The Crystal Structure Of A Hyperthermophilic Archaeal TATA .

The Crystal Structure of Pyrococcus woesei TBPabdyad symmetric and deviate from one another witha root mean squared difference (RMSD) of 1.8 Å forcorresponding Ca atoms. If the N-terminal substructure of PwTBP is superimposed on itscounterparts from ScTBPc and AtTBP, the RMSDsfor corresponding Ca atoms are 1.9 Å and 2.0 Å,respectively (Figure 3a). The same alignment of theC-terminal substructures results in a RMSD of 2.5 Åbetween PwTBP and ScTBPc and 2.6 Å betweenPwTBP and AtTBP (Figure 3b). The sequences ofScTBPc and AtTBP are 81% identical, which isreflected in a higher structural similarity betweenthem than between either and PwTBP. A compari-1075Figure 3. Ca carbon traces ofPwTBP com ared to that of ScTBPc.a, Superposition of N-terminal substructure of PwTBP (filled blackbonds) and ScTBPc (outlinedbonds) numbered according toPwTBP sequence and orientedroughly like the N-terminal substructure in the right-hand side ofFigure 2a. Pro53 and the corresponding ScTBPc residue are in thecis configuration. Residues 33 and48 are connected by a disulfide bondin the PwTBP structure. Extendedloop in PwTBP structure betweenresidues 90 and 102 is the result ofa sequence insertion in this region(Figure 1). b, C-terminal substructure and a short sequence of the Nterminus of PwTBP (filled blackbonds) superimposed uponScTBPc(outlined bonds) oriented as in a.Numbered according to PwTBPsequence. Pro144 and the corresponding ScTBPc residue are in thecis configuration.son of the two substructures of the eukaryotic TBPsresults in 1.0 Å and 1.5 Å RMSD between corresponding Ca atoms for the N-terminal andC-terminal repeats, respectively.Proline residues 53 and 144 of PwTBP are in thecis configuration as are their eukaryotic counterparts. Cysteine 33 and cysteine 48 are unique to thetwo sequenced hyperthermophilic archaeal TBPs,P. woesei and T. celer (Figure 1), and form a disulfidebond (Figures 2 and 4). Electron density insimulated annealing omit maps (Brünger, 1993), inwhich both cysteine residues and an area encompassing a radius of 8 Å surrounding them wasFigure 4. Stereo pair showing 2Fo Fc electron-density map contoured at 2.0s. The disulfide bond between Cys33and Cys48 is shown.

1076The Crystal Structure of Pyrococcus woesei TBPacbdFigure 5. Electrostatic potential surrounding PwTBP (a) and ScTBPc (b) calculated in 100 mM salt and rendered withthe program GRASP (Nicholls et al., 1993). Orientation of the protein is the same as the red molecule of Figure 2a.Positive potentials are blue and negative potentials are red with both contoured at a magnitude of 1 kT. Chargednitrogen atoms are shown in blue, charged oxygen atoms red, and hydrophobic residues green. Electrostatic potentialof PwTBP (c) and ScTBPc (d) labeled the same as above and oriented as the red molecule in Figure 2c.omitted, confirmed a disulfide bond between them.Based on this density they were refined as cystine.Proline 232 (yeast numbering), which is highlyconserved in eukaryotic TBPs and is responsible fora kink in the H2' helix, is not present in the PwTBPsequence. This allows the H2' helix of PwTBP toremain linear, as is the H2 helix in all TBPstructures, archaeal and eukaryotic.The electrostatic potential surrounding PwTBP isdramatically different than that of its eukaryoticcounterparts (Figure 5). This is due to more acidicresidues on the surface of the PwTBP molecule thatresult in more ion-pairs (Table 2) and a corresponding neutralization of the positive potential seen inthe eukaryotic structures. The possible implicationsof this on DNA affinity is discussed below.shown). Consistent with the dimer formation seenin solution, the crystal structures of all TBPs reveala dimer in the asymmetric unit, with the monomersrelated by a non-crystallographic dyad (Figure 2).In all crystal structures (eukaryotic and archaeal)the C-terminal repeats make up most of the