH3)6

3� as has been observed in the NMRstructure of a fragment of 16 S rRNA that binds toribosomal protein S8 (Kalurachchi & Nikonowicz,1998). In addition, the same imino to amino, iminoto H5 and imino to H10 NOE crosspeak connectiv-ities were observed for VPK in the presence ofeither Mg2� or Co(NH3)6

3�. Assignments forexchangeable protons in the presence of Mg2� at15 �C and Co(NH3)6

3� at 20 �C are provided in theSupplementary Material.

Analysis of non-exchangeable proton resonances

The sugar H10 to base aromatic H6/H8 NOEcrosspeak connectivities that are typical of A-formhelical structure can be followed through both

stems of the pseudoknot in the presence of eithermetal ion complex. The relative intensities of thesecrosspeaks are the same in both cases. In someregions of the spectra, better dispersion is obtainedin the presence of Co(NH3)6

3�. In addition, connec-tivities observed through the pseudoknot loops inthe presence of Mg2� are also seen in the presenceof Co(NH3)6

3� (Figure 5(a) and (b)). As notedabove, signi®cant structural changes do occurupon metal binding in VPK, particularly in thejunction region. The comparison of spectra in Mg2�

and in Co(NH3)63� demonstrates that these changes

occur in the presence of either metal ion complex,and that there is not much structural differencebetween the structure in the presence of Mg2� orthe presence of Co(NH3)6

3�. Assignments for non-exchangeable protons in the presence of Mg2� at30 �C and Co(NH3)6

3� at 35 �C are provided in theSupplementary Material.

Intermolecular NOE crosspeak assignment

NOESY experiments in H2O were used to detectintermolecular NOE crosspeaks between the Co

Figure 5. Aromatic to H10 regionof a 400 ms 2H2O NOESY of theVPK pseudoknot in the presence of(a) 5 mM Mg2� and (b) 2 mMCo(NH3)6

3�. The continuous andbroken lines indicate standard A-form walks for the two strands ofstem 2. The dotted line indicatesthe A6H2 to G7H8 connectivitythat is observed in loop 1. Arrowsdenote the A6 and the G28 intranu-cleotide H8 to H10 NOE. (c) TheCo(NH3)6

3� proton to H6/H8 regionof a 300 ms H2O NOESY exper-iment. Intermolecular NOE cross-peaks are observed betweenCo(NH3)6

3� protons and the H6/H8proton of the denoted nucleotide.(d) The Co(NH3)6

3� proton to H6/H8 region of a 13C-resolved HSQC-NOESY on a VPK sample selec-tively 13C-labeled at the adenine C8and the uracil C6. Only one cross-peak, to the A6H8, is observed.Sample conditions in (a) are 2 mMVPK, 5 mM Mg2�, 10 mM sodiumphosphate, 200 mM NaCl, 100 mMEDTA (pH 6.4) at a temperature of30 �C. Sample conditions in (b), (c)and (d) are identical, with theexception of 2 mM Co(NH3)6

3�

instead of 5 mM Mg2�, and atemperature of 35 �C.

Table 1. Summary of NMR data and structural con-straints

ConstraintsIntraresidue NOEs 69Interresidue NOEs 117Intermolecular NOEs 15Hydrogen bonding 45Torsion angle 279

Convergence 26/100Violations

NOE >0.2 AÊ 0Torsion angle >10 � 0

RMS deviations from ideal covalent geometryBond lengths (AÊ ) 0.004Bond angles (deg.) 0.83Impropers (deg.) 0.23

RMS deviations for converged structuresAll atoms (AÊ ) 3.62 (2.41 for family 1,

2.81 for family 2)a

All atoms except loop 2 (AÊ )b 2.42Binding site (AÊ )c 2.01

a Families were de®ned based on the two general geometriesadopted by loop 2 as it enters the junction region. See Figure 6.There are 14 structures in family 1 and 12 in family 2.

b Residues 1-19, 28-34.c Residues 6-13, 28-34.


(NH3)63� protons and the RNA protons. A total of

15 positive crosspeaks (same sign as diagonalpeaks) were detected with varying intensities andline widths. Crosspeaks from the Co(NH3)6

3� pro-tons to the G10, G11, and G29 imino (Figure 4) andC12, C30, and C31 amino (Figure 5(c)) protonswere assigned from the H2O NOESY data.

Intermolecular NOE crosspeaks to aromaticRNA protons were more dif®cult to assign due toresonance overlap. The H2O NOESY experimentsat different concentrations of Co(NH3)6

3� were usedto determine which crosspeaks appeared ®rst andwith what intensity. Titration with Co(NH3)6

3�

gave rise to a set of peaks whose intensitiesincreased with increasing concentrations of Co(N-H3)6

3�; these peaks were assigned to a strong ionbinding site. At higher concentrations of ligand,crosspeaks due to non-speci®c Co(NH3)6

3� bindingbegan to be observed. These results are consistentwith one relatively strong binding site.

