The Effect of Ligand Binding on the Galactokinase Activity of Yeast Gal1p and Its Ability to Activate Transcription
2008; Elsevier BV; Volume: 284; Issue: 1 Linguagem: Inglês
10.1074/jbc.m807878200
ISSN1083-351X
AutoresChristopher Sellick, Thomas A. Jowitt, Richard J. Reece,
Tópico(s)Protein Structure and Dynamics
ResumoThe galactokinase from Saccharomyces cerevisiae (ScGal1p) is a bifunctional protein. It is an enzyme responsible for the conversion of α-d-galactose into galactose 1-phosphate at the expense of ATP but can also function as a transcriptional inducer of the yeast GAL genes. For both of these activities, the protein requires two ligands; a sugar (galactose) and a nucleotide (ATP). Here we investigate the effect of these ligands on the stability and conformation of ScGal1p to determine how the ligands alter protein function. We show that nucleotide binding increases the thermal stability of ScGal1p, whereas binding of galactose alone had no effect on the stability of the protein. This nucleotide stabilization effect is also observed for the related proteins S. cerevisiae Gal3p and Kluyveromyces lactis Gal1p and suggests that nucleotide binding results in the formation of, or the unmasking of, the galactose-binding site. We also show that the increase in stability of ScGal1p does not result from a large conformational change but is instead the result of a smaller more energetically favorable stabilization event. Finally, we have used mutant versions of ScGal1p to show that the galactokinase and transcriptional induction functions of the protein are distinct and separable. Mutations resulting in constitutive induction do not function by mimicking the more stable active conformation but have highlighted a possible site of interaction between ScGal1p and ScGal80p. These data give significant insights into the mechanism of action of both a galactokinase and a transcriptional inducer. The galactokinase from Saccharomyces cerevisiae (ScGal1p) is a bifunctional protein. It is an enzyme responsible for the conversion of α-d-galactose into galactose 1-phosphate at the expense of ATP but can also function as a transcriptional inducer of the yeast GAL genes. For both of these activities, the protein requires two ligands; a sugar (galactose) and a nucleotide (ATP). Here we investigate the effect of these ligands on the stability and conformation of ScGal1p to determine how the ligands alter protein function. We show that nucleotide binding increases the thermal stability of ScGal1p, whereas binding of galactose alone had no effect on the stability of the protein. This nucleotide stabilization effect is also observed for the related proteins S. cerevisiae Gal3p and Kluyveromyces lactis Gal1p and suggests that nucleotide binding results in the formation of, or the unmasking of, the galactose-binding site. We also show that the increase in stability of ScGal1p does not result from a large conformational change but is instead the result of a smaller more energetically favorable stabilization event. Finally, we have used mutant versions of ScGal1p to show that the galactokinase and transcriptional induction functions of the protein are distinct and separable. Mutations resulting in constitutive induction do not function by mimicking the more stable active conformation but have highlighted a possible site of interaction between ScGal1p and ScGal80p. These data give significant insights into the mechanism of action of both a galactokinase and a transcriptional inducer. The yeasts Saccharomyces cerevisiae and Kluyveromyces lactis are both capable of utilizing galactose as a source of carbon. To do so, they activate the genes encoding the enzymes of the Leloir pathway, collectively termed the GAL genes (1Sellick C.A. Reece R.J. Trends Biochem. Sci. 2005; 30: 405-412Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The transcriptional switch controlling GAL gene expression is composed of an activator (Gal4p), an inhibitor (Gal80p), and a ligand sensor/transcriptional inducer (Gal3p in S. cerevisiae or Gal1p in K. lactis). The GAL regulatory proteins from the two yeasts are at least in part, interchangeable. For example, Gal4p from both S. cerevisiae (ScGal4p) and K. lactis (KlGal4p) will complement a gal4 mutation in either yeast (2Salmeron J.M. Johnston S.A. Nucleic Acids Res. 1986; 14: 7767-7781Crossref PubMed Scopus (99) Google Scholar, 3Webster T.D. Dickson R.C. Nucleic Acids Res. 1988; 16: 8011-8028Crossref PubMed Scopus (33) Google Scholar) despite the two proteins sharing comparatively little overall sequence similarity (28% amino acid identity and 57% similarity over their entire length). Gal80p from either yeast are highly related (58% amino acid identity and 82% similarity) and will inhibit the transcriptional activity of either version of Gal4p (4Salmeron J.M. Langdon S.D. Johnston S.A. Mol. Cell. Biol. 1989; 9: 2950-2956Crossref PubMed Scopus (28) Google Scholar). However, although KlGal1p can complement both a Scgal1 (galactokinase-defective) and Scgal3 (ligand sensor-defective) mutation (5Meyer J. Walker-Jonah A. Hollenberg C.P. Mol. Cell. Biol. 1991; 11: 5454-5461Crossref PubMed Scopus (99) Google Scholar), ScGal3p cannot complement the non-inducible phenotype of a Klgal1 deletion mutant unless the KlGAL80 gene is also replaced by ScGAL80 (6Zenke F.T. Engles R. Vollenbroich V. Meyer J. Hollenberg C.P. Breunig K.D. Science. 1996; 272: 1662-1665Crossref PubMed Scopus (131) Google Scholar).ScGal1p and ScGal3p are extraordinarily similar proteins (∼70% amino acid identity and ∼90% similarity). Both proteins require galactose and ATP to function. ScGal1p utilizes the ligands as a galactokinase that converts galactose into galactose 1-phosphate at the expense of ATP. ScGal3p, on the other hand, requires the ligands to promote an interaction with ScGal80p. This interaction ultimately results in the activation of GAL gene expression (1Sellick C.A. Reece R.J. Trends Biochem. Sci. 2005; 30: 405-412Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The enzymatic mechanism of ScGal1p has been shown to proceed via an ordered tertiary complex mechanism in which ATP binds first (7Timson D.J. Reece R.J. Biochimie (Paris). 2002; 84: 265-272Crossref PubMed Scopus (46) Google Scholar). Although ScGal1p is primarily a galactokinase, it is able to weakly interact with ScGal80p (8Timson D.J. Ross H.C. Reece R.J. Biochem. J. 2002; 363: 515-520Crossref PubMed Scopus (48) Google Scholar) and serves as an inducer of GAL gene expression in the absence of ScGal3p. ScGal3p, however, does not possess a detectable galactokinase activity although ScGal3p can be converted into a galactokinase through the insertion of two amino acids (a serine and an alanine immediately after amino acid 164) (9Platt A. Ross H.C. Hankin S. Reece R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3154-3159Crossref PubMed Scopus (91) Google Scholar).Recently, the three-dimensional structures of ScGal1p, ScGal80p, and KlGal80p have been solved (10Thoden J.B. Sellick C.A. Reece R.J. Holden H.M. J. Biol. Chem. 2007; 282: 1534-1538Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 11Thoden J.B. Sellick C.A. Timson D.J. Reece R.J. Holden H.M. J. Biol. Chem. 2005; 280: 36905-36911Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 12Kumar P.R. Yu Y. Sternglanz R. Johnston S.A. Joshua-Tor L. Science. 2008; 319: 1090-1092Crossref PubMed Scopus (43) Google Scholar). In addition, the structure of KlGal80p in the presence of a peptide representing Gal4p has been solved (13Thoden J.B. Ryan L.A. Reece R.J. Holden H.M. J. Biol. Chem. 2008; 283: 30266-30272Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). ScGal1p, whose structure was solved in the presence of galactose and a non-hydrolysable ATP analogue, displays a marked bilobal appearance, with the active site located between distinct N- and C-terminal domains (11Thoden J.B. Sellick C.A. Timson D.J. Reece R.J. Holden H.M. J. Biol. Chem. 2005; 280: 36905-36911Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The nucleotide and galactose binding sites are buried between the two domains, and this led to the proposal of a mechanism by which the binding of the ligands results in a conformational change. It has not been possible to crystallize ScGal1p in the absence of its ligands. 