solution structure of the dna- and rpa-binding domain of the human repair factor xpa

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Solution structure of theDNA- and RPA-bindingdomain of the humanrepair factor XPA

tions were obtained which were related by an approximate non-crystallographic two-fold axis observed in self-rotation functions.Phases calculated from the native MUG structure positionedaccording to the molecular replacement solutions were improvedusing solvent flattening and averaging with DM, and used to cal-culate Fourier maps. Strong electron density for a single duplex ofDNA was visible in these maps, and allowed the construction ofan initial model for the DNA dodecamer. Although the DNAduplex is formed by a self-complementary sequence, the twostrands are not structurally identical, and the non-crystallograph-ic two-fold symmetry that relates the two protein molecules doesnot relate the two strands of the DNA. The structure has beenrefined to 2.8 Å resolution by cycles of simulated annealing andpositional refinement using X-PLOR, alternated with manualadjustment using ‘O’. Omit maps have been used to minimizemodel bias in the definition of the central nucleotides of the DNAduplex which do not have a canonical B-DNA structure. The finalmodel consisting of 2,466 protein atoms, 472 DNA atoms and 93well defined solvent atoms has an R-factor of 0.206 and an R-freeof 0.286 (Table 1).

Coordinates. Coordinates for the refined MUG structure havebeen deposited with the Brookhaven Protein Data Bank (acces-sion code 1mug).

AcknowledgmentsWe thank our colleagues for assistance with data collection and the LudwigInstitute for Cancer Research and the CLRC Daresbury Laboratory for provisionof X-ray diffraction facilities. We are particularly grateful to P. Hopkins and S.Thor Sigurdsson for making the coordinates of the cisplatin adduct availableto us. This work was supported by the Cancer Research Campaign, as well asby the Schweizerische Krebsliga (J.J.) and the Julius Müller Stiftung (J.J.).

Tracey E. Barrett1, Renos Savva1,4, Tom Barlow2, TomBrown2, Josef Jiricny3 and Laurence H. Pearl

1Department of Biochemistry and Molecular Biology, University CollegeLondon, Gower Street, London WC1E 6BT, UK. 2Department of Chemistry,University of Southampton, Highfield, Southampton, SO17 1BJ, UK. 3Instituteof Medical Radiobiology, August Forel-Strasse 7, 8029 Zürich, Switzerland.4Present address: Laboratory of Molecular Biology, Department ofCrystallography, Birkbeck College London, Malet Street, London WC1E 7HX,UK.

Correspondence should be addressed to: L.H.P. email: [email protected]

Received 1 April, 1998; accepted 3 July, 1998.

1. Lindahl, T. & Karlström, O. Biochemistry 12, 5151–5154 (1973).2. Lindahl, T. & Nyberg, B. Biochemistry 13, 3405–3410 (1974).3. Nakabeppu, Y., Kondo, H. & Sekiguchi, M. J. Biol. Chem. 259, 3723–3729 (1984).4. Sakumi, K. et al. J. Biol. Chem. 261, 5761–5766 (1986).5. Bjoras, M., Klungland, A., Johansen, R.F. & Seeberg, E. Biochemistry 34,

4577–4582 (1995).6. Nedderman, P. & Jiricny, J. J. Biol. Chem. 268, 21218–21224 (1993).7. Dianov, G. & Lindahl, T. Curr. Biol. 4, 1069–1076 (1994).8. Barrett, T.E. et al. Cell 92, 117–129 (1998).9. Gallinari, P. & Jiricny, J. Nature 383, 735–738 (1996).

10. Seeberg, E., Eide, L. & Bjørås, M. Trends Biochem. Sci. 20, 391–397 (1995).11. Mol, C.D., Kuo, C.F., Thayer, M.M., Cunningham, R.P. & Tainer, J.A. Nature 374,

381–386 (1995).12. Gorman, M.A. et al. EMBO J. 16, 6548–6558 (1997).13. Slupphaug, G. et al. Nature 384, 87–91 (1996).14. Cuniasse, P. et al. Nucleic Acids Res. 15, 8003–8022 (1987).15. Kalnik, M.W., Chang, C.N., Johnson, F., Grollman, A.P. & Patel, D.J. Biochemistry

