Structural and Energetic Factors of the Increased Thermal Stability in a Genetically Engineered Escherichia coliAdenylate Kinase

Abstract

Several variants of Escherichia coliadenylate kinase, designed to bind a Zn2+ ion, were produced by site-directed mutagenesis. The metal binding and enzymatic properties of the engineered variants have been described (Perrier, V., Burlacu-Miron, S., Bourgeois, S., Surewicz, W. K., and Gilles, A.-M. (1998) J. Biol. Chem. 273, 19097–19101). Here we report the structural properties and stability changes in a 4-Cys variant which binds a Zn2+ ion and has an increased thermal stability. CD studies indicate a very similar secondary structure content in the wild type and the engineered variant. NMR analysis revealed that the topology of the parallel β-sheet, belonging to the protein core, and of the peripheral antiparallel β-sheet are also conserved. The small local changes observed in the neighborhood of the substitution sites reflect a more compact state of the metal-binding domain. The Zn2+-bound quadruple mutant shows an increased thermal stability, reflected in a 9 °C increase of the mid-temperature of the first cooperative unfolding step. Binding of a bisubstrate analogP1,P5-di(adenosine-5′)-pentaphosphate increases, by about 7 °C, the midpoint of this transition in both wild type and modified variant. The NMR data suggest that the peripheral domains involved in substrate binding unfold during the first denaturation step. Urea denaturation experiments indicate an increased resistance against chemical unfolding of the Zn2+-binding variant. In contrast, the Gibbs free energy of unfolding (at physiologically relevant conditions) of the quadruple mutant is lower than that of the wild type. Several variants of Escherichia coliadenylate kinase, designed to bind a Zn2+ ion, were produced by site-directed mutagenesis. The metal binding and enzymatic properties of the engineered variants have been described (Perrier, V., Burlacu-Miron, S., Bourgeois, S., Surewicz, W. K., and Gilles, A.-M. (1998) J. Biol. Chem. 273, 19097–19101). Here we report the structural properties and stability changes in a 4-Cys variant which binds a Zn2+ ion and has an increased thermal stability. CD studies indicate a very similar secondary structure content in the wild type and the engineered variant. NMR analysis revealed that the topology of the parallel β-sheet, belonging to the protein core, and of the peripheral antiparallel β-sheet are also conserved. The small local changes observed in the neighborhood of the substitution sites reflect a more compact state of the metal-binding domain. The Zn2+-bound quadruple mutant shows an increased thermal stability, reflected in a 9 °C increase of the mid-temperature of the first cooperative unfolding step. Binding of a bisubstrate analogP1,P5-di(adenosine-5′)-pentaphosphate increases, by about 7 °C, the midpoint of this transition in both wild type and modified variant. The NMR data suggest that the peripheral domains involved in substrate binding unfold during the first denaturation step. Urea denaturation experiments indicate an increased resistance against chemical unfolding of the Zn2+-binding variant. In contrast, the Gibbs free energy of unfolding (at physiologically relevant conditions) of the quadruple mutant is lower than that of the wild type. Adenylate kinases (AKs) 1The abbreviations used are: AK, adenylate kinase; Ap5A,P1,P5-di(adenosine-5′)-pentaphosphate; AKe, wild-type E. coli adenylate kinase; AKC4, adenylate kinase variant with four amino acid substitutions (H126C, S129C, D146C, and T149C); AKHC3, three-substitution variant (S129C, D146C, and T149C); LID, domain closing over ATP of AKe (residues 122–159); DQF-COSY, double-quantum filtered correlation spectroscopy; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement two-dimensional spectroscopy; TOCSY, total correlation spectroscopy. represent a class of small (20–26 kDa) and abundant phosphotransferases involved in the reversible transfer of the terminal phosphate group from ATP to AMP. They contribute to the maintenance of a constant level of cellular adenine nucleotides, necessary for energetic metabolism and nucleic acid synthesis. From the structural point of view (Fig. 1) AKs belong to the α/β class (a five-stranded β-sheet surrounded by several α-helices), characteristic for many nucleotide-binding proteins (1Schulz G.E. Curr. Opin. Struct. Biol. 1992; 2: 61-67Crossref Scopus (299) Google Scholar). In addition to this protein core, they contain generally two smaller domains involved in the substrate binding. One is composed by two helices (α2 + α3) and contributes to the mononucleotide