An ancient oxidoreductase making differential use of its cofactors.

Biol. Chem. 2014; 395(7-8): 855–869

Doreen Blüher, Annekathrin Reinhardt-Tews, Martin Hey, Hauke Lilie, Ralph Golbik, Karin D. Breunig* and Alexander Andersa,*

An ancient oxidoreductase making differential use of its cofactors Abstract: Many transcription factors contribute to cellular homeostasis by integrating multiple signals. Signaling via the yeast Gal80 protein, a negative regulator of the prototypic transcription activator Gal4, is primarily regulated by galactose. ScGal80 from Saccharomyces cerevisiae has been reported to bind NAD(P). Here, we show that the ability to bind these ligands is conserved in KlGal80, a Gal80 homolog from the distantly related yeast Kluyveromyces lactis. However, the homologs apparently have diverged with respect to response to the dinucleotide. Strikingly, ScGal80 binds NAD(P) and NAD(P)H with more than 50-fold higher affinity than KlGal80. In contrast to ScGal80, where NAD is neutral, NAD and NADP have a negative effect in KlGal80 on its interaction with a KlGal4peptide in vitro. Swapping a loop in the NAD(P) binding Rossmann-fold of ScGal80 into KlGal80 increases the affinity for NAD(P) and has a significant impact on KlGal4 regulation in vivo. Apparently, dinucleotide binding allows coupling of the metabolic state of the cell to regulation of the GAL/LAC genes. The particular sequences involved in binding determine how exactly the metabolic state is sensed and integrated by Gal80 to regulate Gal4. Keywords: Gal80; Gal4; GAL switch; Kluyveromyces lactis; nicotinamide adenine dinucleotide; transcription regulation; metabolic sensing. DOI 10.1515/hsz-2014-0152 Received February 20, 2014; accepted May 1, 2014

a Present address: Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany *Corresponding authors: Karin D. Breunig and Alexander Anders, Institut für Biologie, Martin-Luther-Universität Halle-Wittenberg, D-06099 Halle, Germany, e-mail: [email protected]. de; [email protected] Doreen Blüher, Annekathrin Reinhardt-Tews and Martin Hey: Institut für Biologie, Martin-Luther-Universität Halle-Wittenberg, D-06099 Halle, Germany Hauke Lilie and Ralph Golbik: Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, D-06099 Halle, Germany

Introduction Gene specific activators regulate eukaryotic transcription by transmitting intra- or extracellular signals to the transcription apparatus. Many of them are directly or indirectly affected by the metabolic or energy status of the cell. Remarkably, a number of nuclear co-regulators resemble metabolic enzymes and bona fide enzymes participate in transcriptional regulation (Meyer et al., 1991; Aravind and Koonin, 1998; Rolland et al., 2001; Messenguy and Dubois, 2003; Zheng et al., 2003; Kim and Dang, 2005; Moreno et al., 2005; Cho et al., 2006). In these cases, substrate binding sites may be used as sensor domains in metabolic signaling. A striking number of proteins that function in gene repression depend on NAD(H): glycolysis-derived NADH is known to be an allosteric regulator of the co-repressor CtBP (Zhang et al., 2002). NmrA, a repressor of nitrogen metabolism in Aspergillus is a member of the short-chain dehydrogenase reductase superfamily and binds NAD and NADP (Lamb et al., 2003). Sirtuins are deacetylases that use NAD as co-substrate in the deacetylation reaction and the establishment of silenced chromatin (Imai et al., 2000; Vaziri et al., 2001; Lin and Guarente, 2003; Liou et al., 2005). A classic transcription switch module regulating expression of the yeast galactose (GAL) genes consists of three proteins, two of which have evolved from metabolic enzymes. Gal3 is closely related to galactokinases; Gal80 resembles NAD(P)/H dependent oxidoreductases of the GFOR family (glucose-fructose oxidoreductase) (Aravind and Koonin, 1998; Thoden et al., 2007). Interaction between these two proteins regulates the activity of the transcription activator Gal4 (Zenke et al., 1996; Reece and Platt, 1997; Yano and Fukasawa, 1997; Sil et al., 1999). Gal3 has lost catalytic activity but retains the ability to bind the galactokinase substrates galactose and ATP. As such, it functions as a galactose sensor and signal transducer. Binding of both metabolites apparently induces a conformational change in Gal3, which stimulates the formation of a Gal3-Gal80 complex resulting in activation of Gal4 (Zenke et al., 1996; Yano and Fukasawa, 1997; Sil et al., 1999; Thoden et al., 2005; Lavy et al., 2012). Gal80