2J. B. Thoden and H. M. Holden, unpublished data.2J. B. Thoden and H. M. Holden, unpublished data.Here we probe the mechanism by which ScGal1p is converted into an active galactokinase/transcriptional inducer by the presence of its ligands. Using a variety of biophysical techniques, we show that in the presence of its ligands ScGal1p becomes a substantially more stable protein. This effect is also observed for the related proteins ScGal3p and KlGal1p. Analysis of the conformation of ScGal1p in the absence and presence of ligands by velocity analytical ultracentrifugation (AUC) 3AUC, analytical ultracentrifugation; DSC, differential scanning calorimetry.3AUC, analytical ultracentrifugation; DSC, differential scanning calorimetry. demonstrated that the binding of ligands does not introduce a wholesale conformational change within the protein. Instead, it simply stabilizes the structure of the protein. We also investigated the effect of introducing ScGal3p constitutive mutations into ScGal1p on the stability of the protein to determine whether constitutive activation results from the formation of an active conformation in the absence of ligands. We demonstrate that four of these mutations, when introduced into ScGal1p, result in constitutive transcriptional activation but that these mutations have negative effects on the galactokinase activity of the protein. There is, however, still a difference in their stability in the presence and absence of ligands, which would suggest that constitutive transcriptional induction does not occur through the stabilization of the protein in the absence of ligands.EXPERIMENTAL PROCEDURESEscherichia coli and Yeast Strains—The production of ScGal1p, ScGal80p, and KlGal80p was performed in HMS174(DE3), BL21(DE3), and Rosetta(DE3) E. coli cells (obtained from Novagen), respectively. MC2 yeast cells (MATa, trp1, ura3-52, leu2-3, prc1-407, prb1-112, pep4-3) (9Platt A. Ross H.C. Hankin S. Reece R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3154-3159Crossref PubMed Scopus (91) Google Scholar) were used for Gal3p production and purification. β-Galactosidase assays were performed in JPY5::ΔGAL1::ΔGAL3::RY131 cells (MATα ura3-52 his3Δ200 leu2Δ1 trp1Δ63 lys2Δ385) (9Platt A. Ross H.C. Hankin S. Reece R.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3154-3159Crossref PubMed Scopus (91) Google Scholar). Disruption of GAL1 and GAL3 from JPY5 was achieved by using PCR-generated blaster cassettes (14Alani E. Cao L. Kleckner N. Genetics. 1987; 116: 541-545Crossref PubMed Scopus (745) Google Scholar).Protein Expression and Purification—Protein purification from E. coli was performed after the appropriate gene was cloned into pET28a (Novagen). E. coli cells harboring these plasmids were grown in 3 liters of LB medium supplemented with 30 mg/liter kanamycin at 37 °C to an A600 of 0.8. The cells were then induced with 1 mm isopropyl 1-thio-β-d-galactopyranoside for 16 h at 16 °C. The induced cells were pelleted by centrifugation at 1800 × g for 5 min, resuspended in buffer A (20 mm Tris-HCl (pH 8.0), 300 mm NaCl, 30 mm imidazole, 10% (v/v) glycerol), and lysed by sonication (3 × 30 s). The lysate was clarified by centrifugation at 75,000 × g for 20 min. The supernatant was then loaded onto a 2 ml Ni2+-NTA-agarose column (ProBond resin; Invitrogen) prewashed with buffer A. The column was then washed with buffer A, and protein was eluted from the column with buffer A containing 250 mm imidazole. The protein was then dialyzed into several changes of buffer B (20 mm Tris-HCl (pH 8.0), 200 mm NaCl, 1.4 mm β-mercaptoethanol, 10% (v/v) glycerol) at 4 °C overnight.For the purification of Gal3p, MC2 yeast cells were transformed with pAP45 (pYEX-BX (Clontech) containing the GAL3 gene (15Platt A. Reece R.J. EMBO J. 1998; 17: 4086-4091Crossref PubMed Scopus (150) Google Scholar)) and grown in 6 liters of Leu–-selective medium at 30 °C to an A600 of 1.0. The cells were then induced with 0.5 mm CuSO4 for 24 h. The induced cells were pelleted by centrifugation at 1800 × g for 5 min, resuspended in buffer A, frozen in liquid nitrogen, and lysed in a SPEX CertiPrep 6850 Freezer/Mill using 5 cycles with 2 min of grinding and 2 min of cooling per cycle (16Ansari A. Schwer B. EMBO J. 1995; 16: 4001-4009Crossref Scopus (114) Google Scholar). The cells were thawed at room temperature, and the lysate was clarified by centrifugation at 75,000 × g for 20 min. The protein was purified on a 2-ml Ni2+-NTA-agarose column as described above.Mutagenesis—Mutations were introduced into pET28-GAL1 using the QuikChange method (17Wang W. Malcolm B.A. Biotechniques. 