Characterization of Metal and Nucleotide Liganded Forms of Adenylate Kinase by Electrospray Ionization Mass Spectrometry

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 339, No. 2, March 15, pp. 291–297, 1997 Article No. BB979877

Characterization of Metal and Nucleotide Liganded Forms of Adenylate Kinase by Electrospray Ionization Mass Spectrometry Gilbert Briand,*,1 Ve´ronique Perrier,† Mostafa Kouach,* Masayuki Takahashi,‡ Anne-Marie Gilles,† and Octavian Baˆrzu† *Laboratoire d’Application de Spectrome´trie de Masse, Universite´ de Lille II, 59045 Lille Cedex, France; †Unite´ de Biochimie des Re´gulations Cellulaires, Institut Pasteur, 75724 Paris Cedex 15, France; and ‡Groupe d’Etude de Mutagene`se et Cance´rogene`se, URA1342, Institut Curie, Orsay, France

Received October 9, 1996, and in revised form December 19, 1996

Complexes of adenylate kinase from Escherichia coli, Bacillus subtilis, and Bacillus stearothermophilus with the bisubstrate nucleotide analog P 1,P 5-di(adenosine 5*)-pentaphosphate and with metal ions (Zn2/ and/or Mg 2/) were analyzed by electrospray ionization mass spectrometry. P 1,P 5-di(adenosine 5*)pentaphosphateradenylate kinase complex was detected in the positive mode at pH as low as 3.8. Binding of nucleotide to adenylate kinase stabilizes the overall structure of the protein and preserves the Zn2/ chelated form of the enzyme from the gram-positive organisms. In this way, it is possible in a single mass spectrometry experiment to screen metal-chelating adenylate kinases, without use of radioactively labeled compounds. Binding of Mg 2/ to enzyme via P 1,P 5-di(adenosine 5* )-pentaphosphate was also demonstrated by mass spectrometry. Although no amino acid side chain in adenylate kinase is supposed to interact with Mg 2/, Asp93 in porcine muscle cytosolic enzyme, equivalent to Asp84 in the E. coli adenylate kinase, was proposed to stabilize the nucleotiderMg 2/ complex via water molecules. q 1997 Academic Press Key Words: adenylate kinase; nucleotide binding; metal binding; mass spectrometry; circular dichroism.

Recent advances in structural analysis of proteins result from association of recombinant DNA technology with fast, sensitive, and resolutive methods of purification and characterization. Mass spectrometry, a new 1 To whom correspondence should be addressed at Laboratoire d’Application de Spectrome´trie de Masse, Universite´ de Lille II, Place Verdun, 59045 Lille Cedex, France. Fax: 33 03 20 62 68 68.

and powerful tool in protein chemistry (1–4) has extended its application from molecular mass determination to analysis of noncovalent complexes of various origins (5–8). As the stability of these complexes depends on the properties of each partner, and on the chemical nature and pH of the solvent, the choice of conditions compatible with efficient ionization of target molecules and detection of the complexes is critical (9, 10). On the other hand, by extending the limits under which noncovalent complexes could be observed by mass spectrometry, new insights on protein stability or on ligand-induced conformational changes might be revealed. The first application of electrospray ionization mass spectrometry (ESI– MS)2 to the study of proteinrligand noncovalent interactions was reported by Ganem et al. (8, 11). Baca and Kent (12) showed by ESI– MS complex formation between the dimer of HIV-1 protease and an inhibitor, while Ganguly et al. (5, 13) explored buffers or pH ranges compatible with the formation of RasrGTP or RasrGDP complexes. In this study, we identified by ESI–MS complexes of bacterial adenylate kinases with the bisubstrate nucleotide analog Ap5 A and with Zn2/ and/or Mg 2/. Ap5 ArAK complexes can be evidenced in the positive ion mode at pH as low as 3.8, conditions under which the Zn2/containing bacterial enzymes are stabilized and the metal is specifically detected. In the negative-ion mode 2 Abbreviations used: AK, adenylate kinase; AK e , AK st , AK sub , E. coli, B. stearothermophilus, and B. subtilis adenylate kinases; AK eC4 and AKeC2 are modified forms of AK e in which His126 , Ser129 , Asp146 , and Thr149 were substituted with four Cys residues or in which Asp146 and Thr149 were substituted with two Cys residues; Ap5 A, P 1,P 5di(adenosine-5*)-pentaphosphate; ESI–MS, electrospray ionization mass spectrometry; CD, circular dichroism.

