29Halo-ATP and -GTP analogues: Rational phasing toolsfor protein crystallography

MATHIAS GRUEN, CHRISTIAN BECKER, ANDREA BESTE, JOCHEN REINSTEIN,AXEL J. SCHEIDIG,and ROGER S. GOODYMax-Planck-Institut für molekulare Physiologie, Abteilung Physikalische Biochemie, Otto-Hahn-Straße 11,44227 Dortmund, Germany

~Received June 8, 1999;Accepted September 2, 1999!

Abstract

The solution of the crystallographic macromolecular phase problem requires incorporation of heavy atoms into proteincrystals. Several 29-halogenated nucleotides have been reported as potential universal phasing tools for nucleotidebinding proteins. However, only limited data are available dealing with the effect of 29-substitution on recognition bythe protein. We have determined equilibrium dissociation constants of 29-halogenated ATP analogues for the ATPbinding proteins UMP0CMP kinase and the molecular chaperone DnaK. Whereas the affinities to UMP0CMP kinase areof the same order of magnitude as for unsubstituted ATP, the affinities to DnaK are drastically decreased to undetectablelevels. For 29-halogenated GTP analogues, the kinetics of interaction were determined for the small GTPases p21ras~Y32W!~fluorescent mutant! and Rab5. The rates of association were found to be within about one order of magnitude of thosefor the nonsubstituted nucleotides, whereas the rates of dissociation were accelerated by factors of;100 ~p21ras! or;105 ~Rab5!, and the resulting equilibrium dissociation constants are in the nm ormM range, respectively. The datademonstrate that 29halo-ATP and -GTP are substrates or ligands for all proteins tested except the chaperone DnaK. Dueto the very high affinities of a large number of GTP binding proteins to guanine nucleotides, even a 105-fold decreasein affinity as observed for Rab5 places the equilibrium dissociation constant in themM range, so that they are still wellsuited for crystallization of the G-protein:nucleotide complex.

Keywords: 29I0Br-ATP; 29I0Br-AppNHp; 29I0Br-GppNHp; fluorescence spectroscopy; phase problem; X-raycrystallography

A large number of proteins interact with ATP or GTP. While thethree-dimensional~3D! structures of many of these proteins areknown, a large number remain to be determined. Despite homol-ogies between many proteins and classes of proteins that interactwith ATP or GTP, 3D structural homology is seldom high enoughto allow structural determination from X-ray crystallographic datausing molecular replacement only. Thus, the usual problem ofdetermining the phases of the reflections remains, and the mostcommonly used method~multiple isomorphous replacement~MIR!!still involves the incorporation of heavy atoms into protein crys-tals. After crystallization, finding such derivatives is the secondmajor bottle neck in the determination of the 3D structure of bio-

macromolecules. In recent years, a second method, that of multiplewavelength anomalous dispersion~MAD !, has become frequentlyused~Ogata, 1998!.

Whereas most labeling procedures focus on the protein itself,only a few attempts have been made to incorporate foreign atomsinto the ligands of proteins. In principle, advantage can be taken ofthe natural affinity of the ligand to the protein. Optimally, themodified ligand should fulfill three criteria:~1! The incorporated~heavy! atom~s! should be suitable for the common phasing meth-ods~MIR or MAD !; ~2! the incorporation of the heavy atom mustnot decrease the ligand’s affinity to the protein below levels thatallow formation of the complex at concentrations suitable for crys-tallization; and~3! for wide application, the ligand has to be boundby a large number of proteins. The 29-halogenated ATP and GTPanalogues depicted in Figure 1~Naber et al., 1995; Gruen et al.,1999! potentially fulfill these requirements. The element iodinehas been widely used as a heavy atom for macromolecular phasingby MIR, whereas bromine is especially suitable for MAD phasing.Recent simulations based on the experimental parameters of so-phisticated new synchrotron facilities indicate that a single bro-mine atom should provide sufficient phasing power for proteins ofup to 50 kDa~Bourenkov, 1999!.