1999; 26: 680-682Crossref PubMed Scopus (485) Google Scholar). Oligonucleotide sequences are available on request. The GAL1 open reading frame of each mutated plasmid was sequenced to confirm the presence of the mutation and that no other mutations had been introduced during the PCR. All mutant proteins were expressed and purified using the same method as described for the wild-type protein above.Galactokinase Kinetics—Galactokinase activity was measured by using an enzyme-linked assay system (7Timson D.J. Reece R.J. Biochimie (Paris). 2002; 84: 265-272Crossref PubMed Scopus (46) Google Scholar, 18Ali J.A. Jackson A.P. Howells A.J. Maxwell A. Biochemistry. 1993; 32: 2717-2724Crossref PubMed Scopus (308) Google Scholar). Briefly, reaction mixtures (150 μl) were set up in 96-well microtiter dishes containing 20 mm HEPES-KOH (pH 8.0), 150 mm NaCl, 5 mm MgCl2, 400 μm phosphoenol pyruvate, 1 mm dithiothreitol, 1 mm NADH, 1.1 units pyruvate kinase, 1.5 units lactic dehydrogenase (Sigma). Reactions were supplemented with Gal1p or mutant Gal1p and various concentrations of galactose and ATP. The plates were incubated at 30 °C, and the decrease in absorbance at 340 nm was measured using a Multiskan Ascent plate reader.Differential Scanning Calorimetry (DSC)—DSC was performed using a VP-DSC instrument (MicroCal Inc., Amherst, MA) with a scan rate of 60 °C/h from 15 to 100 °C. Protein samples (0.3 mg/ml) were dialyzed into buffer C (20 mm Tris-HCl (pH 8.0), 200 mm NaCl, 10% (v/v) glycerol) and run with buffer C as a reference. Ligands were added at a concentration of 2.5 mm. All thermal transitions were irreversible under the conditions used in this study. Data were analyzed using ORIGIN software (Microcal).Velocity AUC—ScGal1p was further purified by gel filtration on a Sephadex 200 10/300 GL column (GE Healthcare) which had been pre-equilibrated in buffer D (20 mm Tris-HCl (pH 8.0), 150 mm NaCl). The column was run at 0.5 ml/min with 0.5-ml fractions collected, and the peak fractions, as determined by SDS-PAGE, were pooled. Samples were loaded into two-sector cells in either a Beckman XLA or XL1 rotor at concentrations ranging between 0.3 and 1.7 mg/ml. Sedimentation of the proteins was achieved at 115,000 × g collecting data for 200 scans at 280 nm at 20 °C. Both interference optics and absorbance were used to determine concentration dependence of the sedimentation coefficient (s), and the sedimentating boundaries were analyzed using both Is-g*(s) and Lamm-equation solution modeling with the program Sedfit (19Schuck P. Biophys. J. 2000; 78: 1606-1619Abstract Full Text Full Text PDF PubMed Scopus (3003) Google Scholar).β-Galactosidase Assays—Yeast cells (JPY5::ΔGAL1::ΔGAL3:: RY131) were transformed with pRJR374, which contains the GAL1 coding sequence under the control of the GAL3 promoter or versions of the same plasmid containing mutated versions of the GAL1 gene. The cells were grown in 3 ml of the appropriate yeast selective dropout medium at 30 °C to an A600 of 0.8–1.0. The cells were pelleted by centrifugation at 4000 × g for 5 min and resuspended in 250 μl of buffer E (100 mm Tris-HCl (pH 8.0), 1 mm dithiothreitol, 20% (v/v) glycerol). Theβ-galactosidase assays were performed as described previously (20Sellick C.A. Reece R.J. J. Biol. Chem. 2006; 281: 17150-17155Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar).RESULTSThe Galactokinase Activity of ScGal1p Was Inhibited by Gal80p, and This Inhibition Was Relieved by Gal4p—ScGal1p can function both as a galactokinase and a transcriptional inducer. We investigated the effect of the interaction of ScGal1p with ScGal80p on its galactokinase activity (Fig. 1). The presence of ScGal80p inhibits the galactokinase activity of ScGal1p. The galactokinase activity of ScGal1p was reduced by 50% at a 1:1.5 molar excess of ScGal80p (Fig. 1A). It is possible that the interaction with ScGal80p locks ScGal1p into a conformation that prevents it from catalyzing the formation of galactose 1-phosphate or