binding, NMP domain closing over bound AMP (residues 30–59). The other is involved in binding of ATP and, due to its large movement, closing over the bound nucleotide, it was called LID domain. In the mitochondrial and most of the bacterial variants of AK, the LID domain has 38 residues (residues 122–159 in AKe) while smaller cytosolic enzymes have only an irregular loop of 11 residues (2Schulz G.E. Müller C.W. Diederichs K. J. Mol. Biol. 1990; 213: 627-630Crossref PubMed Scopus (188) Google Scholar, 3Gerstein M. Schulz G. Chotia C. J. Mol. Biol. 1993; 229: 494-501Crossref PubMed Scopus (114) Google Scholar). In the AKe (a “long” kinase) the LID domain contains a small four-stranded antiparallel β-sheet (4Müller C.W. Schlauderer G.J. Reinstein J. Schulz G.E. Structure. 1996; 4: 147-156Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). X-ray structures of the enzyme in complex with various ligands revealed that this domain has significantly different positions relative to the protein core, depending on the nature and number of bound ligands. Therefore, it was inferred that complexation (particularly with ATP) induces a large scale rotation/translation movement from a remote position into a close contact with the main part of the protein (3Gerstein M. Schulz G. Chotia C. J. Mol. Biol. 1993; 229: 494-501Crossref PubMed Scopus (114) Google Scholar, 4Müller C.W. Schlauderer G.J. Reinstein J. Schulz G.E. Structure. 1996; 4: 147-156Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar, 5Berry M.B. Meador B. Bilderback T. Liang P. Glaser M. Phillips Jr., G.N. Proteins Struct. Funct. Genet. 1994; 19: 183-198Crossref PubMed Scopus (135) Google Scholar, 6Vonrhein C. Schlauderer G.J. Schulz G.E. Structure. 1995; 3: 483-490Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). Solution studies, using resonance energy transfer measurements (7Bilderback T. Fulmer T. Mantulin W.W. Glaser M. Biochemistry. 1996; 35: 6100-6106Crossref PubMed Scopus (31) Google Scholar), confirmed the existence of such hinge motion of the LID domain upon binding of Ap5A, AMP, and ATP. Nevertheless, the short AKs conserve their catalytic activity despite the absence of the LID domain, but seems to loose the high specificity for ATP (8Miyoshi K. Egi Y. Shioda T. Kawasaki T. J. Biochem. 1990; 108: 267-270Crossref PubMed Scopus (31) Google Scholar). A screening of bacterial AKs has revealed that the enzymes from Gram-positive organisms bind a Zn2+ ion viacoordinative bonds to four side chains (Cys and Asp) situated in the β-hairpin loops of the LID domain (9Glaser P. Presecan E. Delepierre M. Surewicz W.K. Mantsch H.H. Bârzu O. Gilles A.-M. Biochemistry. 1992; 31: 3038-3043Crossref PubMed Scopus (57) Google Scholar). Based on sequence comparison with AK from the Gram-positive, thermophilic Bacillus stearothermophilus, we have designed several variants of theEscherichia coli enzyme involving three- or four-amino acid substitutions in the LID domain, at positions His126, Ser129, Asp146, and Thr149. In the companion study (10Perrier V. Burlacu-Miron S. Bourgeois S. Surewicz W.K. Gilles A.-M. J. Biol. Chem. 1998; 273: 19097-19101Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) we showed that some of these variants bind a Zn2+ ion and conserve the enzymatic activity. In this study we report the results of NMR and CD experiments aimed to characterize in more detail the structural consequences of the amino acid substitutions and zinc binding. The 4-Cys variant has an increased thermal and chemical (urea) stability, but the structural stability at physiologically relevant conditions is slightly reduced. All chemicals were of highest purity available. Protein design, expression, and purification were described in our companion study (10Perrier V. Burlacu-Miron S. Bourgeois S. Surewicz W.K. Gilles A.-M. J. Biol. Chem. 1998; 273: 19097-19101Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The far-UV CD spectra were recorded on a Jasco 700 spectropolarimeter in a 0.1-cm quartz cells. The spectra were obtained as an average of five runs and were corrected for the contribution of the buffer. For the thermal denaturation experiments we followed the ellipticity at 222 nm as a function of temperature, programmed to rise uniformly at 60 °C/h. Urea denaturation was monitored by the changes in ellipticity at 222 nm. Aliquots of concentrated solutions of protein were added to urea solutions (0–8.8 m) and the samples were incubated until equilibrium was reached at the temperature chosen for determining the unfolding curve (20 °C). The Gibbs free energy of denaturation was estimated by Equation 1 ΔG=−RT lnKEquation 1 where K is the equilibrium constant of denaturation at a given reaction temperature (RT) and urea concentration. The measured free energy difference is considered (11Pace C.N. Methods Enzymol. 