Reprint requests to: Mathias Gruen, Max-Planck-Institut für molekularePhysiologie, Abteilung Physikalische Biochemie, Otto-Hahn-Straße 11,44227 Dortmund, Germany; e-mail: grun@mpi-dortmund.mpg.de.

Abbreviations:MABA-ADP, N8-~4-N9-methylanthraniloylaminobutyl!-8-aminoadenosine-59-diphosphate; AP5A, P1,P5-~diadenosine!-59-penta-phosphate; UP5A, P1-~adenosine!-59-P5-~uridine!-59-pentaphosphate;mant,N-methylanthraniloyl; MIR, multiple isomorphous replacement; MAD,multiple wavelength anomalous dispersion; FRET, fluorescence resonanceenergy transfer.

Protein Science~1999!, 8:2524–2528. Cambridge University Press. Printed in the USA.Copyright © 1999 The Protein Society

2524

The only question remaining is the effect of the 29-substitutionon recognition by the protein. Naber et al.~1995! investigated thiseffect for 29I-ATP and the enzymes creatine kinase, hexokinase,pyruvate kinase, and myosin and foundKm andvmax values similarto those for ATP. Furthermore, crystals containing 29I-ADP wereobtained with the proteins kinesin and nonclaret disjunctional pro-tein. These initial results suggest that 29-substitution will be tol-erated by at least some ATP binding enzymes.

In this paper, we present detailed binding kinetics for all sixnucleotide analogues available~Fig. 1! with a selection of variousATPases and GTPases.

Results

We have examined the interaction of halogenated adenosine nu-cleotides with two ATP-dependent proteins~UMP0CMP kinaseand DnaK! and that of halogenated guanosine nucleotides withtwo GTPases, p21ras and Rab5. The results are described sepa-rately below.

29Halo-ATP as ATP analogues

Our aim was to expand the binding studies previously reported byNaber et al.~1995! to representatives of other families of ATP-binding proteins especially using the nonhydrolyzable AppNHpanalogues that are extremely useful for the crystallization of pro-teins with ATPase activity. Therefore, binding studies were per-formed with UMP0CMP kinase, a nucleotide monophosphate kinasefrom Dyctyostelium discoideumas well as with DnaK fromEsch-erichia coli, a member of the heat shock protein family involved inprotein folding.

Binding to UMP0CMP kinase

The structure of UMP0CMP kinase has been solved in complexwith the bisubstrate inhibitor UP5A to a resolution of 2.2 Å~Pro-tein Data Bank~PDB! entry 1UKE; Scheffzek et al., 1996!. The 29-and 39-hydroxy groups of the adenosine moiety are exposed to thesolvent and are not in contact with any protein residue. Therefore,29halo substitution of ADP or ATP is not likely to affect the protein-nucleotide interaction significantly. The binding constants of allfour 29halo-ATP analogues were determined by competitive titra-

tion of a complex of UMP0CMP kinase and the fluorophoremantAP5Pmant~Reinstein et al., 1990!. In this procedure, the flu-orophore is displaced and the resulting change of fluorescenceserves as monitor for the progress of the reaction~Fig. 2!. Thebinding constants for ATP~Kd 5 30 mM !, 29I-ATP ~Kd 5 20 mM !,and 29Br-ATP ~Kd 5 10 mM ! were found to be similar withinexperimental error, indicating that 29halo substitution indeed hasno significant effect upon ATP recognition by UK. The imidofunction of the AppNHp analogues, however, leads to a decrease inaffinity by approximately one order of magnitude~Kd 5 180 mMfor 29Br-AppNHp and 29I-AppNHp!, most likely as a consequenceof distortion of a hydrogen bond network in which the oxygens oftheb- and of theg-phosphorus of the triphosphate group are involved.