The 2H2O NOESY experiments at 20 �C and35 �C provided chemical shift assignments foraromatic RNA protons at the two different tem-peratures. These were then compared with H2ONOESY experiments recorded at the same tem-peratures. From these experiments it was possibleto provide assignments for all but one of the inter-molecular NOE crosspeaks. NOE crosspeaks weredetected between the Co(NH3)6

3� protons and theG1H8, G7H8, G10H8, G11H8, G28H8 and C30H6(Figure 5(c)). The G1H8 is attributed to binding atthe 50-terminal triphosphate; addition of smallamounts of Mg2� to the Co(NH3)6

3�-VPK complexdiminished the intensity of this crosspeak signi®-cantly without altering the intensity of the otherpeaks. The unassigned peak was the strongestintermolecular NOE crosspeak observed and, dueto overlap of the A6H8 and the G9H8 resonances,could not be unambiguously assigned from theH2O NOESY data of the unlabeled pseudoknot.This intermolecular crosspeak was unambiguouslyassigned as the A6H8 using an RNA samplespeci®cally 13C-labeled at the adenine C8 and ura-cil C6 position. An HSQC-NOESY experimentdone in 90 % H2O/10 % 2H2O was used to detectNOE crosspeaks from adenine H8 and uracil H6protons. In the intermolecular Co(NH3)6

3� proton toRNA aromatic proton region of this spectrum, onlyone crosspeak was observed and that was to theA6H8 (Figure 5(d)). The fact that no intermolecularcrosspeaks to adenosine nucleotides at or near thejunction are observed is consistent with no Co(NH3)6

3� binding in this region of the pseudoknot.Also, no intermolecular crosspeaks are observed toany of the ®ve adenine H8 s in loop 2 of the pseu-doknot, again consistent with no Co(NH3)6

3� bind-ing in this region of the pseudoknot.

Structure calculations

A total of 100 starting structures were generatedwith randomized backbone torsion angles and ran-domized position of Co(NH3)6

3�. These initial struc-

tures were then subjected to the global foldprotocol as described in Materials and Methods. Ofthe 100 starting structures, 30 structures convergedwith low NOE energies and total energies. Theseconverged structures were then subjected tofurther re®nement and energy minimization (seeMaterials and Methods). After re®nement andenergy minimization, 26 of the 30 structures werejudged as acceptable structures based on low NOEenergies and total energies, as well as lack of NOEviolations greater than 0.2 AÊ . These 26 structureswere used to determine the structure statisticsshown in Table 1.

Discussion

VPK pseudoknot structure

The structure of the VPK pseudoknot in the pre-sence of Co(NH3)6

3� does not vary signi®cantlyfrom the structure determined in Mg2� (Shen &Tinoco, 1995), although there are slight differences.Due to a number of new intramolecular RNA NOEcrosspeaks, the intermolecular Co(NH3)6

3� protonto RNA proton NOE crosspeaks, and a largerinitial set of starting structures, the structure of thepseudoknot is now better de®ned. Stem 1 andstem 2 are fairly well de®ned in the 26 convergedstructures, with RMS deviations from the averagestructure of 0.81 AÊ and 1.09 AÊ , respectively. Inter-molecular NOE crosspeaks from Co(NH3)6

3�

protons to RNA protons have bridged the gapbetween the short, two-nucleotide loop 1 and themajor groove of stem 2, providing indirect loop-to-stem constraints; information that was previouslyunavailable. This has allowed better de®nition ofloop 1, although the A6 and G7 bases in loop 1 canpoint out into solution or in towards stem 2, and


still satisfy the NOE data available. The larger loop2 is much more dif®cult to place with respect tostem 1. One critical, previously unassigned, NOEcrosspeak was detected between the A27 H10 inloop 2 and the G17 H10 in stem 1. This crosspeak isobserved in the presence of Mg2� or Co(NH3)6