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856 D. Blüher et al.: NAD(P) binding of Gal80 can interact with the Gal4 activation domain on the one hand and with Gal3 on the other. Genetic approaches to separate those functions have revealed the extreme sensitivity of Gal80 to single amino acid substitutions (Melcher, 2005). Hence it is not fully understood how Gal4 and Gal3 interactions influence each other and how they modulate the activity of Gal4. As an exciting aspect, evidence for a role of the adenine dinucleotides NAD and NADP in Gal4 regulation has been presented (Kumar et al., 2008). NADP was shown in vitro to interfere with the stability of the Gal4-Gal80 complex whereas NAD appeared to stabilize the complex. The latter evidence is based on the fact that soaking cocrystals of Saccharomyces cerevisiae Gal80 with a Gal4 peptide comprising the Gal80 binding domain with NAD improved the diffraction pattern in X-ray crystallography (Kumar et al., 2008). Much insight into the evolution of the GAL switch has been gained from the comparative analysis of the S. cerevisiae regulatory module with that of Kluyveromyces lactis. K. lactis has diverged from the Saccharomyces lineage before the whole-genome duplication event and hence lacks many paralogous genes including GAL3 (Keogh et al., 1998). Instead KlGAL1 encodes a bifunctional protein, which catalyzes the phosphorylation of galactose and transduces the galactose signal (Meyer et al., 1991). As in Gal3, the KlGal1 regulatory function, i.e., the ability to bind K. lactis Gal80 (KlGal80), is independent of its enzymatic activity (Meyer et al., 1991; Zenke et al., 1996). Cross-complementation studies have shown that KlGal4 can replace Gal4 in S. cerevisiae and vice versa (Salmeron and Johnston, 1986; Riley et al., 1987) and KlGal80 and ScGal80 bind to Gal4 via a highly conserved C-terminal region that overlaps the transcription activation domain (TAD) (Johnston et al., 1987; Salmeron et al., 1989; Dickson et al., 1990; Zenke et al., 1993). However, Gal3 is unable to replace KlGal1 in K. lactis unless KlGal80 is substituted by ScGal80 (Zenke et al., 1996). Hence, despite high sequence conservation, the KlGal1-KlGal80 interaction appears to differ from that of Gal3-Gal80. In line with this hypothesis, not all GAL80S mutations that abolish Gal3 binding and result in a dominant super-repressed phenotype in S. cerevisiae give a similar phenotype when equivalent mutations are introduced into KlGAL80 (Zenke et al., 1999). Moreover, the KlGal80 protein does not shuttle between cytoplasm and nucleus like ScGal80 and thus cytoplasmic sequestration of KlGal80 does not contribute to KlGal4 regulation. Instead, the KlGal1-KlGal80 interaction occurs in the nucleus and competes with the formation of the Gal80Gal4 complex (Anders et al., 2006).

High resolution X-ray structures of KlGal80 and ScGal80 show high similarity (Thoden et al., 2007; Kumar et al., 2008) and confirmed the relationship to enzymes of the glucose-fructose oxidoreductase family [GFOR (Kingston et al., 1996)]. Co-crystals of ScGal80 and KlGal80 with a 22 amino acid Gal4 peptide indicated that the Gal4-TAD binds to a cleft between the N- and C-terminal lobes of Gal80 in both cases but the peptides were poorly resolved and their orientations seemed different (Kumar et al., 2008; Thoden et al., 2008). The ScGal80-TAD cocrystal contained NAD, which was bound in close proximity to the TAD peptide. For KlGal80 no crystals with a bound dinucleotide could thus far be obtained. On the contrary, soaking KlGal80 crystals in NAD abolished diffraction (Thoden et al., 2008). Here, we show that KlGal80, like ScGal80, is able to bind NAD and NADP but with much lower affinity. In contrast to ScGal80, both NAD and NADP have a negative effect on KlGal80-Gal4-TAD interaction. We present evidence that, despite its relatively low affinity for NAD(P), KlGal80 is regulated in vivo by this activity. Furthermore, we show that the effect exerted by the dinucleotide binding function in vivo can be modulated by exchanging a short stretch of amino acids involved in NAD(P) binding. Overall, our data support a model in which both KlGal80 and ScGal80 integrate the metabolic state of the cell via their dinucleotide binding functions to modulate expression of the GAL/LAC genes. We propose that S. cerevisiae Gal80 has evolved divergently from KlGal80 by increasing the affinity for the dinucleotide, eventually leading to a new signaling function of the ligand in the GAL switch.

Results K. lactis Gal80 binds NAD(P) with low affinity NAD(P) has been shown to bind S. cerevisiae Gal80 and in an in vitro binding assay interaction of ScGal80 with Gal4 was impaired by the presence of NADP but not of NAD (Kumar et al., 2008). So far no quantitative data were reported and the role and significance of this binding for Gal4 regulation is poorly understood. Here we have determined binding constants and questioned whether the binding of NAD(P) is conserved in KlGal80. His6-tagged variants of KlGal80, named NHGal80 and IHGal80 (for N-terminal or internally tagged KlGal80, respectively) (Anders et al., 2006), and N-terminally His6-tagged Gal80 from S. cerevisiae (NHScGal80) were


D. Blüher et al.: NAD(P) binding of Gal80 857

Figure 1 Influence of NAD and NADP on NHGal80 monitored by tryptophan fluorescence spectroscopy. Fluorescence spectra from 305 to 380 nm were recorded with an excitation wavelength of 295 nm. 1 μm protein solution was titrated with NAD (closed circles) and NADP (open circles) to a final cofactor concentration of 1.5 mm. Values were corrected for inner-filter effects and unspecific quenching as described in Material and methods and the corrected tryptophan fluorescence at 345 nm is plotted as a function of NAD(P) concentration. Dissociation constants were derived from curve fitting as described in Material and methods and are listed in Table 1.