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the Zn2/rAK complex can be observed even in the absence of Ap5 A. In this way, it is possible to screen metalchelating AKs without use of radioactively labeled ligands. The nature of the metal bound to AKs can be identified in a single ESI–MS experiment. EXPERIMENTAL PROCEDURES Materials. Adenine nucleotides, restriction enzymes, T4 DNA ligase, T4 DNA polymerase, and coupling enzymes were from Boehringer Mannheim. Taq DNA polymerase was from Cetus Corp. T7 DNA polymerase and deaza sequencing mixes were from Pharmacia. Blue Sepharose was from Pharmacia LKB Biotechnologies Inc. Oligonucleotides were synthesized using the phosphoramidate method and a commercial DNA synthesizer (Cyclone, TM Biosearch). Bacterial strains, plasmids, growth conditions, and DNA manipulations. The NMI-5 Escherichia coli strain was used for DNA amplification by PCR. The plasmids were overexpressed in the BLI-5 E. coli strain (derived from the BL21, Novagen, Inc.) harboring a second plasmid, pDIA17, which encodes the lacI gene. This strain expresses the RNA T7 polymerase gene on the chromosome. Bacillus subtilis adk gene carried by the plasmid pDIA5316 (14), and poorly expressed in the FB8 strain, was recloned by PCR in the expression vector pET22b (Novagen). The two oligonucleotides used for amplification were 5*-GGAATTCCATATGAACTTAGTCTTAATGGGGCT-3* and 5*-CCAAGCTTTCATTTTTTTAATCCTCCAAGAAG-3*; two restriction sites NdeI and HindIII shown in bold letters in the oligonucleotide sequences were created at both ends of the amplified fragment. After digestion by NdeI and HindIII the amplified adk gene from B. subtilis was inserted into the pET22b plasmid digested by the same enzymes. One clone containing the B. subtilis adk gene, pPV2005, was kept. The DNA insert was sequenced using the double-stranded dideoxynucleotide sequencing technique (15). The E. coli adk gene from plasmid pEAK91 (kindly provided by A. Wittinghofer) was cut by ClaI and filled in with Klenow polymerase, then digested by SalI. The 920-bp fragment containing the adk gene was cloned in pET22b in DXbaI and SalI. The new plasmid containing the E. coli adk gene, pPV1003, was used for site-directed mutagenesis (Perrier et al., in preparation). Two modified variants of the adk gene from E. coli, a quadruple mutant AK eC4 and a double mutant AK eC2 , are carried by the plasmids pPV1118 and pPV1109, respectively. The obtention of these variants will be described in detail elsewhere. To overexpress the adk genes, the plasmids were transformed into the BLI-5 E. coli strain. Bacteria were grown in LB medium (16) supplemented with 100 mg/ml ampicillin and 30 mg/ml chloramphenicol. When the cultures reached an absorbance of 1.0 at 600 nm the lac promoter was induced with 1 mM isopropyl-b-D-thiogalactoside. Bacteria were harvested by centrifugation 3 h after induction. The adk gene from Bacillus stearothermophilus carried by the plasmid pDIA5309 was overexpressed as previously described (17). Purification of AK and activity assays. The AKs overproduced in E. coli were purified by a two-step procedure involving chromatography on Cibacron blue 3G-A Sepharose CL-6B, and Ultrogel AcA54 (18). Adenylate kinase activity was determined at 307C and 334 nm on an Eppendorf ECOM6122 photometer. One unit of enzyme activity corresponds to 1 mmol of the product formed in 1 min at 307C and pH 7.4 (19). Mass spectrometry. A Sciex API-I simple quadrupole mass spectrometer equipped with a standard atmosphere pressure ionization source (Perkin–Elmer, Toronto, Canada) was used. Samples were delivered to the source at a flow rate of 5 ml/min utilizing a medical infusion pump (Model 11, Harvard apparatus, South Natick, MA). The instrument was calibrated with polypropylene glycol in the positive mode. Experiments were performed at a declustering potential (orifice voltage) of {90 V. The potential of the spray needle was held to /5.0 kV in the positive mode or 04.5 kV in the negative mode.