3�.Although it is clear that loop 2 crosses the minorgroove of stem 1, it is not clear exactly how it doesso. As can be seen in Figure 6 and Table 1, the twostems and loop 1 of the VPK pseudoknot superim-pose relatively well, with an RMSD to the averagestructure of 2.42 AÊ . Loop 2, however, is not wellde®ned and seems to adopt a number of possibleconformations. Although this is mainly due to thelack of NOE and torsion angle constraints, there isevidence to support a dynamic loop 2. In DQF-COSY experiments, H5-H6 crosspeaks for loopresidues C21, U22, and C23 are of higher intensitythan H5-H6 crosspeaks for residues in the stemregions. Because the H5-H6 scalar coupling is con-stant, the observed difference in intensities indicatefast internal dynamics relative to the stem regions.Although this does not rule out the existence ofalternate relaxation pathways, which may lead tobroadening of the stem H5-H6 crosspeaks and thuslower intensity peaks, additional NMR evidencesupport a dynamic loop 2. The adenine H2 reson-ances are usually the sharpest peaks in the NMRspectra of RNA due to favorable relaxation proper-ties, yet the H2 resonances of A26 and A27 in loop2 are not detected. Furthermore, the structures

were analyzed in order to identify protons within3 AÊ and within 5 AÊ of loop 2 nucleotide protons.For each structure, roughly ten unique NOE cross-peaks are predicted involving loop 2 to stem 1 orloop 2 to loop 2 nucleotides that are not observed.The absence of these NOE crosspeaks could becaused by rapid internal motion of loop 2, leadingto smaller local correlation times for these protons.Taken together, these data are consistent with adynamic loop 2.

Co(NH3)63� binding site

The Co(NH3)63� ion binds in a pocket consisting

of loop 1 and stem 2 residues (Figure 7). The short,two-nucleotide loop 1 must cross the major grooveof the six base-pair stem 2, causing a sharp loop-to-stem turn and bringing the loop into close con-tact with the stem. This close contact is particularlyunfavorable because of the repulsion of negativelycharged phosphates on the three strands. TheCo(NH3)6

3� ion shields the electrostatic repulsion,and mediates this close interaction between theloop 1 and stem 2 by forming hydrogen bonds tothe nitrogen and oxygen acceptors that are avail-able on the surface of this binding pocket.

The Co(NH3)63� ion is in the fast exchange regime

within the binding pocket as well as in fastexchange between the bound and free forms as evi-denced by a single average resonance frequencyfor all 18 Co(NH3)6

3� protons. The structure calcu-

Figure 6. Superposition of the 26converged VPK structures on thebasis of all atoms for all residuesexcepting loop 2 residues. Loop 2is observed to adopt a number ofpossible conformations as it crossesthe minor groove of stem 1. Thereare, however, two general geome-tries adopted by loop 2 as it entersthe junction region, allowing forthe de®nition of two generalfamilies of structures. Loops andthe junction A14 nucleotide areshown in magenta and stems incyan. The RMS deviation from theaverage structure is 2.42 AÊ .

Figure 7. Lowest energy struc-ture of the VPK pseudoknot withCo(NH3)6

3� shown in the bindingpocket. Loops and the junction A14nucleotide are shown in magenta,stems in cyan, and the Co(NH3)6

3�

ion is shown in red. Co(NH3)63�

binds in a binding pocket formedby nucleotides in loop 1 and themajor groove of stem 2.


lation protocol produced a family of structureshaving different positions and orientations of theCo(NH3)6

3�. Each of the 26 ®nal structures was ana-lyzed for hydrogen-bonding interactions (Figure 8).Hydrogen bonds were de®ned as bond lengthsbetween the proton on the donor atom and theacceptor atom shorter than 2.5 AÊ with a bondangle of 180(�60)�. Hydrogen bonds were foundfrom the Co(NH3)6

3� protons to the pro-Sp oxygenatom of A6, G7, U8, G9, G10, G29, the pro-Rp oxy-gen atom of C5, A6, G7, U8, and the 20 hydroxyl ofC5, A6, G7, G28. Hydrogen bonds from Co(NH3)6

3�

protons to the bases were observed to the N7 ofA6, G7, G9, G10, G11, G28 and G29 and the O6 ofG7, G10, G11, G28 and G29, as well as the N3 ofA6 and G7. All of these hydrogen bond acceptorsline the pocket formed by the sharp turn of loop 1into stem 2, and are the major determinants of thestabilization of this turn by the metal ion. Therapid tumbling of Co(NH3)6

3� within the bindingpocket allows it to interact with any of thesehydrogen bond acceptors.

Comparison with other metal ion binding sites

General trends that have been recently observedin divalent metal ion binding to RNA are alsoobserved in the VPK pseudoknot. The Co(NH3)6

3�

ion binds in a somewhat distorted major grooveand interacts with non-bridging phosphate oxygenatoms and purine bases, especially the O6 and N7of guanine residues (Correl et al., 1997; Cate &

Doudna, 1996; Pan et al., 1993; Scott et al., 1995;Pley et al., 1994). The divalent ion binding sitereported here, however, is substantially differentfrom the site that has been identi®ed in tandemG �U base-pairs in the P5b (Cate & Doudna, 1996;Kieft & Tinoco, 1997), P5 (Cate & Doudna, 1996;Colmenarejo & Tinoco, 1999), and P1 (Allain &Varani, 1995) helices of the T. thermophila group Iintron. In this type of site the divalent ion binds inthe major groove to tandem G �U base-pairs byhydrogen bonding to the O6 and N7 of the gua-nine bases. The uracil carbonyl group increases theelectronegativity of the surface of the major grooveand provides a concave surface for metal ion bind-ing. The ion does not interact with the ribose phos-phate backbone. Substituting the G �U pairs withG �C base-pairs diminishes binding to Co(NH3)6

3�,presumably due to the disruption of the electrone-gative surface and greater steric hindrance uponthe replacement of the U carbonyl with the Camino group (Kieft & Tinoco, 1997).