Binding to the chaperone protein DnaK

The situation is quite different for the chaperone DnaK. The crystalstructure of the nucleotide binding domain of its homologue, bo-vine Hsp70, has been solved in complex with ADP at a resolutionof 1.93 Å ~PDB entry 3HSC; Flaherty et al., 1990!. Hsp70 is 49%identical and 66% similar toE. coliDnaK and serves as a structuralmodel for this discussion. The ATP 29OH group in Hsp70 is in-volved in a hydrogen bonding network with Glu268 and Lys271.Hence, 29-substitution with a halogen is expected to interferestrongly with the DnaK-nucleotide interaction.

Since DnaK hydrolyses ATP, the binding was investigated forthe nonhydrolyzable compounds 29I-AppNHp and 29Br-AppNHpby competitive titration of a complex of DnaK and the fluorophoreMABA-ADP ~Theyssen et al., 1996!. Even an;2,000-fold excessof the halo analogue over a 2mM DnaK:MABA-ADP complexdoes not result in measurable dissociation~data not shown!, indi-cating that 29halo-substitution strongly decreases the affinity of theanalogues to aKd value that is, at best, in the mM range~comparedto 1.6mM for nonsubstituted AppNHp; Theyssen et al., 1996!.

29Halo-GppNHp as GTP analogues

The nonhydrolyzable GppNHp has found widespread applicationin crystallography of numerous GTP-binding proteins. We haveinvestigated the kinetics of 29I- and 29Br-GppNHp with p21ras andRab5, members of the ras superfamily of small GTPases~Bourneet al., 1991!.

Binding to p21ras

The crystal structure of p21ras in the GppNHp form is known ingreat detail~Pai et al., 1990!. In p21ras:GppNHp there is a hydro-gen bond formed between the 29-OH group~as a donor! and themain-chain oxygen of Val29~as an acceptor! ~d 5 2.8 Å! as wellas a second H-bond with a water molecule which interacts with theside chain carboxyl group of Asp30. Therefore, the analogue bind-ing should be less restrained compared to Hsp70, since, for exam-ple, removal of the water molecule should be possible.

In contrast to the ATP analogues and the respective ATPases,affinities of guanine nucleotides to most GTPases cannot easily bedetermined by simple competitive titration due to the very slowrates of dissociation~koff! ~John et al., 1990; Rensland et al., 1995!.Therefore, in the case of p21ras, the kinetics of association~kon! ofthe nucleotide-free form of the protein were investigated with mix-tures of the fluorophore mantGDP and 29halo-GppNHp using astopped flow method~John et al., 1990!. The mutant p21ras~Y32W!

A B

CFig. 1. Structures of 29halo-ATP and -GTP analogues available.A: 29I-and 29Br-ATP. B: 29I- and 29Br-AppNHp. C: 29I- and 29Br-GppNHp. A5adenine-9-yl; G5 guanine-9-yl; X5 I or Br; PPP5 triphosphate residue;PPNHP5 b,g-imidotriphosphate residue.

29Halo-nucleotides for phasing in X-ray crystallography 2525

~which displays almost identical kinetic properties to those of thewild-type; Rensland et al., 1995! was used, since this allowedfluorescence measurements by FRET from tryptophan to the mantgroup, and from whichkobs, the pseudo-first-order rate constantfor association, could be determined as a function of concentrationof the 29halo-nucleotides. Plots of this dependence were linear~Fig. 3A! and the slopes of fitted lines yieldedkon ~Table 1!. Whilethese values are in the range found earlier for guanosine nucleo-tides, the rates of dissociation were found to be unusually fast.They were determined by displacement of the 29-halo-GppNHpderivatives from their complex with p21rasby an excess of mantGDP~Fig. 3B!. Single exponential fitting yielded the values ofkoff

~Table 1!.While the values ofkon for the halo derivatives were within a

factor of 2–3 of the nonsubstituted nucleotides, the rates of disso-ciation are significantly affected by the substitution: whereas dis-sociation of the 29halo analogues is complete within;1,000 s,little displacement of nonsubstituted GppNHp by mantGDP couldbe observed in a time range of 30,000 s~not shown!. This unusu-ally fast dissociation of 29halo-GppNHp leads to a drop in affinityto p21ras by two orders of magnitude, resulting in equilibrium