28, 3373–3383 (1989).16. Manoharan, M., Ransom, S.C., Mazumder, A. & Gerlt, J.A. J. Am. Chem. Soc. 110,

1620–1622 (1988).17. Goljer, I., Kumar, S. & Bolton, P.H. J. Biol. Chem. 270, 22980–22987 (1995).18. Wang, K.Y., Parker, S.A., Goljer, I. & Bolton, P.H. Biochemistry 36, 11629–11639 (1997).19.Huang, H., Zhu, L., Reid, B.R., Drobny, G.P. & Hopkins, P.B. Science 270, 1842–1845

(1995).20. Leslie, A.G.W. Mosflm users guide (MRC-Laboratory of Molecular Biology,

Cambridge, UK, 1995).21. CCP4. Acta Crystallogr. D 50, 760–763 (1994).22. Navaza, J. Acta Crystallogr. A 50, 157–163 (1994).

The solution structure of the central domain of the humannucleotide excision repair protein XPA, which binds to dam-aged DNA and replication protein A (RPA), was determined bynuclear magnetic resonance (NMR) spectroscopy. The centraldomain consists of a zinc-containing subdomain and a C-ter-minal subdomain. The zinc-containing subdomain has a com-pact globular structure and is distinct from the zinc-fingersfound in transcription factors. The C-terminal subdomainfolds into a novel α/β structure with a positively chargedsuperficial cleft. From the NMR spectra of the complexes, DNAand RPA binding surfaces are suggested.

Nucleotide excision repair is the ubiquitous pathway by whicha broad spectrum of structurally unrelated DNA damage isremoved from the genome. The importance of nucleotide exci-sion repair has been highlighted by studies on the human inher-ited disease, xeroderma pigmentosum (XP)1. Cells from XPpatients have defects in nucleotide excision repair and thereforeare hypersensitive to UV irradiation. Complementation analysis

have identified seven complementation groups (A–G) and avariant form in the XP cells. The gene that complements XPgroup A cells encodes XPA, which preferentially binds to UV- orchemically damaged DNA, suggesting that XPA is involved in thedamage recognition step of nucleotide excision repair2–6. XPAhas also been shown to bind to other repair factors7–15.Experiments in vitro have shown that XPA has a moderatelyhigher affinity for damaged DNA than for undamaged DNA.Moreover, its binding to damaged DNA is enhanced in the pres-ence of RPA7,8. It has also been shown that the binding activity ofXPA both to damaged DNA and to undamaged DNA is increasedby interaction of XPA with the excision repair cross-comple-menting rodent repair deficiency group 1 protein, ERCC114.Thus, it has been suggested that XPA may play a role in loadingthe incision protein complex onto a damaged site, as a multi-functional protein that coordinates the early steps of nucleotideexcision repair processes9,11.

XPA consists of several distinct functional domains. Its N-termi-nal portion (residues 4–97) contains RPA34- and ERCC1-bindingregions9,10,12–14. The C-terminal portion (residues 226–273) hasbeen shown to bind to TFIIH10,11. The central domain (residues98–219) was identified as the minimal polypeptide essential for thebinding to damaged DNA, using a combination of limited proteol-ysis and deletion analysis6. The domain also includes the regionessential for binding to RPA70, the largest single-strand (ss)DNA-binding subunit of the RPA trimer, which is indispensable for theearly step of nucleotide excision repair8,9. The central domain con-

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tains the zinc-binding sequence of Cys-X-X-Cys-(X)17-Cys-X-X-Cys in its N-terminal portion16 (Fig. 1a).

Structure determinationThe solution structure of the central domain of XPA was solved bymultidimensional double and triple resonance NMR spectroscopy.The structure calculations, using simulated annealing, were basedon a total of 1,336 nuclear Overhauser effect (NOE)-derived inter-proton distance restraints and 83 φ dihedral angle NMR-derivedrestraints. A summary of the structural statistics for a set of the final30 structures, which show the lowest energy and no violationgreater than 0.3 Å is listed in Table 1.