The binding of Co(NH3)63� to the VPK pseudo-

knot more closely resembles the binding site seenin the P5c region of the group I intron (Cate &Doudna, 1996) and the binding sites of the ®vemetal ions seen in the recently solved crystal struc-ture of a 5 S rRNA domain (Correll et al., 1997). Inthese binding sites the metal ion complex simul-taneously interacts with non-bridging phosphateoxygen atoms and base functional groups. Bothdirect coordination and outer sphere coordinationof the metal ion are observed in these binding

Figure 8. Close up view of the binding pocket for the lowest energy structure showing energetically equivalentorientations of the Co(NH3)6

3� ion and the hydrogen-bonding pattern observed. Loop 1 and the junction A14 nucleo-tide are shown in magenta, stem 2 in cyan, Co(NH3)6

3� in red, hydrogen bond acceptor atoms are shown as whiteballs, and hydrogen bonds are green. Hydrogen bonds are seen from Co(NH3)6

3� to the (a) A6 pro-Rp oxygen atom,(b) C5 20 hydroxyl group, (c) G29 pro-Sp oxygen atom, (d) G28 20 hydroxyl group, (e) G29 N7, (f) G7 O6, (g) G7 N7,and (h) A6 N7. Similar analysis for other members of the structural family, reveals slightly different hydrogen-bond-ing patterns.


sites. The metal in Co(NH3)63� cannot directly coor-

dinate to RNA groups, and therefore serves as aprobe of sites that do not involve direct coordi-nation. In the case of the Co(NH3)6

3� binding to theVPK pseudoknot, the placement of loop 1 in themajor groove of stem 2, as well as the perturbationof stem 2 to accommodate this short loop, creates aunique pocket where the interactions betweenCo(NH3)6

3� and the loop 1 phosphate, 20 hydroxyl,and base functional groups makes up for the gen-erally unfavorable binding to the G �C base-pairs inthe major groove. The substitution of G �U for G �Cbase-pairs in stem 2 should create a higher af®nitybinding site.

Comparison with metal binding predictions

Calculation of the electrostatic surface of theVPK pseudoknot reveals the pocket formed byloop 1 and stem 2 to be among the most electrosta-tically negative regions of the pseudoknot.Hermann & Westhoff (1998), have demonstratedthe use of Brownian dynamics simulations to pre-

dict metal-ion binding sites within RNA foldsdetermined by NMR, including the VPK pseudo-knot. This approach simulates the trajectories ofpositively charged spheres subject to the effect ofboth random Brownian movement and the electro-static ®eld of the RNA molecule in order to ident-ify metal-ion binding pockets. The application ofthis simulation to the VPK pseudoknot predictedthree metal-ion binding sites (Hermann & Westh-off, 1998). The ®rst metal ion is predicted to bindto G4 and C5 in stem 1 and A25 and A26 in loop2, the second is predicted to bind to A6 and G7 inloop 1, and the third to G7 in loop 1 and G9 andG10 in stem 2. The last two predicted sites prob-ably correspond to the single Co(NH3)6

3� site ident-i®ed by NMR. The Co(NH3)6

3� binding site can infact contact all the residues in these two predictedsites simultaneously. There is no NMR evidencethat a Co(NH3)6

3� binding site exists at the pre-dicted binding site of G4 and C5 in stem 1 andA25 and A26 in loop 2. It is important to note thatthe Brownian dynamics simulation was applied tothe previously published VPK structure (Shen &


Tinoco, 1995). We ®nd that the position of loopresidues vary considerably within the family ofstructures compatible with the NMR data. Repeat-ing the simulation with other low energy membersof the structural family would show whetherpredicted metal-ion sites involving loops are in¯u-enced by the choice of structure within a family.