A

B

Fig. 2. Competitive titrations of a complex of UMP0CMP kinase~0.2mM !and mantAP5Amant~0.2mM ! with (A) 29halo-ATP or(B) 29halo-AppNHp.The fluorophore is displaced by the added nucleotides and binding ismonitored as a decrease in fluorescence. Data were fitted as described~Thrall et al., 1996! using a Turbo Pascal computer program. Experimentalconditions: 50 mM Tris0HCl ~pH 7.5!, 20 mM DTE, 100 mM KCl,1.6 mM MgCl2.

A

B

C

Fig. 3. A: Nucleotide free p21ras~Y32W! ~0.15 mM ! was mixed rapidlywith a mixture of mantGDP~fixed at 2.0mM ! and 29halo-GppNHp~var-ious concentrations! in the stopped-flow machine. This resulted in singleexponential fluorescence transients, from which values ofkobs were ob-tained and plotted against the concentration of 29halo-GppNHp. The slopeof the fitted straight line gives the second order rate constant for theassociation of the halo-nucleotide.B: Displacement of 29-halo-GppNHp~2 mM ! from its complex with p21ras~Y32W! ~1 mM ! by mantGDP~10 mM !. The data are well described by a single exponential fit fromwhich the rate constant for dissociation of the halo-nucleotide is obtained.C: Determination ofkobs for the binding of the 29halo-GppNHp analoguesto nucleotide-free Rab5 using the same experiment as forA. The results forA–C are summarized in Table 1. Experimental conditions: 64 mM Tris0HCl ~pH 7.6!, 5 mM DTE, 10 mM MgCl2.

2526 M. Gruen et al.

dissociation constants of;30 nM compared to 0.07 nM for non-substituted GppNHp~Rensland et al., 1995! ~Table 1!.

Binding to Rab5

The 3D structure of Rab5 has not yet been reported and hence nodetailed structural analysis and no prediction can be made for theeffect of 29-substitution. The kinetics of the interaction were stud-ied by examining the association of nucleotide-free Rab5 with theGTP analogues monitoring intrinsic tryptophan fluorescence usinga stopped flow machine. The values ofkobswere derived by singleexponential fitting and were found to vary linearly with the nu-cleotide concentration~Fig. 3C!. As for p21ras, 29-substitution seemsto have only a moderate effect on the rate of association~slope ofthe linear plot, Fig. 3!, which is about one order of magnitudeslower for 29halo-GppNHp than for GppNHp. There was, how-ever, a more dramatic affect on the rates of dissociation of 29I- and29Br-GppNHp. In fact, the dissociation reaction of the 29halo-analogues is so fast that it can be determined by extrapolation ofthe linear plot for the dependence of the apparent association rateconstant to zero concentration~Table 1; Fig. 3C!, which cannot bedone for the nonsugar-modified nucleotides because of the smallmagnitude of this constant~Simon et al., 1996!. This rapid disso-ciation results in a decrease of;105 in affinity compared to GTPor GDP~Simon et al., 1996!.

Discussion

The data reported here and elsewhere demonstrate that introduc-tion of a halogen in place of the 29-hydroxyl group in ATP or GTPhas a varied effect on the interaction with triphosphate-binding andutilizing proteins. Thus, while there appears to be little effect onthe interaction of ATP with creatine kinase, hexokinase, pyruvatekinase, myosin, and UMP0CMP kinase, binding to the chaperoneprotein DnaK appears to be completely abolished by the modifi-cation. This can be readily understood on the basis of the available3D structures of the respective proteins with adenosine nucleo-tides, since they show no strong interactions with the nucleotide29-hydroxyl group, whereas there is an interaction between thishydroxyl group and residues Glu268 and Lys271 of Hsp70, themammalian homologue of the bacterial DnaK~Flaherty et al.,1990!.