The central domain of XPA consists of a zinc-containing subdo-main (residues 102–129) and a C-terminal subdomain (residues138–209), connected by an eight amino acid linker sequence (Fig.1b,c). Backbone and hydrophobic side chains within each subdo-main are well defined except for parts of the loop regions (Table 1).As a total of 42 NOE contacts between the zinc-containing subdo-main, the C-terminal subdomain, and the linker sequence wereobserved in the NOESY spectra, the relative orientation of the twosubdomains, as well as that of the linker sequence, are relatively welldefined (Fig. 1b and Table 1). Since we observe few medium- andno long-range NOEs for the C-terminal sequence (residues211–219) this region was excluded from the structure calculations.

The zinc-containing subdomainThe overall structure of the zinc-containing subdomain, which iscomposed of an antiparallel β-sheet and a helical turn, is differentfrom those of other zinc-fingers so far reported (Fig. 2a,b), while itslocal structure around the four cysteine residues, including hydro-gen bond networks, is common to the (Cys)4 type zinc-fingers ofGATA-1 and other zinc fingers17. Residues 103–112 form a β-hair-pin structure which encompasses Cys 105 and Cys 108, whose sidechains coordinate a zinc ion. The helical turn ranges from Tyr116–Phe 121, and the following loop (residues 122–129) providesthe other two cysteine residues, Cys 126 and Cys 129, whose sidechains coordinate the zinc ion. The structure of this subdomain isdominated by the zinc ion, which is coordinated to the Sγatoms ofthe four cysteine residues in an S configuration as defined by Berg18.The subdomain is also stabilized by the hydrophobic core formedby Val 103, Phe 112 and Met 118 (Fig. 2a).

The zinc-containing subdomain has a hydrophobic patchmade up of Tyr 116, Leu 117, Phe 121 and Leu 123 on the outersurface of the helical turn (Fig. 2b). These hydrophobic residuesalso serve as part of a hydrophobic core which contains residuesin the C-terminal subdomain, Leu 138, Ile 165, Leu 182 and Leu184. The core is formed by packing the three-stranded β-sheet ofthe C-terminal subdomain against the helical turn of the zinc-containing subdomain (Fig. 2c). A total of 101 NOE contacts

Fig. 1 a, Sequence alignment of the centraldomains of human XPA and other XPAs. Thenumbering is shown for human XPA. Theasterisks indicate the zinc-coordinated cys-teine residues16. The residues identical tohuman XPA are boxed. The secondary struc-ture of human XPA is indicated. Importantresidues discussed in the text are colored(green, basic; yellow, acidic; grey, hydropho-bic). b, Best-fit backbone superpositions ofthe 30 final structures of the central domainof human XPA (residues 98–210) in stereo.The backbone atoms of residues 102–155,163–165 and 180–209 are superimposed.Loops L1 (residues 148–163) and L2 (residues166–179) are colored green. c, Schematic rib-bon drawing of the NMR structure of thecentral domain of human XPA in stereo,drawn with the programs MOLSCRIPT29 andRASTER3D30. α-helices are green and β-strands are red. Secondary structure ele-ments are indicated.

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among the hydrophobic residues that form the core wereobserved in the NOESY spectra. Therefore, the side chainrotamers of Tyr 116, Leu 117, Phe 121, Leu 123, Leu 138 and Leu182 are well defined.

The zinc-containing subdomain and the subsequent linkersequence both include many glutamate and aspartate residues(9/36) (Figs 2b, 4a). This highly acidic feature, which is also seenin the zinc-binding sequences of XPAs from other species, is alsodistinct from those of the zinc-fingers of transcription factors.

The C-terminal subdomainThe C-terminal subdomain consists of a sheet-helix-loop region(residues 138–182) and a helix-turn-helix region (residues183–209) (Figs. 3a,b). The sheet-helix-loop region is composedof an antiparallel β-sheet (strands β3–5), helix α1, and two longloops, L1 and L2. Loop L1 (residues 148–163) connects the α1helix and the β4 strand, and loop L2 (residues 166–179) con-nects strands β4 and β5. While the structure of loop L1, exceptfor residues 156–162, has been relatively well defined byhydrophobic contacts with residues in helices α1 and α2, loop L2apparently does not form a single definite structure based on theobservation of few long-range NOEs. The signals for residues171–174 could not be observed in (15N, 1H) correlation (HSQC)

spectra, possibly due to local exchange broadening and/or rapidexchange of the amide protons with solvent. Steady-state heteronuclear 15N{1H}-NOE values19 smaller than 0.68 wereobserved for the residues 157–162 in loop L1, for all the residuesin loop L2 except residues 171–174 whose signals could not beobserved in the HSQC spectra, and for the nine C-terminalresidues of the central domain (data not shown). This indicatesthat L2, part of L1 and the C-terminal flanking sequence arehighly mobile in solution.