Metal ions in frameshifting pseudoknots

Numerous mutational studies have been done todeduce the structural properties of frameshiftingpseudoknots that are important for function(Chamorro et al., 1992; Brierley et al., 1991; Brierley,1995; ten Dam et al., 1994). Three solution NMRstructures (Shen & Tinoco, 1995; Kang & Tinoco,1997; Du et al., 1997) and one X-ray crystal struc-ture (Su et al., 1999) of frameshifting pseudoknots,as well as an NMR structure of one non-frameshift-ing mutant (Kang et al., 1996) have been obtainedin an attempt to deduce a frameshifting mechan-ism. It has been dif®cult to identify conservedstructural features among different frameshiftingsequences that provide insight into the frameshift-ing mechanism. Precise maintenance of frameshiftlevels (Dinman & Wickner, 1992), as well as vari-ation in frameshifting levels among differentviruses, suggests that viruses may use modi®cationof the pseudoknot frameshifting signal to ®ne tunethe level of frameshifting required by each virus(ten Dam et al., 1995). If this is true, then the gener-al requirement of a pseudoknot as a frameshift sig-nal may be conserved, while special features ineach pseudoknot regulate the level of frameshift-ing. Nevertheless, several structural features areconserved. One of the highly conserved structuralfeatures of pseudoknots involved in high ef®ciencyretroviral ÿ1 frameshifting is a very short loop 1crossing the major groove of stem 2. The length ofloop 1 is generally one or two residues and stem 2is generally ®ve to seven base-pairs (Brierley,1995). A sharp turn at the junction of loop 1 andstem 2 is therefore required with close packingbetween loop 1 residues and the major groove ofstem 2. This turn can be stabilized by an RNA-RNA interaction such as a loop residue to stemresidue hydrogen bond, or through the involve-ment of a metal ion, both of which may be director water mediated. In the case of the VPK pseudo-knot this turn is stabilized by a divalent metal ion.

The only other frameshifter pseudoknot struc-ture that demonstrates detailed interactions ofmetal ions is the X-ray crystal structure of thepseudoknot from beet western yellow virus(BWYV; Su et al., 1999). In the BWYV pseudoknot,this sharp turn from loop 1 into stem 2 is mediatedby a hydrogen bond between a loop phosphateoxygen atom (corresponding to the A6 non-bridging phosphate oxygen atom in VPK) and theN4 of a cytosine in the 30 closing base-pair of stem2 (corresponding to the U8 in VPK). The turn isstabilized by an organized network of water mol-ecules which hydrogen bond to non-bridging phos-

phate oxygen atoms throughout the turn. It is notclear that stabilization of this turn in BWYV is ageneral feature of frameshifter pseudoknots, sincethe sequence of loop 1 can vary from one virus toanother and the identity of the ®rst base-pair ofstem 2 also varies. Also, the structure of the BWYVpseudoknot does not reveal the role of Mg2� instabilization of the pseudoknot structure, sinceMg2� is observed to bind at the 50-terminal tripho-sphate group, where it does not mediateany pseudoknot-speci®c interactions. Su andco-workers rationalize that the large concentrationof Na� (relative to Mg2�) present in their crystalli-zation buffer leads to stabilization of the pseudo-knot structure by Na�, rather than Mg2�. In theBWYV pseudoknot, a Na� is seen to bind in theminor groove of stem 1, interacting with bases instem 1 and loop 2. It is rather unlikely that thisNa� ion is exactly mimicking the role of a divalention considering that the Na� has a tetrahedralcoordination geometry while the Mg2� coordi-nation geometry is octahedral. Furthermore, diva-lent metal ions have not been shown to generallybind in the minor groove of RNA and in this casewould probably interfere with the minor groovetriplex that is formed between loop 2 and stem 1(Su et al., 1999).

The stabilization of the loop 1 to stem 2 turn andof the close proximity of loop 1 and stem 2 by adivalent metal ion may be a more general featureof this type of frameshifter pseudoknot regardlessof sequence, as is evident from the extensive non-sequence-speci®c interactions to the phosphateoxygen atoms and 20-hydroxyl groups of the back-bone throughout the turn. Interactions with thebase functional groups in stem 2, particularly theO6 and N7 of guanine bases will, for the mostpart, be conserved due to high G �C content ofstem 2 in frameshifting pseudoknots. In fact, G �Ubase-pairs may form a higher af®nity binding siteby replacing the C amino group with a U carbonylgroup. Furthermore substitution of G �C base-pairswith A �U base-pairs will provide a major groovesurface very similar to the surface formed by G �Cbase-pairs. In this case the G carbonyl group hasbeen replaced by the A amino, but the C aminohas been replaced by the U carbonyl group.