dimerinterface and mask the hydrophobic DNA-bindingsurface of each molecule. As shown in Figure 2,the two eukaryotic TBPs dimerize in a similarmanner with a solvent-excluded interface for eachmonomer of 1600 Å2. The interface between the twomonomers in the asymmetric unit of PwTBP alsoburies 1600 Å2 of solvent accessible surface area(Table 2), but the nature of the interface is distinctlydifferent (Figure 2), and thus a specific dimerarrangement is not evolutionarily conserved.Homodimer formationHyperthermostabilityAs seen for the eukaryotic TBPs, HsTBP, AtTBP,and ScTBPc (Coleman et al., 1995; Nikolov &Burley, 1994), the archaeal TBP is a dimer insolution as determined by gel filtration (data notTo test the thermostability of PwTBP, the meltingtemperature was determined by differential scanning calorimetry (Figure 6). The midpoint ofthe thermal denaturation was 101 C in 50 mM

1077The Crystal Structure of Pyrococcus woesei TBPTable 2. Structure statisticsaPwTBPScTBPcAtTBPTotal number of atoms140314151421Hydrogen bonds 010,100/10,4001500Ion pairs (no.)cMonomersDimer interfaceSolvent-accessible surface area (Å2 )dMonomersBuried in dimer interfaceSolvent-accessible surface area composition (%)Non-polar56/55Polar18/19Charged26/26Of charged % 3)60/5917/1623/25(69/70)46/4545/4346/46Protein density0.818/0.8150.796/0.7980.793/0.796Void volume (Å3 )g4100/42004800/47004900/4800Atoms buried (%)efaCalculations include both molecules in the asymmetric unit (A/B) and residues 5 to 184 of thePwTBP structure, 19 to 198 of the AtTBP structure (Nikolov et al., 1992), 61 to 240 of the ScTBPcstructure (J. H. Geiger, crystal structure refined at 2.6 Å).bHydrogen bonds were defined by the method of Kabsch & Sander (1983).cIon-pairs were defined as two ionizable groups E4 Å apart (Barlow & Thornton, 1983).dThe algorithm of Lee & Richards (1971) using a probe radius of 1.4 Å.eAtoms with no solvent accessible surface.fMolecular volumes using the program PQMS (Connolly, 1985), and the atomic radii reportedby Rashin et al. (1986). Data for all structures compared were collected on frozen crystals. van derWaals volume, volume calculated with zero Å radius probe. Solvent-excluded volume, volumecalculated with 1.4 Å radius probe. Protein density, van der Waals volume/solvent-excludedvolume.gVoid volume solvent-excluded volume van der Waals volume.potassium phosphate (pH 7.0). Under reducingconditions the transition decreased to 97 C and inhigh salt (800 mM potassium phosphate) thetransition increased to 109 C. Thus, pure bacteriallyexpressed PwTBP is, indeed, extremely thermostable indicating that neither endogenous cellular factors nor in vivo modifications are needed forthis property.Pw TBP-DNA interactionA thorough analysis of the salt and temperaturedependence of PwTBP-DNA interactions ispresently under way. Preliminary results indicate asalt and temperature dependence for the binding ofPwTBP to its DNA target that is distinctly differentfrom that observed for its eukaryotic counterparts.At room temperature gel mobility shift assays showweak affinity of PwTBP for the elongation factor 1apromoter, AAGCTTTAAAAAGTAA (box A sequence underlined; Rowlands et al., 1994). Atapproximately the intracellular salt, concentrationfound in P. woesei, 800 mM potassium phosphate(Scholz et al., 1992), titration calorimetry experiments show that PwTBP binds specifically to thesame box A sequence with a dissociation constant(Kd ) of 13 mM at 25 C (data not shown). Increasingthe salt concentration to 1.3 M potassium phosphate enhances affinity 24 times while decreasing itto 50 mM potassium phosphate abolishes anydetectable enthalpy of binding. Earlier gel mobilityshift assays by Rowlands et al. (1994), indicatedenhanced affinity at 55 to 60 C, and titrationcalorimetry experiments corroborate this by showing that affinity is enhanced 30 times when thetemperature is raised to 45 C. If both the saltconcentration and temperature are