The helix-turn-helix region in the C-terminal subdomainconsists of two long helices, α2 (residues 183–194) and α3(residues 197–209), connected by the well-conserved Gly195–Ser 196 sequence forming a short turn. This region ispacked tightly against helix α1, loop L1 and the β-sheet of thesheet-helix-loop region, stabilized by a hydrophobic core con-taining Ala 144, Tyr 148, Leu 149, Leu 150, Leu 155, Leu 162, Tyr181 and Ile 186 (Fig. 3a).

The C-terminal subdomain has a large cleft between the sheet-helix-loop region and the helix-turn-helix region. The surfaceelectrostatic potentials show that many positively charged sidechains, which are contributed by well-conserved Lys 141, Lys145, Lys 151, Lys 179, Lys 204 and Arg 207, are present in the cleft(Figs 3b, 4a).

Table 1 Structural statistics1 for the central domain of XPA

Total number of distance constraints 1,389Intraresidual 293Sequential ( |i - j | = 1 ) 376Medium-range ( |i - j ≤4 ) 286Long-range ( |i - jl > 4 ) 381Hydrogen bonds 47Sulphur–sulphur 6

Number of dihedral angle constraints 83Distance constraint violations greater than 0.3 Å 0Dihedral angle constraint violations greater than 5° 0R.m.s. deviations from experimental constraints

Distance (Å) 0.0130 ± 0.0011Angle (o) 0.325 ± 0.061

R.m.s. deviations from idealized covalent geometryBonds (Å) 0.0020 ± 8.02 × 10–5

Angles (o) 0.487 ± 0.008Impropers (o) 0.300 ± 0.011

X-PLOR potential energy (Etotal) 171.5 ± 8.3PROCHECK Ramachandran plot statistics (residues 102–209, ∆156–162, ∆166–179)

Residues in most favored regions 67.2%Residues in additional allowed regions 30.3%Residues in generously allowed regions 2.2%Residues in disallowed regions 0.3%

R.m.s. deviations to the mean coordinate positionsZinc-containing subdomain (residues 102–129)

Backbone heavy atoms 0.23 ÅAll heavy atoms 0.75 Å

C-terminal subdomain excluding part of loop L1 and all of loop L2 (residues 138–209, ∆156–162, ∆166–179)


Central domain excluding part of loop L1 and all of loop L2 (residues 102–209, ∆156-162, ∆166–179)


1These statistics comprise the ensemble of the final 30 simulated annealing structures from Met 98 to Asn 210 calculated with X-PLOR version 3.126.All variances are quoted ± one standard deviation.

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Interestingly, the genomic structure of the xpa gene shows a goodcorrelation to the tertiary structure elements20. Exon 3 (residues96–130) encodes the entire zinc-containing subdomain, exon 4(residues 131–185) encodes the linker and the sheet-helix-loopregion, and exon 5 (residues 186–224) encodes most of the helix-turn-helix region and the C-terminal flanking sequence.

DNA and RPA binding sitesTo identify the DNA binding surface of the central domain of XPA,we performed chemical shift perturbation experiments. Selectivechemical shift perturbation and/or broadening were observed forthe signals in the (15N, 1H) HSQC spectrum on mixing with anequimolar amount of the oligonucleotide treated with thechemotherapeutic agent, cisplatin, which reacts with DNA to formintrastrand cross-links21. Almost all of the signals that showedremarkable chemical shift perturbation or broadening were attrib-uted to the amide residues in the basic cleft of the C-terminal sub-domain (Fig. 4b). The internal curvature of the basic cleft fits wellto the diameter of standard B-form double-stranded DNA. On the