Conclusion

Using Co(NH3)63� as a probe for Mg2�, this study

describes the detailed interaction of a divalentmetal ion with an RNA pseudoknot in solution.The structure is generally well de®ned, althoughinternal dynamics in loop 2 do not allow the pre-cise positioning of loop 2 relative to the otherregions of the pseudoknot. The metal ion bindingpocket formed by loop 1 residues and the majorgroove of stem 2 provides a surface lined withphosphate oxygen atoms, 20 hydroxyl groups, andhydrogen bond acceptors in the bases. Binding ofthe metal ion stabilizes the sharp turn of loop 1


into stem 2 and the close proximity between loop 1and the major groove of stem 2. Although thebinding is weak (Kd � 5(�3) mM), binding of Mg2�

to VPK is likely to be biologically signi®cant due tothe high intracellular concentration of Mg2�

(30 mM) (Cowan, 1995) and the absence of signi®-cant concentrations of other multivalent ions (Feig& Uhlenbeck, 1999). A one to two nucleotide loop1, ®ve to seven base-pair stem 2, and the avail-ability of numerous non-sequence-speci®c hydro-gen bond acceptors, all conserved features of thistype of frameshifter pseudoknot, de®ne the site ofMg2� in stabilization of the pseudoknot andsuggests that this is a general feature of this typeof frameshifter pseudoknot. It is becoming increas-ingly clear that in order to understand RNA fold-ing, we will have to learn the ``rules'' by whichmetal ions bind to RNA and facilitate theformation of compact and complex three-dimen-sional structures that are essential for function. Inthe VPK pseudoknot, a divalent metal ion bringstogether a single-stranded region of the RNA mol-ecule with a helical region, and stabilizes a sharpturn in the molecule. Electrostatic attraction to thephosphates plus water-mediated hydrogen bondsto acceptors on the backbone and the bases pro-vide the binding energy. Because pseudoknots,sharp turns, and close packing of helices andsingle-stranded regions are general features ofRNA structures, we expect that metal ion stabiliz-ation of this type of tertiary interaction will be acommon feature of RNA folds.

Materials and Methods

Sample preparation

RNA oligonucleotides were transcribed in vitrousing phage T7 RNA polymerase and synthetic DNAtemplates (Milligan et al., 1987; Wyatt et al., 1991).RNA from transcription reactions was puri®ed bydenaturing 20 % (w/v) polyacrylamide gel electrophor-esis, removed from gel slices by electro-elution (Simon& Schuster), and concentrated by ethanol precipitation.RNA pellets were dissolved in a 5 mM EDTA solutionand then dialyzed in two steps. The ®rst step con-sisted of dialysis at 4 �C against 5 mM EDTA to che-late any divalent cations. In the second step, RNAsamples were extensively dialyzed at 4 �C against abuffer solution consisting of 10 mM sodium phos-phate, 200 mM NaCl, 100 mM EDTA (pH 6.4). Cobalt(III) hexammine chloride was purchased from Sigma.Stock solution concentrations of cobalt (III) hexamminewere determined by mass and veri®ed by absorbanceat 470 nm (e � 56(�1) Mÿ1cmÿ1) (Kieft, 1997).

Selective 13C-labeling of RNA

Selectively 13C-labeled nucleoside triphosphates werechemically synthesized and enzymatically phosphory-lated as described (SantaLucia et al., 1995). RNA oligo-nucleotides selectively 13C-labeled at adenine C8 anduracil C6 were in vitro transcribed using 13C8-labeledATP and 13C6-labeled UTP and unlabeled GTP and CTPas described (Shen & Tinoco, 1995). Selectively labeled

RNA oligonucleotides were puri®ed and dialyzed asdescribed above.

UV absorbance melting curves

UV absorbance melting curves were recorded ona Gilford 250 spectrophotometer connected to a PC.Heating was controled by a Gilford 2527 Thermo-pro-grammer also connected to the PC. RNA sample concen-trations for melting experiments were 4 mM. Sampleswere heated to 70 �C for 30 seconds and then cooled to0 �C for two minutes prior to data acquisition. Foursamples were heated simultaneously at a heating rate of0.5 deg. C per minute. The data were subsequently pro-cessed using Kaleidagraph software. Data weresmoothed over a 5 deg. C window and absorbance wasplotted against temperature. Melting temperatures weredetermined from the maximum values of the derivativeof absorbance with respect to temperature. Standardenthalpy changes were estimated from the full widths athalf height of the derivative curves.

Equilibrium dialysis

The large amount of RNA needed for equilibriumdialysis experiments prevented us from studying thepseudoknot. Instead we used the P5b hairpin RNAstudied by Kieft & Tinoco (1997) as a model molecule.P5b RNA in 10 mM sodium phosphate, 200 mM Na�,100 mM EDTA (pH 6.4) was placed into one half-cell, andCo(NH3)6

3� in the same buffer was placed into the otherhalf-cell. Equilibrium dialysis experiments were per-formed in 150 ml half-cells separated by a 2000 molecularmass cut off dialysis membrane. The samples wereallowed to equilibrate at 4 �C for 24 hours. This equili-bration temperature and time were determined by moni-toring Co(NH3)6

3� concentration as it diffused across themembrane in the absence of any RNA. Free Co(NH3)6

3�

concentrations were determined using inductivelycoupled plasma (ICP) spectroscopy to measure the con-centration of cobalt in the half-cell without RNA. BoundCo(NH3)6

3� concentration was determined using ICP spec-troscopy to determine the concentration of cobalt in thehalf-cell containing RNA and subtracting the free cobaltconcentration. Equilibrium dissociation binding constantswere determined by Scatchard analysis of the data.