1986; 131: 266-280Crossref PubMed Scopus (2420) Google Scholar) a linear function of the denaturant concentration ΔG=ΔGH2O−m[urea]Equation 2 where ΔG(H2O), the extrapolated value of ΔG at zero molar denaturant, represents the intrinsic stability of the protein in absence of urea. Samples were prepared by dissolving the lyophilized protein in potassium phosphate buffer (50 mm) in 2H2O or in 1H2O containing 7% 2H2O at pH 6.5. NMR spectra were obtained on a Varian Unity 500 NMR spectrometer, using standard methods for pure absorption DQF-COSY (12Rance M. Sorensen O.W. Bodenhausen G. Wagner G. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 117: 479-485Crossref PubMed Scopus (2597) Google Scholar), NOESY (13Kumar A. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1980; 95: 1-6Crossref PubMed Scopus (2030) Google Scholar), and TOCSY experiments with 30–60-ms spin lock times (14Davis D.G. Bax A. J. Am. Chem. Soc. 1985; 107: 2820-2821Crossref Scopus (1073) Google Scholar). Proton chemical shifts were referenced relative to the water signal which, in our conditions, at 308 K resonates at 4.69 ppm from the sodium 2,2-dimethyl-2-silapentane sulfonate signal. CD spectroscopy in the far-UV region is a sensitive measure of the secondary structure content in polypeptides and is particularly useful in comparing the backbone structural differences between related proteins (15Johnson Jr., C.W. Proteins Struct. Funct. Genet. 1990; 7: 205-214Crossref PubMed Scopus (894) Google Scholar). The similarity between the CD spectra of the wild-type and mutated enzymes (data not shown) indicates that the two proteins have roughly the same secondary structure content. NMR analysis of a protein such as AKe (23,587 Da, flexible domains, high α-helix content) is a difficult task. The proton resonance assignment was facilitated by the large spectral dispersion in the fingerprint region corresponding to β-strand residues and by the strong sequential connectivities between CαHi and NHi+1 in this type of secondary structure (16Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, New York1986Crossref Google Scholar). Using several two-dimensional homonuclear NMR spectra (DQF-COSY, TOCSY, and NOESY) we assigned the majority of the resonances corresponding to the two β-sheet domains in the AKe and AKC4. The quality of the two-dimensional spectra and some of the sequential NOE pathways of AKC4 are shown in Fig. 2. The chemical shift values for the assigned resonances in the proton spectra of AKC4 and AKe are available upon request from the authors. Resonance assignment enabled us to identify a large number of sequential and long range dipolar interactions between specific protons of the protein. Observation of strong sequential connectivitiesd αN(i, i + 1) indicates very short distances between the corresponding protons (CαHi, NHi+1), as is the case for a backbone in an extended conformation. The local geometry was further characterized by the analysis of the chemical shift values. Indeed, statistical analysis of experimental NMR data on proteins, showed that the Cα protons experience a mean upfield shift of 0.4 ppm if the residue belongs to an α-helix and almost the same downfield shift value in the β-sheet or extended structures (17Wishart D.S. Sykes B.D. Richards F.M. Biochemistry. 1992; 31: 1647-1651Crossref PubMed Scopus (2015) Google Scholar). Together, the sequential connectivities and the secondary shift of α protons provided a reliable delineation of the β-strands in AKe and AKC4. In addition, analysis of long range NOEs between resonances corresponding to the backbone protons revealed the relative arrangement of the secondary structure elements. In particular, the presence of long range d αα(i, j) NOE cross-peaks is characteristic for antiparallel β-sheets. The synthesis of the present NMR data is the global topology of the polypeptide chain in the two β-sheet domains, as represented in Fig. 3 for AKC4. A number of other long range dipolar interactions involving all the protons support this topology. For instance, methyl protons of Leu153 give NOE connectivities with amide protons of Tyr133, Gly144, and Lys145 and with aromatic protons of Tyr133. The secondary and tertiary structure descriptions of AKC4, based on the present experimental data, are very similar to that obtained for the wild-type enzyme and are completely compatible with the crystallographic structure of AKe (4Müller C.W. Schlauderer G.J. Reinstein J. Schulz G.E. Structure. 