elevated to1.3 M potassium phosphate and 45 C, then theaffinity is enhanced 370 times to give a Kd of 35 nM,as shown by titration calorimetry. By contrast, theaffinity of ScTBP for the TATA-box (Kd 2.4 nM;Hahn et al., 1989), is diminished 300 times when thesalt concentration is increased from 50 mM to300 mM potassium chloride and also decreases attemperatures exceeding 30 C (Petri et al., 1995).Although the temperatures for these in vitro studiesare well below the optimal growth temperature ofP. woesei (105 C), it is clear that elevatedtemperature and increased salt concentrationenhance the stability of the archaeal complex atlevels which destabilize the eukaryotic interaction.Interface with general and specifictranscription factorsThe crystal structure of AtTBP complexed withHomo sapiens TFIIB and DNA demonstrated thatTFIIB interacts with the C-terminal ‘‘stirrup’’ ofAtTBP (Nikolov et al., 1995). Of the eight residuescontacted by TFIIB, all but two are conserved or aresimilar in PwTBP, and there are no charge reversals(Figure 1). Not surprisingly the TFIIB homolog in

1078The Crystal Structure of Pyrococcus woesei TBPactivators have been shown to bind PwTBP(Rowlands et al., 1994). Since PwTBP contains abasic region on the H2 helix similar to that ofeukaryotic TBPs, it is possible that PwTBP’sinteractions with some eukaryotic activators arealso similar to that of eukaryotic TBPs.DiscussionHyperthermostabilityFigure 6. Thermal transitions of PwTBP and ScTBPcmeasured by differential scanning calorimetry. a,Exothermic denaturation/aggregation of ScTBPc (50 mMpotassium phosphate pH 7.0, 10% glycerol) withtransition and presumed unfolding occurring about 60 C.b to d, Unfolding transition of PwTBP in (b) reducingconditions (2 mM dithiothreitol (DTT), 50 mM potassiumphosphate, pH 7.0), (c) oxidizing conditions (50 mMpotassium phosphate, pH 7.0) which is identical with10% glycerol added; and (d) high salt and oxidizingconditions (800 mM potassium phosphate, pH 7.0).archaea, TFB, forms strong interactions with botharchaeal and eukaryotic TBP/DNA complexes(Rowlands et al., 1994; Ghosh et al., unpublished).This is consistent with the fact that well orderedcrystals of the PwTFB/PwTBP/A-box have beenreported (Kosa et al., 1996).Yeast TFIIA (ScTFIIA) is a negatively chargedmolecule that interacts with four basic residuesalong the N-terminal ‘‘stirrup’’ of ScTBPc (Geigeret al., 1996; Tan et al., 1996). None of these basicresidues in ScTBPc are conserved in PwTBP, andtwo are of opposite charge. In fact, most of the TBPresidues that contact TFIIA in the ScTBPc/ScTFIIA/TATA complex are not conserved (Figure 1).A negative potential surrounds the N-terminalstirrup of PwTBP (Figure 5) and this negativepotential would repel the negative charge of a yeastlike TFIIA molecule. Additionally, the disulfidebond in PwTBP alters the conformation of the S2strand of PwTBP relative to that of the eukaryoticTBPs. This S2 strand forms the predominantinteracting surface of ScTBPc with ScTFIIA. Anarchaeal TFIIA homolog has not yet been identified,and if one were to exist, its binding surface to TBPwould have to be significantly different.Basic residues project from the surface of the H2helix of PwTBP in a manner similar to that foundin eukaryotic TBPs (Figure 5c and d). This region,in eukaryotic TBPs, has been implicated in bindingthe activation domains of eukaryotic transcriptionfactors such as p53 and E1A (Lee et al., 1991; Liuet al., 1993). Although endogenous activators havenot been found in Archaea, these eukaryoticHyperthermophilic organisms are those whichgrow optimally at temperatures exceeding 90 C(Adams, 1993). Based on phylogenetic studies, mosthyperthermophilic genera have been classified asArchaea, while two are Bacteria (Woese et al., 1990).P. woesei is a hyperthermophilic archaeal species,and as expected, the TBP molecule from thisorganism is stable at the high temperatures. PwTBPhas an unfolding