NMR spectroscopy

Sample concentrations for NMR experiments, deter-mined by UV absorbance at 260 nm, were approximately2 mM with the exception of one dimensional (1D)titration experiments where the RNA concentrationswere typically 400 mM. Samples containing Mg2� wereprepared by directly adding 1 M MgCl2 to the dialyzedsample to a ®nal Mg2� concentration of 5 mM. ThisMg2� concentration has been previously determined tobe suf®cient for pseudoknot stabilization (Shen &Tinoco, 1995). Samples containing Co(NH3)6

3� were pre-pared by directly adding 0.5 M Co(NH3)6

3� to the dia-lyzed sample to a ®nal Co(NH3)6

3� concentration whichwas equimolar to the RNA concentration. Samples weretitrated with 1 M MgCl2 or 0.5 M Co(NH3)6

3� to achievethe desired concentrations. Samples for exchangeableproton experiments were lyophilized to dryness andredissolved in 90 % H2O/10 % 2H2O. Samples used fornon-exchangeable proton experiments were lyophilizedto dryness, lyophilized twice from 99.96 % 2H2O, and


then dissolved in 99.96 % 2H2O. Trace amounts of TSP(3-(trimethylsilyl)propionate-d4) were added to allsamples as a chemical shift reference standard. All NMRspectra were processed using FELIX 95.0 (Biosym Tech-nologies, Inc.).

Exchangeable proton 1D spectra were recorded on aGE GN-500 spectrometer equipped with a Bruker HXprobe. Solvent suppression was achieved by the jump-return method with the excitation maximum setbetween the imino and amino/aromatic resonances(Plateau & GueÂron, 1982). A spectral width of10,000 Hz was used to collect 4096 complex datapoints with a relaxation delay of two seconds.

Two-dimensional spectra were collected on a BrukerAMX-600 spectrometer equipped with a Bruker tripleresonance triple-axis gradient probe or a Bruker DRX-500 spectrometer equipped with a Nalorac triple reson-ance z-axis gradient probe. Unless otherwise stated, 400-512 blocks of 32-80 scans consisting of 2048-4096complex points were collected over a spectral width of10,000 Hz (DRX-500) in H2O, or over spectral widths of4000 Hz (DRX-500) or 5000 Hz (AMX-600) in 2H2O. Therelaxation delay was set to two seconds for all exper-iments.

NOESY experiments in H2O were recorded at150 ms, 300 ms, and 400 ms mixing times at tempera-tures of 15 and 30 �C in Mg2� and 20 �C and 35 �C inCo(NH3)6

3�. Solvent suppression was accomplished byreplacing the last pulse in the NOESY sequence witha jump-return sequence and applying a z-gradientpulse during the mixing time. NOESY experiments in2H2O were recorded using a presaturation pulse forsuppression of the residual HDO peak. Zero anddouble quantum artifacts at short mixing times weresuppressed by applying a z-gradient pulse during themixing time and incrementing the mixing time fromone block to the next for a total of 5 ms incrementa-tion over the full experiment time. The 2H2O NOESYspectra was acquired at 60 ms, 150 ms, 300 ms, and400 ms mixing times at temperatures of 15 and 30 �Cfor Mg2� and 20 �C and 35 �C for Co(NH3)6

3�.DQF-COSY experiments were recorded using a presa-turation pulse for suppression of the residual HDOpeak. The 13C-resolved HSQC-NOESY experiment wasdone in 90 % H2O/10 % 2H2O. The pulse sequence ofMajumdar & Zuiderweg (1993) was used to providewater suppression via pulsed ®eld gradients withoutintroducing an unfavorable excitation pro®le.