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Ion spray mass spectra were acquired from m/z 600 to 2400 with a step size of 0.1 Da and a dwell time of 2 ms. Mac Bio spec was the computer program for calculation of the molecular mass of adenylate kinases. For investigations of the noncovalent complexes in the positive ionization mode, 0.2 mg AK was dissolved in 85–90 ml of distilled water, then 5 ml of Ap5 A (2 mg/ml in water) was added. To facilitate protonation of the complex, 5–10 ml of an acid solution (20% acetonitrile and 0.1% formic acid in water, pH 2.9) was added to make a final volume of 100 ml. To promote deprotonation of proteins in the negative ionization mode, the solvent used for infusion was 1% triethylamine in water. Other analytical procedures. Circular dichroism (CD) spectra were measured with an F-710 spectropolarimeter (Jasco) at room temperature (227C). The band width was 2 nm and the spectra were averaged over 10 scans at 100 nm/min with an integration time of 0.5 s. The samples were prepared as for ESI–MS measurements, spectra being measured in a 0.1-cm optical path cuvette. Estimation of secondary structure from CD spectra was performed with commercial software using Yang’s data as reference (20). Protein concentration was determined according to Bradford (21) using a Bio-Rad kit or by amino acid analysis. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed as described by Laemmli (22) and gels were stained with Coomassie blue.

RESULTS

Circular dichroism analysis of AK e and AK st at acidic pH. The far-uv spectrum of AK e at neutral pH is characterized by negative minima at 222 and 208 nm and by a positive maximum at 190 nm, indicating over 50% of a-helical structure (23, 24). By lowering the pH to 3.8, 3.4, and 2.9, there is a progressive decrease in ahelical content of AK e (Fig. 1A). However, even at the lowest pH the protein still preserves a significant proportion of secondary structure elements. Ap5 A exerted a stabilizing effect on AK e , most visible at pH 3.4 (Fig. 1B), which means that at pH §3.4, the Ap5 ArAK e complex could be visualized by ESI–MS. AK st , which has a much higher stability against thermal or GdmCl denaturation than AK e or AK sub (14, 17), exhibited at pH 3.8 CD spectra very similar to those recorded at neutral pH, in either the absence or presence of Ap5 A (Figs. 1C and 1D). Molecular mass determination of recombinant AKs. ESI–MS in the positive-ion detection was first carried out at pH 2.9 where AK e exhibited a coherent series of multiply charged ions centered around /24 charges (Fig. 2A). At pH 3.8, the ‘‘bell-shaped’’ distribution of ion signals was shifted upward, centered around /18 charges (Fig. 2B). The average molecular mass of AK e (23,586.9 { 1.0), determined at these two pH values, was in agreement with the value calculated from the sequence (23,586.1 Da). From several variants of AK e obtained by site-directed mutagenesis and analyzed by ESI–MS in the positive mode (pH 2.9 or 3.8), the AK eC2 mutant exhibited a slight but reproducible difference (02.6 Da) with the mass calculated from the sequence. This mutant, active in crude extracts, was inactivated after purification and dialysis against ammonium bi-

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sponding to multiply protonated ions of the enzymer nucleotide complex (average molecular mass Å 24,503.6 { 1.2) and of the free AK e were observed (Fig. 2C). The protein mass increment of 916.7 corresponded to the molecular mass of Ap5 A (916.4). AK eC2 , although inactive upon formation of disulfide bridge, binds Ap5 A at pH 3.8 as efficiently as the wild-type protein (data not shown).

FIG. 1. Far-uv circular dichroism spectra of AK e (A and B) and AK st (C and D) at different pH values in the absence or presence of Ap5 A. Spectra were recorded at a protein concentration of 16 mM. Ap5 A when present (B and D) was at 20 mM. (rrr) pH 2.9; (–r–) pH 3.4 ; (---) pH 3.8; (—) pH 6.4.

carbonate. Inactivation and loss of mass of AK eC2 mutant (Table I) results most probably from intramolecular disulfide bridge formation between Cys146 and Cys149 . The molecular mass of AKs from the gram-positive organisms B. subtilis and B. stearothermophilus, two zinc-binding proteins (14, 17), corresponded to that calculated from the sequence (Table I). Under acidic conditions required for ionization in the positive mode, the Zn2/ was lost upon evaporation of charged droplets. It should be noted, however, that at pH 3.8 the ion signals of AK st were centered around /15 charges (Fig. 3A), indicating lower accessibility of this protein to protonation compared to AK e . Interaction of bacterial adenylate kinases with Ap5 A at acidic pH analyzed by ESI–MS. When AK e and a slight excess of Ap5 A over the protein were analyzed by ESI–MS at pH 3.8, two envelopes of peaks corre-

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FIG. 2. Electrospray mass spectrum of AK e in the absence or presence of Ap5 A. AK e (80 mM) in acetonitrile/formic acid/water at pH 2.9 (A) or 3.8 (B and C) was analyzed in the absence (A and B) or presence (C) of Ap5 A (100 mM). Signals from the nucleotide-free AK e (*) and Ap5 ArAK e complex (s) are indicated in C.