that it blocks product release. The inhibition of ScGal1p galactokinase activity in the presence of ScGal80p is similar to that previously reported for KlGal1p in the presence of KlGal80p (21Anders A. Lilie H. Franke K. Kapp L. Stelling J. Gilles E.D. Breunig K.D. J. Biol. Chem. 2006; 281: 29337-29348Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The same effect is, however, not observed if the experiment is repeated using KlGal80p (Fig. 1B). The lack of effect seen with KlGal80p is perhaps expected as KlGal80p is able to interact with and inhibit ScGal4p, but this inhibition cannot be relieved by ScGal3p (6Zenke F.T. Engles R. Vollenbroich V. Meyer J. Hollenberg C.P. Breunig K.D. Science. 1996; 272: 1662-1665Crossref PubMed Scopus (131) Google Scholar). This indicates that ScGal1p is also unable to interact with KlGal80p.The inhibition of ScGal1p galactokinase activity by ScGal80p is relieved efficiently by ScGal4p (Fig. 1C) but only modestly by ScGal3p (Fig. 1D). An equimolar concentration of ScGal4p was found to completely alleviate the inhibitory effect of ScGal80p upon the galactokinase activity of ScGal1p (Fig. 1C), whereas a 2-fold molar excess of ScGal3p only increased the relative galactokinase activity from 0.27 to 0.5 (Fig. 1D). Taken together, these data indicate that the form of ScGal1p that interacts with ScGal80p is incapable of performing its normal enzymatic role. Disrupting the interaction between ScGal1p and ScGal80p (by the addition of ScGal4p, which will favor the formation of the more stable Gal4p-Gal80p complex) leads to the restoration of galactokinase activity. The weak restoration of activity observed in the presence of ScGal3p presumably results from the competitive formation of Gal3p-Gal80p complexes, thereby allowing ScGal1p to again function as an enzyme.ScGal1p Is Stabilized in the Presence of ADP and Galactose—When the crystal structure of ScGal1p was solved, we suggested that ligand binding resulted in a conformational change in the protein leading to a more stable structure (11Thoden J.B. Sellick C.A. Timson D.J. Reece R.J. Holden H.M. J. Biol. Chem. 2005; 280: 36905-36911Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). To investigate the effect of ligand binding on ScGal1p we used DSC on highly purified ScGal1p to estimate the temperature at which the protein unfolds in the presence and absence of ligands (Fig. 2A). In the absence of nucleotide and galactose, the protein was found to have a melting temperature (Tm) of 56.4 °C. The peak consisted of two distinct and separate unfolding events that most likely result from the N- and C-terminal domains of the protein unfolding at different temperatures. One domain unfolds at 54.3 °C and the other at 57.2 °C (Table 1, ScGal1p data); however, it was not possible to determine which domain unfolds first. In the presence of ADP (used instead of ATP to prevent enzyme catalysis) and galactose the Tm of ScGal1p was increased to 59.1 °C, an increase of 2.9 °C, indicating significant stabilization of the protein structure in the presence of ligands (Fig. 2A and Table 1, ScGal1p + ADP + Gal data). Similar experiments performed with highly purified KlGal1p also showed an increase in the Tm of the protein in the presence of its ligands from 55.9 °C to 60.4 °C (Table 1, KlGal1p and KlGal1p + ADP + Gal data). The presence of ligands also stabilizes ScGal3p. In the absence of ATP and galactose, the protein consistently precipitated during DSC analysis, making a determination of the Tm impossible. However, in the presence of the ligands, ScGal3p does not precipitate and has a Tm of 57.1 °C (Table 1, ScGal3p + ADP + Gal data). This demonstrates that this family of proteins adopts a more stable conformation upon ligand binding, whether it is for galactokinase activity or for transcriptional induction (which is the only role of ScGal3p).FIGURE 2DSC analysis of ScGal1p. Protein samples in the absence or presence of galactose and/or ADP were heated from 15 to 100 °C. A, DSC of ScGal1p in the absence and presence of ligands showing the maximum of thermal transition (Tm) and ΔHv in each case. B, DSC of ScGal1p in the absence or presence of one or both ligands. For clarity, only the Tm is indicated. Full