Structural constraints

Distance constraints were used to maintain hydrogenbonds for base-pairs which clearly formed as seen in theH2O NOESY experiments. Four distance constraintswere used for the G �C base-pairs, three for the A �Ubase-pairs, and ®ve for the G �U base-pairs. The hydro-gen bond range was set to �0.1 AÊ . All observed imino toimino NOE crosspeaks were from G iminos in G �C base-pairs, and were assigned a distance range of 3.2 to 4.5 AÊ .Intra- and intermolecular NOE crosspeaks for non-exchangeable protons were classi®ed into distance rangecategories based on their intensities in 60 ms, 150 ms,and 300 ms mixing time NOESY experiments in 2H2O.Co(NH3)6

3� protons were treated as non-exchangeable,since they do not exchange on the time-scale of oneblock of scans (Anderson et al., 1943). NOE crosspeaks tothe Co(NH3)6

3� were referenced to a pseudo-atom at theposition of the cobalt atom, with appropriate distance

corrections. NOE crosspeaks were classi®ed as strong(1.8-3.2 AÊ ), medium (2.0-4.0 AÊ ), weak (2.5-5.0 AÊ ), andvery weak (3.0-6.0 AÊ ). Crosspeaks which could arisefrom a spin diffusion pathway were put into a distancerange of 2.0-5.0 AÊ .

Ribose sugar conformations, or d torsion angles, weredetermined based on H10-H20 scalar couplings observedin DQF-COSY experiments. Backbone torsion angles (a,b, g, e, and z) were constrained to standard A-form(Saenger, 1984) in the helical stems of the pseudoknotbased on observed scalar couplings and NOE connectiv-ities that indicated A-form structure. The backbone tor-sion angles of the opening and closing base-pairs of stem2, closing base-pair of stem 1, and all loop nucleotideswere not constrained.

Structure calculations

The molecular dynamics program X-PLOR was usedto do the structure calculations (BruÈ nger, 1993). TheCo(NH3)6

3� complex ion was built using the Buildermodule within the INSIGHT II suite of programs (Bio-sym Technologies, Inc.). Partial charges and Lennard-Jones potentials for the Co(NH3)6

3� ion were taken fromthe ESFF force®eld values. The structure of the Co(NH3)6

3� ion prepared in this manner is in good agree-ment with the published crystal structure (Kruger &Reynhardt, 1978). A total of 100 starting structures weregenerated with randomized backbone torsion angles a,b, g, d, e, and z, and randomized positioning of theCo(NH3)6

3� complex ion. The structure calculation wasperformed in three stages. The global fold protocol con-sisted of terms including bond lengths, bond angles,improper angles, and the NMR-derived distance and tor-sion angle constraints. The bond angle and bond lengthforce constants were set to 1000 kcal molÿ1 AÊ ÿ2 and500 kcal molÿ1 radÿ2, respectively. The NOE force con-stant was set to 50 kcal molÿ1 AÊ ÿ2 and the torsion angleforce constant was initially set to 5 kcal molÿ1 radÿ2 andincreased throughout the calculation to a ®nal value of50 kcal molÿ1 radÿ2. The protocol consisted of 500 cyclesof energy minimization of the randomized starting struc-ture. This was followed by restrained moleculardynamics (rMD) at 1000 K followed by rMD duringcooling to 300 K. Finally 100 cycles of energy minimiz-ation were done. All NOE distance constraints wereused during the global fold protocol, however intermole-cular Co(NH3)6

3� to the RNA distance constraints weregiven a loose, initial range of 0-10 AÊ . Only torsion anglesspecifying the sugar puckers and keeping amino groupsin the plane of the base-pairs were used during theglobal fold protocol. The second stage of structure calcu-lation was a re®nement of the converged structures. Allof the previous terms used in the global fold were usedfor the structure re®nement with the addition of thebackbone torsion angles. The intermolecular Co(NH3)6

3�

to RNA distance constraints were now assigned semi-quantitative values according to the NOE data using thesame criteria and categories as for the RNA non-exchangeable protons. The re®nement protocol consistedof 500 cycles of energy minimization, rMD at 1000 Kduring which the backbone torsion angles were intro-duced in two stages: ®rst, b,g, and e were introduced,followed by a and z, rMD while cooling to 300 K, and100 cycles of energy minimization. In the ®nal stage ofstructure calculation, these re®ned structures underwentrMD at 300 K followed by 3000 cycles of energy mini-mization which now included all the terms used in thestructure re®nement with the addition of Lennard-Jones


potentials. This structure calculation protocol resulted ina family of converged structures which were viewedusing INSIGHT II.

Acknowledgments

This research was supported in part by National Insti-tute of Health Grant GM 10840, by the Department ofEnergy Grant DE-FG03-86ER60406, and through instru-mentation grants from the Department of Energy (DE-FG05-86ER75281) and from the National Science Foun-dation (DMB 86-09305). We thank Ms Barbara Denglerfor managing the laboratory, Mr David Koh for synthe-sizing DNA oligonuceotides, and Dr Jeffrey Pelton forvaluable NMR advice.

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Edited by D. E. Draper

(Received 12 February 1999; received in revised form3 May 1999; accepted 4 May 1999)

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Supplementary material for this paper comprisingtwo Tables is available from JMB Online

solution structure and thermodynamics of a divalent metal ion … · 2015-12-29 · berkeley and...

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