other hand, smaller and more uniform sig-nal losses with no detectable chemical shiftchange were observed for the residues in thezinc-containing subdomain. The peakintensities for the 22 well resolved signals inthe subdomain became 61.3 ± 7.2 (1σ) % ofthe original values. These small and uni-form losses are caused mainly by an increaseof the effective molecular mass upon forma-tion of the XPA–DNA complex. These find-ings suggest that the basic cleft and thesurrounding region are involved in theinteraction with the DNA, but that the zinc-containing subdomain is not. We also founda similar pattern of chemical shift perturba-tion upon complex formation with a non-damaged oligonucleotide (data not shown),suggesting that the same binding surface isshared for both the non-damaged and thedamaged DNA. This is probably due to thelow binding specificity of XPA alone fordamaged DNA compared to undamagedDNA6. Previous filter binding experimentsdetermined the affinities of the centraldomain of XPA for DNA molecules of 2,686

base pairs with and without multiple sites of damage6. In theseexperiments, the dissociation constants for the damaged DNA andXPA98–219, and for the non-damaged DNA and XPA98–219, are esti-mated to be ~4 x 10–8 M and ~6 x 10–7 M respectively.

The interaction between XPA and RPA is essential for nucleotideexcision repair7,8,22. Although XPA binds to RPA70 and RPA347,8,the deletion analyses of XPA showed that RPA70 contributes morepredominately to the XPA-binding than RPA34 does9. The residues181–422 of RPA70, RPA70181–422 have been shown to form a com-pact structural domain that has single-stranded DNA bindingactivity23. We examined the interaction of the central domain ofXPA with RPA70181–422. Selective signal losses were observed in the(15N, 1H) HSQC spectrum after mixing with an equimolar amountof RPA70181–422. Signal losses are observed when the chemical shiftsare perturbed considerably by binding, the exchange rate betweenthe free and bound states is comparable to the chemical shift differ-ence between the two states, or the ligand binding enhances theamide proton exchange rate with water. Almost all of the signals forwhich the peak intensity decreased to <65% of the original values

Fig. 2 Structures of the zinc-containing subdo-main. a, Best-fit backbone superpositions of the30 final structures of the zinc-containing subdo-main (residues 102–129). The backbone atomsof these residues are superimposed. The sidechains of the residues in the hydrophobic coreare colored red, and those of the zinc-bindingcysteines are colored green. b, Stick representa-tion of the zinc-containing subdomain. Thelocations of residues that contribute to theacidic patch in the vicinity of the β-hairpin areindicated in red. The locations of residues thatcontribute to the hydrophobic patch in thevicinity of the helical turn are indicated ingreen. c, The hydrophobic core formedbetween the zinc-containing subdomain andthe C-terminal subdomain. The hydrophobicside chains of the zinc-containing subdomainthat form the core are colored green, those ofthe C-terminal subdomain are colored red. Allthe figures were produced with the programMOLMOL27.

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were attributed to the residues in the zinc-containing subdomain(Fig. 4b), suggesting that this subdomain serves as an RPA70-bind-ing surface. All of the amide residues with peak intensities thatdecreased to <20% are located in the β-hairpin and its vicinity(residues 101–114). This region contains five acidic residues, andthe observed changes in the spectra upon the binding are sensitiveto ionic strength (data not shown), suggesting that the interactionmay be due to electrostatic interactions. There is also a possibilitythat the sheet-helix-loop region of XPA serves as another bindingsurface with the N- or C-terminal regions of RPA70, which are notpresent in RPA70181–422. Smaller and more uniform signal losseswere observed for the residues of the C-terminal subdomain. Thepeak intensities for the 30 well-resolved signals in the subdomain

became 73.6 ± 5.2 (1σ) % of the original values. These small anduniform losses are due mainly to an increase of the effective molec-ular mass upon formation of the XPA–RPA complex.