details of the ΔHv values are shown in Table 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Summary of the thermodynamic data for ScGal1pSampleTmTm1ΔH1Tm2ΔH2°C°Ckcal/mol°Ckcal/molScGal1p56.40 ± 0.1254.34 ± 0.5260.4 ± 13.157.15 ± 0.1249.6 ± 12.7ScGal1p + ADP + Gal59.17 ± 0.0657.02 ± 0.3865.0 ± 11.959.57 ± 0.0956.2 ± 11.5ScGal1p + ADP59.25 ± 0.0557.18 ± 0.2973.5 ± 9.259.70 ± 0.0657.4 ± 8.9ScGal1p + Gal56.78 ± 0.0555.07 ± 0.4051.6 ± 11.357.51 ± 0.1635.3 ± 11.1ScGal3pNDNDNDNDNDScGal3p + ADP + Gal57.11 ± 0.20NDNDNDNDKlGal1p55.90 ± 0.1153.02 ± 0.3249.6 ± 12.256.16 ± 0.1525.4 ± 12.4KlGal1p + ADP + Gal60.42 ± 0.1057.91 ± 0.4993.4 ± 4.8160.91 ± 0.2180.7 ± 14.7 Open table in a new tab ScGal1p Is Converted to a More Stable Form in the Presence of ADP Alone—It has previously been shown that the catalytic mechanism of yeast galactokinase proceeds through the ordered formation of a tertiary complex in which ATP binds to the enzyme first and galactose second (7Timson D.J. Reece R.J. Biochimie (Paris). 2002; 84: 265-272Crossref PubMed Scopus (46) Google Scholar). This suggests that the binding of the nucleotide is essential either for the formation of, or the unmasking of, the galactose-binding site. DSC with ScGal1p was repeated in the presence of either galactose or ADP alone. In the presence of galactose the Tm remained essentially unchanged (Fig. 2B and Table 1, ScGal1p + Gal data). However, in the presence of ADP the Tm was increased to 59.1 °C (Table 1, ScGal1p + ADP data), which was the same as the increase observed in the presence of both ligands (Fig. 2B). This suggests that the binding of ADP does indeed result in the formation or the unmasking of the galactose-binding site and also demonstrates that the binding of galactose does not have a major effect on the stability or conformation of the protein.The Formation of the Kinetically Active Form of ScGal1p Is Not Mediated by a Significant Conformational Change—The increased stability of ScGal1p in the presence of ligands suggested that ScGal1p may undergo a conformational change upon ligand binding which results in the kinetically active form of the protein seen in the crystal structure (11Thoden J.B. Sellick C.A. Timson D.J. Reece R.J. Holden H.M. J. Biol. Chem. 2005; 280: 36905-36911Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). To investigate this, ScGal1p in the absence or presence of one or both ligands was analyzed by velocity AUC. Velocity sedimentation of ScGal1p at a concentration of 1 mg/ml gave a sedimentation value (sapp) of 2.34 in the absence or presence of ligands (Fig. 3). Sedimentation of ScGal1p using various protein concentrations and extrapolation to zero concentration gave ans20,W0 value of 3.98 S, and a hydrodynamic radius of 3.3 nm. By modeling the crystal structure of ScGal1p (11Thoden J.B. Sellick C.A. Timson D.J. Reece R.J. Holden H.M. J. Biol. Chem. 2005; 280: 36905-36911Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) with beads of known hydrodynamic parameters (SOlution MOdeler) (22Rai N. Nollmann M. Spotorno B. Tassara G. Byron O. Rocco M. Structure. 2005; 13: 723-734Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), we were able to determine whether the crystal structure matched the hydrodynamic properties of ScGal1p in solution. The bead model suggests that the crystal structure would have as20,W0 value of 4.16 S and a hydrodynamic radius value of 3.1 nm, which is similar to, if slightly more compact than, the protein in solution. The addition of galactose and/or ADP had no effect on the sedimentation value of the protein, which clearly demonstrates that the protein does not undergo a large conformational change upon ligand binding (Fig. 3). It is likely, therefore, that ligand binding results in a small, more energetically favorable conformational change that stabilizes the protein and forms or unmasks the galactose-binding site. There is an unpublished structure of an unliganded galactokinase in the Protein Data Bank (accession number 2CZ9) arising from the crystallization of the Pyrococcus horikoshii enzyme (23Inagaki E. Sakamoto K. Obayashi N. Terada T. Shirouzu M. Bessho Y. Kuroishi C. Kuramitsu S. Shinkai A. Yokoyama S. Acta Crystallogr. F Struct. Biol. Cryst. Commun. 