More subtle differences were observed in the interaction of thehalo-derivatives with two different small GTPases. With p21ras,there was little effect on the association rate but a relatively largeeffect ~about 2 orders of magnitude! on the dissociation rate. WithRab5, much more dramatic consequences of the halo-modificationare seen~Table 1!. There is a somewhat larger effect on the asso-ciation rate than with p21ras, but the major effect is on the disso-ciation. With guanosine nucleotides, the rate constant for dissociationis so slow that it is difficult to obtain accurate estimates. The effecton the affinity is about 5 orders of magnitude, bringing theKd

value into the micromolar range. The reason for this effect isprobably a more serious steric clash than with p21ras, presumablywith the backbone. Despite this dramatic loss of affinity, the dis-sociation constant is in a range which is about 2 orders of magni-tude below typical protein concentrations used for crystallization,so that the use of these nucleotides for this purpose is not excludedsolely on the grounds of low affinity. However, generation of a 1:1complex without use of excess nucleotide, which is easily achievedwith GTPases and strongly-bound nucleotides, will be less conve-nient in this case.

Materials and methods

Proteins

All proteins were expressed and purified as described previously:UMP0CMP kinase~Wiesmüller et al., 1995!; DnaK ~Theyssenet al., 1996!; Rab5 nucleotide-free~Simon et al., 1996!,p21ras~Y32W! essentially as described for wild-type p21ras ~Johnet al., 1988!.

Nucleotides

The 29-halogenated nucleotides were synthesised as described~Gruen et al., 1999!. The fluorophores MABA-ADP~Theyssenet al., 1996!, mantAP5Amant~Reinstein et al., 1990!, and all othermethylanthraniloyl nucleotides~John et al., 1990! were preparedand purified as previously reported.

Fluorescence measurements

Static and slow time scale fluorescence measurements were per-formed at 258C with an SLM-AB2 spectrophotometer~Amicon,

Table 1. Association and dissociation kinetics of GTP analogues with p21ras and Rab5

Nucleotide

Protein GppNHp 29Br-GppNHp 29I-GppNHp

p21ras

Rate constant of association,kon ~3 105 M21 s21! 3 1.3 1.5Rate constant of dissociation,koff ~3 1023 s21! — 4.2 5.0Dissociation constant,Kd ~nM! 0.07a 32 33

Rab5Rate constant of association,kon ~3 104 M21 s21! 15 1.7 0.75Rate constant of dissociation,koff ~3 s21! — 0.014 0.024Dissociation constant,Kd ~mM ! 2.93 1025b 0.82 3.2

aRensland et al.~1995!.bMeasured with GTP; Simon et al.~1996!.

29Halo-nucleotides for phasing in X-ray crystallography 2527

Silver Spring, Maryland!. Rapid kinetics were measured with astopped flow apparatus~High Tech Scientific, Salisbury, UnitedKingdom!. Excitation of tryptophan fluorescence was at 290 nm,with detection through a 320 nm cut-off filter. Fluorescene ofmantGDP was excited via FRET at 290 nm, with emission througha 389 nm cut-off filter. Data collection and primary analysis fordetermination of rate constants were performed with the manufac-turer’s software package, while secondary analysis was with theprogram Grafit 3.0~Erithacus software! unless stated otherwise.The displacement titration with UMP0CMP kinase and the ana-logues were analyzed with the known dissociation constant ofmAP5Pm~Kd 5 0.16mM; Wiesmüller et al., 1995! and the cubicsolution that allows extraction of the realKd for the displacingligand ~Thrall et al., 1996!. An exact value for the affinity of thehalogenated ATP analogues for the molecular chaperone DnaKcould not be determined likewise with the knownKd of MABA-ADP ~Kd 5 0.09mM; Theyssen et al., 1996! since no displacementwas observed even at 2,000-fold excess of the displacing ligands.

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2528 M. Gruen et al.