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Molecular Masses (Da) of Various Forms of AKs Measured by ESI–MS in the Positive-Ion Modea

Enzyme

Calculated from sequence

AK e AK eC2 AK eC4 AK sub AK st

23,586.1 23,576.2 23,558.2 24,119.5 24,143.0

Measured 23,586.9 23,573.6 23,557.4 24,119.7 24,143.1

{ { { { {

1.0 1.2 1.5 1.2 2.1

detection mode, shows a series of peaks attributable to the AK eC4rZn2/ complex (average molecular mass 23,619.7 { 2.6) (Fig. 5C). DISCUSSION

Electrospray ionization is a relatively mild procedure for obtaining multiply charged gas-phase ions from a

a The solvent used was 20% acetonitrile and 0.1% formic acid in water (pH 2.9).

AK st supplemented with Ap5 A revealed no free enzyme in ESI–MS at pH 3.8 (Fig. 3B). The average molecular mass of single species was 25,121.9 { 1.3. The difference between the molecular mass of nucleotidecomplexed and nucleotide-free enzyme (978.8) corresponded fairly well to that of Ap5 A and Zn2/. If MgCl2 was included in the mixture of AK st and Ap5 A (Fig. 3C), the molecular mass of the complex (25,145.9 { 1.1) corresponds to the sum of the protein, nucleotide, and two metals. In other words, under acidic conditions not only was AK st able to bind Ap5 A strongly, but the enzymernucleotide complex stabilizes coordination of Zn2/ to the protein, forming single species detectable in ESI–MS experiments. The mass spectrum of AK sub in mixture with Ap5 A (Fig. 4B) at lower pH (3.4) than used for analysis of AK e and AK st showed signals corresponding to apo-AK sub as a major component (average molecular mass 24,119.7 { 1.2) and weaker signals corresponding to the Ap5 Ar AK subrZn2/ complex (molecular mass 25,098.7 { 1.4). Detection of Zn2/ chelated form of AK eC4 by ESI–MS in the positive and in the negative mode. AK eC4 is a mutagenized variant of AK e with an increased thermodynamic stability over the wild-type protein (Perrier et al., unpublished). The substitution of four amino acid residues (His126 , Ser129 , Asp146 , and Thr149) by Cys creates a Zn2/-binding site in the protein very similar to that found in AK st . The metal, not removable by overnight dialysis against buffer, binds less tightly to the protein compared to AK st or AK sub . Thus, while under native conditions both AK sub and AK st reacted very slowly with 5,5*-dithiobis(2-nitrobenzoic acid) (14), the cysteines of AK eC4 reacted readily with this reagent (data not shown). The ESI–MS spectrum of AK eC4 in mixture with Ap5 A revealed three species: the nucleotide-free apo-AK eC4 , the metal-free nucleotiderenzyme complex (average molecular mass 24,472.1 { 1.3), and the Ap5 ArAK eC4rZn2/ complex (average molecular mass 24,538.2 { 2.1) (Fig. 5B). The spectrum of the protein in 1% triethylamine, in the negative-ion

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FIG. 3. Electrospray mass spectrum of AK st in the absence or presence of Ap5 A. AK st (80 mM) in acetonitrile/formic acid/water at pH 3.8 was analyzed in the absence (A) or presence (B and C) of 100 mM Ap5 A. MgCl2 when present (C) was at 200 mM.

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of protein unfolding by an acidic environment is essentially electrostatic (30, 31), it is understandable why in its nucleotide-bound state AK e is ‘‘protected’’ against

FIG. 4. Electrospray mass spectrum of AK sub in the absence or presence of Ap5 A. AK sub (80 mM) in acetonitrile/formic acid/water at pH 3.4 was analyzed in the absence (A) or presence (B) of 100 mM Ap5 A. Signals from the apo-AK sub (*) and AK subrAp5 ArZn2/ (s) complex are indicated in B.