ConclusionNucleotide excision repair is a major DNA repair pathway with ahighly conserved mechanism. However, until now, only one ofthe three-dimensional structures of the proteins involved in theearly steps of nucleotide excision repair was known, that of thessDNA-binding domain of RPA7021. The data presented heresuggest that the zinc-containing subdomain of XPA forms aninterface with RPA70, and that the C-terminal subdomain isinvolved in DNA binding. Analysis of these interactions should

Fig. 3 Structures of the C-terminal subdo-main. a, Best-fit backbone superpositionsof the 30 final structures of the C-termi-nal subdomain (residues 138–209). Thebackbone atoms of the residues 138–155,163–165 and 180–209 are superimposed.Loops L1 (residues 148–163) and L2(residues 166–179) are colored light blue.The side chains of the residues in thehydrophobic core are colored red. b, Thestick representation of the C-terminalsubdomain. The well conserved residuesthat contribute to the positive charges inthe basic cleft are colored blue. The acidicresidues in helices α2 and α3 are coloredred. Both figures were produced with theprogram MOLMOL27.

Fig. 4 a, Distribution of the electrostatic potential (displayed with GRASP31) on the solvent-accessible surface of the central domain of XPA (residues98–210). Blue corresponds to positive potential and red to negative potential. The presence of a positively charged cleft is evident in the C-terminalsubdomain. In the zinc-containing subdomain negatively charged patches are dominant. b, Mapping of the XPA residues with chemical shift pertur-bation or broadening effects in the (15N, 1H) HSQC spectra. The residues of which the amide resonances were perturbed upon complex formationwith the cisplatin-damaged oligonucleotide (chemical shift perturbation defined by more than 0.08 p.p.m. for 15N or 0.02 p.p.m. for 1H, or broaden-ing defined by peak intensities decreased to <50 % of their original values) are indicated in magenta, and those of which the amide resonancesshowed specific broadening upon complex formation with RPA70181–422 (the peak intensities decreased to <65 % of their original values) are coloredgreen. The molecular orientation is the same as in (a)

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facilitate further understanding of the nucleotide excision repairmolecular machinery which, when disrupted by mutation, cancause serious genetic diseases, such as xeroderma pigmentosum.

MethodsSample preparation. XPA98–219 was expressed as described6. For theNMR experiments, a uniformly 15N or 15N-, 13C-labeled protein was pro-duced by growing E. coli in M9 medium containing 15NH4Cl, without orwith [U-13C]-glucose respectively. A uniformly 15N-labeled and fraction-ally deuterated XPA98–219 (C153S) point mutant16 was prepared by grow-ing E. coli in 80% 2H2O. The recombinant proteins were purified bySP-Sepharose (Pharmacia), Mono-S (Pharmacia) ion-exchange and gel-filtration (Pharmacia Sephacryl S-100) chromatographies. RPA181–422 wasprepared as described23. A duplex 24-mer oligonucleotide, 5'-TCTTCTTCTGTGCACTCTTCTTCT-3', treated with cis-diamminedi-chloroplatinum (II) (cisplatin), was prepared as described21. Samples forNMR measurements typically comprised 1.2 mM protein in 50 mMdeuterated Tris-HCl (pH 7.3 at 30 oC), 150 mM KCl, 10 mM DTT, and20 µM Zn(CH3COO)2 in H2O/2H2O (9:1).

NMR spectroscopy. NMR spectra were acquired at 30 °C with a BrukerDMX500, DRX500, or DRX800 NMR spectrometer. A series of three-dimensional experiments (15N-edited NOESY, 15N-edited TOCSY, HNCA,HNCO, HN(CA)CO, CBCA(CO)NH, CBCANH, C(CO)NH, H(CCO)NH andHCCH-TOCSY) were performed for the resonance assignments19. Thestereospecific assignments of the methyl groups of all the Leu and Valresidues were achieved as described24. Distance information wasobtained by 2D, 15N- or 13C-edited 3D or 13C, 15N- or 13C, 13C-edited 4DNOESY experiment with a mixing time of 100 ms19. In addition, 14restraints were obtained through a 15N, 15N-edited 4D NOESY experi-ment on the uniformly 15N-labeled and fractionally deuteratedXPA98–219 (C153S), since this mutant was shown to have an identical con-formation with the wild type XPA98–219 based on the (15N, 1H) HSQCspectra. For torsion angle constraints, HMQC-J and HNHA spectra weremeasured to obtain the backbone vicinal coupling constants (3JHN,Hα)19.The torsion angles χ1 of Tyr 116 and His 136 were estimated from the3JC’Cγ and 3JNCγ coupling constants25.