transition of 101 C which is 40 Chigher than ScTBPc under comparable conditions(Figure 6). While the free energy level of both theunfolded and native forms contribute to thethermostability of a protein (Dill & Shortle, 1991),this study can only address contributions tostability from the native state since the denaturationis irreversible.In the crystallographic study of the hyperthermophilic enzyme aldehyde ferredoxin oxidoreductase, the authors conclude that an unusually highfraction of buried atoms (55%) and a correspondingreduced solvent accessible surface area correlateswith the thermostability of that molecule (Chanet al., 1995). The amount of solvent accessiblesurface area of PwTBP, although less than itseukaryotic homologs is still 7% greater than theaverage protein of its size (Miller et al., 1987). Inaddition, PwTBP does not bury an unusual largefraction of its atoms and is identical in this respectto the mesophilic TBPs (Table 2).One unusual feature of the PwTBP structure thatcould contribute to its thermostability, is its singledisulfide bond. Typical of intracellular proteins,disulfide bonds have not been found in themesophilic TBPs. However, when the unfoldingtransition temperature of PwTBP under oxidizingconditions was compared with the transition in areducing environment (Figure 6), only a modestdecrease of 4 C in the transition temperature wasobserved under reducing conditions.Four recent studies comparing the crystalstructures of hyperthermophilic proteins to those oftheir mesophilic homologs revealed an increase inthe number of ion-pairs in the hyperthermophilicversions (Day et al., 1992; Hennig et al., 1995;Korndörfer et al., 1995; Yip et al., 1995). The numberof ion-pairs, defined as two ionizable groups E4 Åapart (Barlow & Thornton, 1983), were calculatedfor the known TBP structures. PwTBP containseight ion-pairs on its surface and in comparison, theeukaryotic TBPs have four and five surfaceion-pairs (Table 2). Although PwTBP contains moresurface ion pairs than the mesophilic TBPs, the

1079The Crystal Structure of Pyrococcus woesei TBPefficiencies could contribute to the thermostability of PwTBP by increasing the associatedfavorable van der Waals interactions (Richards &Lim, 1994).Rather than a single specific structural attribute,a combination of chemical characteristics known tostabilize mesophilic proteins may work together toincrease the thermostability of PwTBP. Crystalstructures of proteins from hyperthermophilicorganisms, including PwTBP described here, highlight several of these chemical characteristics forincreasing protein thermostability: (1) disulfidebonds; (2) more ion-pair interactions; (3) increasedburied surface area; (4) more compact packing.Pw TBP-DNA interactionFigure 7. Atomic packing in homologous proteinstructures. The mean packing value for each proteinwithout prosthetic groups was calculated from analysisof occluded surface as described in the text. Data pointsare labeled with the respective PDB identifier codes. Thehomolog families shown for comparison are all groups ofhigh resolution crystal structures ( 2.2 Å) available inthe PDB that have high sequence divergence among themembers. The numbers in parentheses are the averagenumbers of amino acid residues per protein in therespective groups. Monomers of the dimeric TATAbinding proteins found in the asymmetric unit arelabeled a and b.observation of increased thermostability at highersalt concentrations for PwTBP (Figure 6) arguesagainst electrostatic interactions as the predominant stabilizing force, since the stabilizing effect ofa surface ion-pair is not likely to be enhanced byraising the ionic strength of the buffer.Richards observed that the interiors of proteinsare tightly packed with densities similar to thoseof crystals of small organic molecules (Richards,1974). Studies of the density of protein interiors haveimplicated ‘‘packing efficiency’’ as a factor in proteinstability (for review, see Richards & Lim, 1994). Amean protein packing value was calculated for theTBP structures and the packing value of PwTBP is6% greater than that of AtTBP, the closest mesophilicTBP (Figure 7). A survey of packing values is shownfor several families of homologous proteins in Figure7. PwTBP is clearly the most efficiently packed; butmore important, its packing value deviates from thatof its homologs by a substantial margin, whereas thepacking values of other families are more tightlyclustered. A complete analysis of the packingefficiency comparing hyperthermophiles and theirmesophilic counterparts is currently under way. Theincreased packing density of PwTBP is due mostly tothe contraction of several segments toward theprotein core as demonstrated in the differencedistance matrix (Figure 8). The contraction of PwTBPhas eliminated about 12% of the void volume foundwithin the solvent-excluded volume of themesophilic proteins (Table 2). The elimination of thisvoid space and consequent increase in packingThe structure of PwTBP gives a plausibleexplanation for the different effects of salt andtemperature on the affinity of eukaryotic andarchaeal TBPs for their respective DNA targets. Themajority of contacts between eukaryotic TBPs andDNA are hydrophobic in nature (J. L. Kim et al.,1993; Y. Kim et al., 1993). Unlike ionic and polarcontacts, hydrophobic interactions are not diminished by high salt and elevated temperature; in fact,they are often strengthened under these conditions.In PwTBP, 21 of the 25 residues which are involvedin hydrophobic contacts between eukaryotic TBPsand the TATA-box are preserved, and modelingsuggests that those which differ would not bedisruptive to the PwTBP/DNA complex. Moreover,these ionic/polar contacts between eukaryotic TBPand the DNA backbone are replaced by residuesthat can only form van der Waals contacts with thesugars. Since the hydrophobic effect is predominantly entropic, an enhanced affinity of TBP forDNA might be expected at higher temperatureswhich is indeed the case for PwTBP. Thateukaryotic TBP’s affinity for DNA decreases atelevated salt concentrations may be explained bythe fact that in the case of the eukaryotic complexthe hydrophobic interactions are supplemented bypolar contacts (J. L. Kim et al., 1993; Y. Kim et al.,1993) and the protein’s positive electrostaticpotential. In addition to providing an attractivebinding force, the positive potential flanks thephosphates of the bound TATA-box helping tosplay open the minor groove of the DNA and,thereby bend the DNA target (J. L. Kim et al., 1993;Y. Kim et al., 1993). Thus, high salt concentrationswould be expected to diminish these electrostaticcontributions to the stability of eukaryotic TBP/DNA complexes. In PwTBP, however, the largenumber of surface acidic side-chains that form thefrequent ion pairs noted above in connection withthermal stability, create a nearly electrostaticallyneutral surface (Figure 5). Thus, the PwTBP/DNAcomplex would not be stabilized by the electrostaticinteractions to the extent seen in the eukaryoticTBP/DNA complexes, and increasing the saltconcentration would not be expected to reducePwTBP’s affinity for DNA as it does to ScTBP’s.

1080The Crystal Structure of Pyrococcus woesei TBPProtein evolutionIn many ways, the hyperthermophilic genera ofthe Bacteria and Archaea appear to be the mostancient of their respective kingdoms (Woese et al.,1990). It has been proposed that the commonancestor of all existent life was a hyperthermophile,and that relatively modern mesophilic biology isadapted from an earlier hot env

Disulfide shown between PwTBP residues 33 and 48. Boxed in blue are residues conserved among eukaryotes and in red are residues conserved between PwTBP and ScTBPc. Residues involved in binding TFIIA, TFIIB, and DNA are labeled a, b, and *, respectively. known archaeal TBPs but is not found in any eukaryotic TBP sequences. Sequence identity