protein solution. It can be performed in positive mode when protein protonation is promoted by organic acids (formic or acetic acid) or in negative mode when ammonia or organic ammonium salts (triethylamine) promote deprotonation. Most investigated adenylate kinases are acidic proteins with isoelectric pH levels between 5 and 6. At pH 2.9 the maximum ionization of AK e is /29, six units lower than the number of total protonable side chains originating from 18 Lys, 13 Arg, 3 His, and the aminoterminal group. At pH 3.8, chosen as a reasonable compromise between efficient ionization and identification of noncovalent complexes, the maximal ionization of AK e is /24, i.e., approximately two-thirds of the total protonable side chains. Binding the Ap5 A to AK e induces large conformational changes in protein as demonstrated by X-ray crystallography (25, 26) or molecular spectroscopy including NMR (27, 28). Complex formation increases the stability of protein against thermal denaturation (29) or against denaturation by low pH. As the mechanism

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FIG. 5. Electrospray mass spectrum of AK eC4 in positive mode (A and B) and in negative mode (C). AK eC4 (80 mM) in acetonitrile/formic acid/water at pH 3.8 was analyzed in the absence (A) or presence (B) of 100 mM Ap5 A. Signals from the apo-AK eC4 (*), apo-AK eC4rAp5 A complex (s), and AK eC4rZn2/rAp5 A complex (l) are indicated in B. C represents a spectrum of AK eC4 (200 mM) in 1% triethylamine (pH 11.4) in negative mode.

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protonation. Electrostatic interactions between oppositely charged groups in Ap5 A and AK e increase the overall stability of protein and provoke an upward shift of ion signals in the Ap5 ArAK e complex. In this respect, two of the three conserved Arg residues (Arg123 , Arg156 , and Arg167) in close contact with the phosphoryl groups of ATP and Ap5 A deserve particular mention. Arg123 and Arg156 are located at the Nand C-terminal ends of a 38-amino-acid residue segment (INSERT domain) containing four stranded antiparallel b-sheets (25). Upon binding of Ap5 A to AK e , Asp158 and Asp159 , which point away from the active center in the nucleotide-free enzyme, interact with the guanidinium group of Arg123 and Arg156 . The Arg123Asp159 and Arg156-Asp158 salt bridges contribute to the large movement of the INSERT domain and assemble the catalytic site of AKs with an increase of the stability of the protein (32). The interaction of bacterial AKs with the two metal ions, Zn2/ and Mg 2/, evidenced by ESI–MS is also worthy of comment. While Zn2/ is a structural component of AKs from gram-positive bacteria (33), Mg 2/ is involved in phosphate transfer. Binding of zinc to adenylate kinase occurs via four Cys residues (AK st) or via three Cys residues plus one Asp residue (AK sub). As the Zn/-binding tendency of proteins is related to the pK a values of the amino acid side chains accepting the metal (4.5 for Asp, 6.5 for His, and 8.5 for Cys), at acidic pH Zn2/ coordination to AKs is considerably affected and not observed by ESI–MS in the positive mode. This effect might be viewed as a competition between hydrogen ions and metal ions for the protein ligand (34). In the presence of Ap5 A stabilization of the overall structure of the protein allows observation of Zn2/ binding to AKs. With regard to Mg 2/, its interaction with AKs is indirect. Mg 2/ binds to the b and g phosphates of ATP forming a bidentate complex or to the second and third phosphates of Ap5 A. When Mg 2/rAp5 A interacts with AK e , Asp84 (which corresponds to Asp93 in the muscle cytosolic variant) assists the proper alignment of the polyphosphate chain in the nucleotide binding pocket of protein, via water molecules (35). Sitedirected mutagenesis of Asp84 (and respectively of Asp93) showed not only an impaired binding of nucleotide substrates but also a markedly decreased affinity for Mg 2/ (35, 36). In conclusion, noncovalent complexes of adenylate kinase with metals or nucleotides can be detected by ESI–MS in both positive and negative mode. The relative ratios of these complexes versus the free enzyme depend on the stability of protein at the selected pH and on the affinity of ligands for adenylate kinase. Despite inherent limitations, mass spectrometry in association with other physical methods such as circular dichroism and infrared or NMR spectroscopy improves

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considerably the resolution of structural analysis with the benefit of sensitivity and rapidity. ACKNOWLEDGMENTS This work was supported by grants from the Centre National de la Recherche Scientifique (URA 1129), the Institut Pasteur, and the Contrats d’Objectifs Re´gion Nord-Pas de Calais, Universite´ de Lille II. We are grateful to M. Goldberg and C. T. Craescu for advice and inspiring discussions, to M. Herdmann for critical comments, and to M. Ferrand for excellent secretarial help.

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