the active site of staphylococcal nuclease: paramagnetic
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Proc. Nat. Acad. Sci. USAVol. 71, No. 7, pp. 2833-2837, July 1974
The Active Site of Staphylococcal Nuclease: Paramagnetic Relaxationof Bound Nucleotide Inhibitor Nuclei by Lanthanide Ions
(nuclear magnetic resonance/3',5'-thymidine diphosphate/gadolinium/calcium-binding proteins/substrate geometry)
BRUCE FURIE*, JOHN H. GRIFFIN*, RICHARD J. FELDMANNt, EDWARD A. SOKOLOSKIT, ANDALAN N. SCHECHTER** Laboratory of Chemical Biology, National Institute of Arthritis, Metabolism, and Digestive Diseases; t Computer Center Branch,Division of Computer Research and Technology; and $ Laboratory of Chemistry, National Heart and Lung Institute, NationalInstitutes of Health, Bethesda, Maryland 20014
Communicated by C. B. Anfinsen, April 12, 1974
ABSTRACT The structure of 3',5'-thymidine diphos-phate bound in the active site of staphylococcal nuclease(EC 188.8.131.52) was studied by measuring the relaxation rateenhancement of substrate analog nuclei by a paramagneticmetal ion. The lanthanide ion, Gd(III), was substituted forCa(II) in the formation of the ternary complex of nuclease-Gd(III)-3',5'-thymidine diphosphate. Measurementswere made of the transverse relaxation rates of protons andthe longitudinal and transverse relaxation rates of thephosphorus nuclei of the bound nucleotide. Internucleardistances between the metal ion and atoms of the 3',5'-thymidine diphosphate nucleotide were determined fromthese data by the Solomon-Bloembergen equation. Ingeneral, these distances corresponded closely to thosedetermined by previous x-ray crystallography of thetbymidine diphosphate complex.These internuclear distances were also used with a
computer program and graphics display to solve formetal-nucleotide geometries, which were consistent withthe experimental data. A geometry similar to the structureof the metal-nucleotide complex bound to nucleasedetermined by x-ray analysis was one of the solutions tothis computer modeling process. For staphylococcalnuclease, the nuclear magnetic resonance and x-raymethods yield compatible high resolution informationabout the structure of the active site. However, differencesof uncertain significance exist between the two structures.
The absence of suitable electronic and magnetic properties ofcalcium ions has limited spectroscopic investigations of theinteraction of these ions with calcium-binding proteins. Re-cently, the use of lanthanide ions as substitutes for calciumand other metal ions has facilitated the study of metal ioninteraction with metal-binding proteins (1-5), in particular,staphylococcal nuclease (6-9).
Staphylococcal nuclease has a single polypeptide chain of16,900 molecular weight (10); its three-dimensional structureis known from x-ray crystallographic studies (11). Nucleaseactivity has an absolute requirement for calcium (10).Trivalent lanthanide ions bind tightly to nuclease, competefor a single Ca(IJ)-binding site, competitively inhibit en-zymatic activity, and substitute for Ca (II) in the formation of
Abbreviations: NMR, nuclear magnetic resonance; 3',5'-dTDP,3',5'-thymidine diphosphate; (1/T2),N and (1/Tj)>1, paramag-netic contribution to the transverse and longitudinal relaxationrates, respectively.
a ternary complex composed of nuclease-metal ion-nucleotideinhibitor (9).A substantial theoretical and experimental foundation
exists for the determination of absolute distances betweenprotein-bound paramagnetic metal ions and nuclei of the pro-tein, substrate, or bound water molecules by the use of nuclearmagnetic resonance (NMR) relaxation techniques (12). Thisapproach has been extended to the use of paramagneticlanthanide ions in biological systems (13-15). We report herethe application of this method to the examination of the solu-tion structure of a nucleotide inhibitor bound to nuclease inthe presence of gadolinium(III). A comparison of the solutionstructure of this nucleotide substrate analog with that de-termined by x-ray crystallography is discussed.
MATERIALS AND METHODS
Staphylococcal nuclease (EC 184.108.40.206; nucleate 3'-oligonucleo-tidohydrolase) was prepared and purified as described (16, 17).Exchangeable protons in nuclease were substituted with deu-terium by successive lyophilization of a nuclease solution from99.7% D20 (Diaprep).
Lanthanide ions were obtained as the anhydrous chloridesalts from K & K Laboratories, dissolved in D20, and keptacidic to prevent hydrolysis. 3',5'-dTDP (CalBiochem) wasprepared in D20. Solutions for a1p studies were passed througha 1 X 7-cm column of Chelex-100 (BioRad) equilibrated with99.7% D20. All solutions were adjusted to pH 6.9 by titrationwith DCl or NaOD.Proton NMR spectra were recorded at 220 MHz at a probe
temperature of 22C 0.50 (unless stated otherwise), with aVarian Associates HR 220 NMR Spectrometer equipped withthe Varian Fourier transform accessory. The solutions, con-tained in a 5-mm NMR tube, were given 25 or 100 transients,each of which corresponded to a 900 pulse. All chemical shiftvalues were measured relative to external tetramethylsilanein CC14. Unless stated otherwise, the solutions studied (500 ul)contained 20 mM\1 nucleotide and 0.1 M NaCl in D20 at pH6.9. Aliquots of lanthanide solutions (1 mM) were added tothe nucleotide-protein solutions in increments of 4-10 Al. Thetemperature dependence of the transverse relaxation rate(1/T2) was determined with a Varian temperature regulatorto change the probe temperature.