1996; 4: 147-156Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar). However, a number of minor spectroscopic modifications were observed between the wild-type protein and AKC4 in the LID domain. Chemical shift changes (up to 0.45 ppm) were noted for residues adjacent to the substituted positions (Ala127, Val125, and Val148). Also, dipolar interactions between amide protons of Tyr133 and Thr154 and δ1,2 methyl protons of Leu153 were observed in AKC4 but not in AKe. This suggests that Zn2+ binding in AKC4 brings closer the two loops containing the cysteine side chains, and the movement extends over the adjacent β-strands, that the methyl of Leu153 into NOE contact with the amide proton of Tyr133. In contrast, i + 1) connectivities for the amide and and the NOE between and were observed in AKe but not in AKC4 The denaturation curve in Fig. was obtained by the ellipticity at 222 which is the in the far-UV CD for an The two proteins a similar an followed by two cooperative by a reflected in the relative ellipticity a of secondary structure is in step. The mid-temperature of the cooperative for the first transition of the wild-type protein is °C which is in with the data M. Gilles A.-M. S. Bârzu O. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. P. A. Biochemistry. 1990; PubMed Scopus Google Scholar) and close to the value (10Perrier V. Burlacu-Miron S. Bourgeois S. Surewicz W.K. Gilles A.-M. J. Biol. Chem. 1998; 273: 19097-19101Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The quadruple mutant shows a significantly increased mid-temperature close to the thermal transition observed by (10Perrier V. Burlacu-Miron S. Bourgeois S. Surewicz W.K. Gilles A.-M. J. Biol. Chem. 1998; 273: 19097-19101Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The four mutant in absence of has an thermal stability between the wild type and AKC4 an at has on the denaturation transition of the thermal and urea unfolding for wild type and engineered adenylate the mid-temperature of the first unfolding is the mean of two In the estimated of the given Urea were obtained with a to the unfolding curve to a the standard are given in concentration at which of the are of linear between the free energy of unfolding and urea the mid-temperature of the first unfolding is the mean of two In the estimated of the given The were obtained with a to the unfolding curve to a the standard are given in Urea concentration at which of the are The of linear between the free energy of unfolding and urea concentration. in a The not observed in studies, is cooperative and shows more similar values for the two proteins and °C for AKC4, The unfolding is reversible for AKC4 and reversible for the wild-type protein the temperature is the first the unfolding is over this temperature, the denaturation is is a bisubstrate which binds at the ATP and AMP sites of a more compact structure in which the LID domain is in close contact with the protein C.W. Schulz G.E. J. Mol. Biol. 1992; PubMed Scopus Google Scholar). the of its binding on the structural stability we the denaturation in the presence of an concentration of The first transition is significantly for AKe, and about 7 °C (Fig. and In contrast, was observed on the A similar thermal was observed for a of J. P. A. Biochemistry. 1990; PubMed Scopus Google Scholar). The unfolding of the wild type and two mutated variants was further by proton NMR spectra at different The to the first equilibrium state is by small changes of some resonances corresponding to of these were in the NMR spectra and were assigned to protons of the protein (Fig. One to the aromatic protons of a slightly high and of is observed in all resonances from protons of a small upfield shift while the high from methyl shift ppm) is the temperature where the first unfolding transition these resonances are for a structure as their chemical shift values are very different to ppm) from the values (16Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, New York1986Crossref Google Scholar). that and are in and of the protein (Fig. chemical shift values by the of aromatic side chains and reflect only small local of the side chains a secondary and tertiary with the wild the spectral changes in the two variants AKC4 and over temperature that the Zn2+ binding in the LID domain the local in the A large number of Cα between and conserve their shift characteristic for β-strand secondary is the Cα proton of a residue from the of the parallel which is in all spectra at ppm (Fig. In the case of AKC4, this is of of secondary structure in the state of the denaturation (at °C). For AKe and AKHC3, the are during the first thermal with the CD data, the NMR suggest that the parallel