2006; 62: 169-171Crossref PubMed Scopus (8) Google Scholar). This structure appears to be very similar to the liganded versions (11Thoden J.B. Sellick C.A. Timson D.J. Reece R.J. Holden H.M. J. Biol. Chem. 2005; 280: 36905-36911Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 24Hartley A. Glynn S.E. Barynin V. Baker P.J. Sedelnikova S.E. Verhees C. de Geus D. van der Oost J. Timson D.J. Reece R.J. Rice D.W. J. Mol. Biol. 2004; 337: 387-398Crossref PubMed Scopus (50) Google Scholar, 25Thoden J.B. Timson D.J. Reece R.J. Holden H.M. J. Biol. Chem. 2005; 280: 9662-9670Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), which supports the notion that a large conformational change does not occur within the galactokinases upon ligand binding.FIGURE 3The effect of ligand binding by ScGal1p on protein conformation. The apparent sedimentation coefficient (sapp) distributions of ScGal1p in the presence and absence of ligands are all similar, indicating that there is no gross conformational change upon binding.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Constitutive Gal3p Mutations Affect the Galactokinase Activity of ScGal1p—Previously, a number of constitutive mutants of ScGal3p have been isolated (26Blank T.E. Woods M.P. Lebo C.M. Xin P. Hopper J.E. Mol. Cell. Biol. 1997; 17: 2566-2575Crossref PubMed Scopus (61) Google Scholar, 27Diep C.Q. Peng G. Bewley M. Pilauri V. Ropson I. Hopper J.E. Genetics. 2006; 172: 77-87Crossref PubMed Scopus (12) Google Scholar, 28Diep C.Q. Tao X. Pilauri V. Losiewicz M. Blank T.E. Hopper J.E. Genetics. 2008; 178: 725-736Crossref PubMed Scopus (17) Google Scholar). These mutant proteins (V69E, V203I, F237Y, D368V, V396A, S509P, and S509L) were found to be able to interact with ScGal80p in the absence of galactose, whereas the wild-type protein only interacts with ScGal80p in the presence of galactose. We surmised that these mutations could either directly alter the ScGal3p-ScGal80p interaction site, or they could mimic the ligand-induced stabilization that occurs upon binding of galactose and ATP. The latter theory was based on the observation that when the constitutive ScGal3p mutations were mapped onto the homology model they were located around the interface between the N- and C-terminal domains (11Thoden J.B. Sellick C.A. Timson D.J. Reece R.J. Holden H.M. J. Biol. Chem. 2005; 280: 36905-36911Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and as expected, this was also the case when the equivalent mutations were mapped onto the structure of ScGal1p (Fig. 4). In an effort to distinguish between these possibilities, we made the equivalent changes to ScGal1p (F75E, V211I, F245Y, D376V, V404A, S517P, and S517L) and monitored the effect on both galactokinase activity and transcriptional induction function. Each protein was overproduced in E. coli and purified to homogeneity, and the relative galactokinase activity of each of these proteins was measured (Fig. 5A). F75E, V211I, and D376V are functional galactokinases with ∼55–75% that of the activity of the wild-type protein. Analysis of kinetic parameters for these proteins (Table 2) indicates that in each case the Km for ATP is altered by ∼3–6-fold, and that the kcat is reduced. The D376V mutation also changes the Km for galactose almost 4-fold. F245Y and V404A possess very weak galactokinase activity (Fig. 5A), and these mutations also predominately affect the Km for ATP and kcat (Table 2). The S517 mutants would appear to have lost all galactokinase activity in our assays. It is possible that the mutations at this position tighten the structure of ScGal1p, thus interfering with binding of the nucleotide.FIGURE 4Ribbon representation of ScGal1p with AMPPNP and α-d-galactose ligands displayed in a ball-and-stick representation (11Thoden J.B. Sellick C.A. Timson D.J. Reece R.J. Holden H.M. J. Biol. Chem. 2005; 280: 36905-36911Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The positions of the constitutive ScGal3p mutations introduced into ScGal1p are indicated as yellow spheres (26Blank T.E. Woods M.P. Lebo C.M. Xin P. Hopper J.E. Mol. Cell. Biol. 1997; 17: 2566-2575Crossref PubMed Scopus (61) Google Scholar, 27Diep
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