Proc. Nat. Acad. Sci. USA 71 (1974)
a1P NMR spectra with proton decoupling were recorded ata probe temperature of 280C i 1 on a Varian XL-100 NMRSpectrometer operating at 40.5 MHz and equipped with aDigilab computer for Fourier transform analysis. Data werecollected for 20 min per spectrum, using 900 900 pulses. AD20 external lock was used. Measurements of longitudinalrelaxation rate (T1) were obtained at 220C by the 180'-r-90'pulse technique (18).The value of 1/T2 was determined from the linewidths at
half-height of assignable resonances and were plotted as afunction of the ratio of Gd(III) concentration to nucleotideconcentration. The values of the paramagnetic contribution tothe transverse and longitudinal relaxation rates [(1/T2)M and(1/T1)M, respectively] were determined by linear extrapola-tion of these data to a 1: 1 complex of Gd(III): nucleotide andsubtraction of the diamagnetic contribution from the (1/T2)observed and (1/Tj)observed.
Equilibrium dialysis was performed with Visking 20 dialysistubing (Union Carbide) prepared in 1% NaHCO3 at 1000 for10 min in a cell with a 2-ml compartment divided by thedialysis membrane. Experiments used 40 mM 3',5'-dTDP inD20 (pH 6.9), GdCl3 in D20, and nuclease. After equilibrationfor 8 hr, samples of each solution were removed, and theproton NMR spectra were obtained.The structure of the bound nucleotide inhibitor was ex-
amined with a DEC PDP-10 computer equipped with anAdage AGT-30 graphics display computer and a DEC-34Dgraphics terminal (19). The structure of 3',5'-dTDP, from thecoordinates taken from x-ray crystallographic data (11), wasmodified by addition of protons using known bond angles andbond lengths. Within the constraints of the atomic Van derWaal radii, the covalent bond lengths, and the distances fromGd(III) to atoms of the nucleotide inhibitor determinedexperimentally, a series of possible conformations wasgenerated. This was accomplished by rotation of the Ni-Cl',C4'-C5', C5'-05', and C3'-03' bonds in increments of 100 orless and the use of a least mean squares analysis for determin-ing the position of Gd(JII) .
RESULTSThe proton magnetic resonance spectrum of 3',5'-dTDP at220 MHz showed a singlet at 7.63 ppm (C6-H), a triplet at6.18 ppm (C1'-H), a singlet at 4.11 ppm (C4'H), a singlet at3.81 ppm (C5',5"H), a broad multiplet centered approxi-mately at 2.5 ppm (C2',2"-H), and a large singlet at 1.74 ppm(CH3). These assignments are based on the analysis by Ts'oet al. (20) of related compounds. The 31p spectrum of 3',5'-dTDP at 40.5 MHz showed two overlapping resonances at110.08 ppm upfield of P406.The effect of temperature on the paramagnetic and diamag-
netic transverse relaxation rate of the nucleotide protons wasdetermined in order to establish whether 3',5'-dTDP was inrapid or slow exchange with the nuclease-metal ion complex.A decrease in the observed transverse relaxation rate withincreasing temperatures was observed for the C6-H protonand C5',5"-H protons of 20 mM 3',5'-dTDP in the presence ofnuclease and Gd(III) or La(III), suggesting conditions of fastchemical exchange compared to the paramagnetic or diamag-
netic relaxation rate. These results imply that the rate ofexchange of the nucleotide between the bound and free statesis greater than any of the relaxation rates of the nucleotidenuclei in the ternary complex and that the observed protonresonances represent a weighted average for the nuclei reso-nances of the bound and unbound nucleotide.
In order to determine the contribution of the paramagneticlanthanide ion to the enhanced relaxation rates of the nucleiof the substrate analog bound in the ternary complex [nu-clease-Gd(III)-nucleotide], we determined the bulk paramag-netic contribution of free Gd(III), the contribution of thebinary complex [nucleotide-Gd(III)], and the diamagneticcontribution to the observed transverse relaxation rates. Insolutions of 20 mM 3',5'-dTDP and 0.1 mM GdCl3 in theabsence of nuclease, extensive broadening was observed forall of the assigned resonances. For example, a linewidth of13.6 and 4.0 Hz was observed for the C6-H resonance in thepresence and absence of Gd(III), respectively. The metal-induced broadening of the C6-H resonance in the presence ofGd(III) could be due to the complexing of Gd(III) and 3',5'-dTDP or to the bulk paramagnetic effect of unbound Gd(III)on the nucleotide protons. No metal-induced broadening ofthe C6