Structure determination. The structures of the central domain ofXPA were calculated with a standard simulated annealing protocol ofX-PLOR 3.126. The NOE connectivities from strong, medium, and weakcross peaks were categorized and assumed to correspond to the upperlimits for proton–proton distances of 3.0, 4.0 and 5.0 Å respectively. Theconstraints for the backbone hydrogen bonds were added for slowlyexchanging amides as 2.7–3.2 or 2.8–3.3 Å for N–O, and 1.7–2.2 or1.8–2.3 Å for H–O. Those in α-helices were estimated based on the shortand medium range NOE patterns. Those in β-sheets were introducedduring the refinement stages. Six sets of hydrogen bond constraintswere introduced between slowly exchanging amides and the Sγof zincbinding cysteines during the final stages of the refinement. The geom-etry of the zinc-binding site was restrained by means of a series of sul-phur–sulphur distance restraints based on the previous metal bindingresults16, in order to ensure the tetrahedral coordination geometry ofthe Cys–Zn bonds. A total of 140 structures were calculated, and 47 ofthem showed no violation greater than 0.5 Å or 5°. Of these, the 30final structures, which showed the lowest energy and no violationgreater than 0.3 Å or 5°, were selected and analyzed with MOLMOL27

and AQUA and PROCHECK-NMR28 software. The linearity of the 41hydrogen bonds of the 30 final structures, to which backbone hydro-gen bond constraints were applied, was checked. For the total 1,230pairs the average values and the standard deviations of the O–HN dis-tance and the angles between N–HN and N–O, and between C' = O andC'–HN, are 2.21 Å +/- 0.11, 23.9 +/- 9.8° and 27.5 +/- 13.7° respectively.

Chemical shift perturbation experiments. An equimolaramount of the 24-mer oligonucleotide duplex treated with cisplatinwas added to 50 µM 15N-labelled XPA98–219 in 77 mM KH2PO4-K2HPO4

(pH 7.8), 77 µM KCl, 7 µM DTT, 15 µM Zn(CH3COO)2, and 5 mMMgCl2. The WATERGATE, water-flip-back (15N, 1H) HSQC spectrum at30 °C was compared with that of the free XPA98–219. For the analysis

of the XPA–RPA70 interaction, an equimolar amount of RPA70181–422

was added to 50 µM 15N-labeled XPA98–219 in 28 mM KH2PO4-K2HPO4

(pH 7.8), 28 mM KCl, 3 mM DTT, 6 µM Zn(CH3COO)2, and 5 mMMgCl2, and the HSQC spectrum was compared with that of the freeXPA98–219.

Coordinates. The coordinates have been deposited in theBrookhaven Protein Data Bank (accession number 1xpa.) The NMRrestraints entry is r1xpamr.

AcknowledgmentsWe thank E.H. Morita, M. Shimizu, T. Shimizu, and M. Maeda for discussions. Wethank M. Wäelchli for a critical reading of the manuscript. This work wassupported by grants to M.S. and K.T. from the Ministry of Education, Science,and Culture of Japan. M.S. was also supported by the Ciba-Geigy (Japan)Foundation for the Promotion of Science. This work was partly supported by aresearch grant to K.T. and K.M. from the Human Frontier Science Program.

Takahisa Ikegami1, Isao Kuraoka2, Masafumi Saijo2,Naohiko Kodo2, Yoshimasa Kyogoku3, KosukeMorikawa4, Kiyoji Tanaka2 and Masahiro Shirakawa1

1Graduate School of Biological Sciences, Nara Institute of Science andTechnology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. 2Institute forMolecular and Cellular Biology, Osaka University, 1-3 Yamadaoka, Suita, Osaka565, Japan. 3Institute for Protein Research, Osaka University, 3-2 Yamadaoka,Suita, Osaka 565, Japan. 4Biomolecular Engineering Research Institute, 6-2-3Furuedai, Suita, Osaka 565, Japan.

Correspondence should be addressed to M.S. email: [email protected]

Received 13 April, 1998; accepted 1 July, 1998.

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solution structure of the dna- and rpa-binding domain of the human repair factor xpa

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