β-sheet and part of the are in the state and only during the thermal due to the NMR study was to the first part of the denaturation The structural stability was further using urea as a chemical and CD spectroscopy. A transition was observed for the two proteins using a enabled us to the the structural stability of the proteins The urea concentration at which of the are is significantly in AKC4 variant a resistance to urea denaturation of this protein. The Gibbs free energy of extrapolated to zero urea concentration is in the wild type relative to the engineered variant. despite an increased thermal stability and a resistance to chemical the quadruple mutant has a lower structural stability in standard conditions denaturant, °C). The of linear of the Gibbs free energy of unfolding on the urea concentration is in AKC4, a of unfolding in the engineered protein. by the CD and NMR of residues and zinc binding in the engineered variant AKC4 conserve the global of the polypeptide chain and only small structural changes in the LID domain. The Zn2+-binding loops closer to other and the domain more The that the catalytic properties are suggests that the domain its to the This is further by the that binding is to this the thermal unfolding mid-temperature with similar in both wild type and AKC4 (Fig. The present experimental data a structural for a of the thermal unfolding of AKe. The CD experiments indicate a complex thermal denaturation with two cooperative analysis of the NMR and CD data suggests that the protein core, the parallel β-sheet and the helices and is in the equilibrium state and in the step. This domain represents about of the total secondary structure and to the CD signal in with the relative of ellipticity during the unfolding transition (Fig. In with this shifts significantly of the first transition but has only a on the Indeed, structural analysis of the binding (3Gerstein M. Schulz G. Chotia C. J. Mol. Biol. 1993; 229: 494-501Crossref PubMed Scopus (114) Google Scholar) revealed that the most are the helices and while the parallel β-sheet is not The present experiments data on the of the LID domain in the denaturation experiments Burlacu-Miron and C. T. revealed that some amide protons in the antiparallel β-sheet more than in the protein β-sheet, and but more than in the peripheral This suggests that the LID domain a more than the parallel β-sheet and unfold in of The large values of the temperature observed in studies in the LID domain of the AKe (4Müller C.W. Schlauderer G.J. Reinstein J. Schulz G.E. Structure. 1996; 4: 147-156Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar) support this of AK with the to a more and compact and induces a structural of the domains antiparallel β-sheet, helices and α3) and of the helices and The total of the protein by about (4Müller C.W. Schlauderer G.J. Reinstein J. Schulz G.E. Structure. 1996; 4: 147-156Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar) and the with several side chains in the C.W. Schulz G.E. J. Mol. Biol. 1992; PubMed Scopus Google Scholar). contribute to the of the first denaturation of the two proteins described The experiments on indicate that the four side chain substitution and zinc binding have a in the thermal stability. of the structures suggests that the stability of the LID domain is due to a of interactions between several side chains the substituted Tyr133, and closer the two small β-sheets. The residues their smaller and are to into this the global thermal stability of the protein. Zn2+ binding has an contribution in the global of this domain. This is of the shown to the proteins from their S. B. Structure. 4: Full Text Full Text PDF Scopus Google Scholar). an the zinc domain also the helices the domain to the protein and the of peripheral structural elements. In contrast, it has on the unfolding of the parallel β-sheet which at The of the AK from the thermophilic B. (9Glaser P. Presecan E. Delepierre M. Surewicz W.K. Mantsch H.H. Bârzu O. Gilles A.-M. Biochemistry. 1992; 31: 3038-3043Crossref PubMed Scopus (57) Google Scholar) is similar to that of the unfolding for AKe and AKC4. This that the contribution of the zinc binding region are other to the related to the Indeed, the metal ion is from the thermophilic to °C, a value which is than in AKe. In with the increased stability against thermal and chemical denaturation of AKC4, its structural stability in standard conditions to lower than that of the wild-type enzyme The of this a reliable of the protein based on of several chemical denaturant, and Also, the NMR spectral the structure and of the of the proteins the necessary structural studies are in in our and for useful