recombinantly expressed in Escherichia coli, highly purified in two chromatographic steps (see Material and methods) and analyzed for dinucleotide binding by fluorescence spectroscopy. The proteins were excited at 295 nm and tryptophan fluorescence spectra were recorded in the presence of increasing NAD and NADP concentrations. The

tryptophan fluorescence was quenched by both dinucleotides in a concentration-dependent manner allowing for determination of binding constants (Figure 1). Fluorescence intensities corrected for inner-filter effects and quenching (Materials and methods) when plotted as a function of the dinucleotide concentrations gave a dissociation constant (KD, app) of 610 μm for the K. lactis NHGal80-NAD complex and of 210 μm for the NHGal80-NADP complex (Table 1). Very similar results (KD, app = 750 and 270 μm for NAD and NADP, respectively) were obtained for internally tagged IHGal80 (Table 1), showing that the position of the His6-tag did not greatly influence the results. To our surprise, despite high structural similarity with KlGal80, the affinity of S. cerevisiae ScGal80 protein for NAD(P) was at least 40-fold higher compared to KlGal80 (KD, app = 15.2 and 2.5 μm for NAD and NADP, respectively; Table 1). Because high autofluorescence meant that fluorescence spectroscopy could not be used to analyze binding of the reduced dinucleotides, we performed isothermal titration calorimetry (ITC) with NHGal80 to compare the binding of reduced and oxidised forms (Figure 2A and B). The dinucleotides were found to bind in an exergonic reaction with a 1:1 stoichiometry of protein to ligand (Figure 2). The KD,app values for NADP and NAD agree well with those determined by fluorescence measurements and confirmed the three- to four-fold higher affinity for NADP compared to NAD (Table 1). The reduced forms were bound at least fivefold less efficiently. For NADH, the affinity was too low for determination of a binding constant. For NADPH, saturation in the titration experiment could not be obtained but the binding isotherms could be fitted assuming a 1:1 stoi-

Table 1 Dissociation constants of Gal80-dinucleotide complexes. Gal80 variant

NHGal80

Apparent KD, app values of dinucleotide binding (μm)a

NAD

NHGal80 and AD-22 IHGal80 NHGal80-W31G NHGal80-W31F NHGal80-SW NHGal80-SW and AD-22 NHScGal80 NHScGal80 and AD-22

610 ± 40 590 ± 35 (ITC)b 1500 ± 440 750 ± 80 – – 7.6 ± 1.5 15.3 ± 0.9 15.2 ± 0.1 11.0 ± 0.1

NADP

210 ± 15 205 ± 16 (ITC)b 170 ± 140 (AUC)c 120 ± 30 (CD)d 960 ± 140 270 ± 20 Not detectable (AUC)c Not detectable (CD)d 3.0 ± 0.1 6.1 ± 0.4 2.5 ± 0.0 9.7 ± 0.2

NADH

Not detectable (ITC)b – – – – – – – –

NADPH 1160 ± 890 (ITC)b

– – – – – – 7.6 ± 0.5 –

a Measured by fluorescence spectroscopy if not otherwise indicated. bIsothermal titration calorimetry. cAnalytical ultracentrifugation. dCircular dichroism spectroscopy.-, not determined.


858 D. Blüher et al.: NAD(P) binding of Gal80

Figure 2 Binding of different dinucleotides to NHGal80 analyzed by isocalorimetric titration. (A) Raw data corrected for dilution effects of 150 μm NHGal80 titrated with 15 mm NAD(H) and 8 mm NADP(H) in a 1.4 ml cell. The titrated dinucleotides are indicated above the panels. The peaks correspond to 1 μl initial injection, followed by 10 × 2.5 μl and 25 × 10 μl injections. (B) Integrated heats, corrected for dilution and mixing, plotted against the ratio between dinucleotide and NHGal80.

chiometry. The resulting KD, app was approximately 1160 μm for NADPH compared to 200 μm for NADP (Table 1). For ScGal80 (NHScGal80), a KD,app of 7.6 μm for NADPH was determined. Thus, the relative differences in affinity for NAD vs. NADP (three- to six-fold) and for reduced vs. oxidised forms of NADP (three- to five-fold) were similar in ScGal80 and KlGal80 but there were almost two orders of magnitude difference in the absolute values.

Tryptophan W31 is responsible for nucleotide-quenched autofluorescence and crucial for ligand binding In the co-crystal structure of ScGal80 with NAD, tryptophan residue 31 (W31) makes stacking interactions with the nicotinamide ring (Kumar et al., 2008) (see also Figure 5B and C). We have analyzed a mutant variant of NHGal80, in which tryptophane 31 was replaced by a glycine (NHGal80-W31G). For this variant, we observed a greatly diminished autofluorescence intensity, which was not further affected by titration with dinucleotides (Table 1). Apparently, W31 is responsible for the quenching of tryptophan fluorescence upon ligand binding. Circular dichroism (CD) spectra for wild type NHGal80 in the near-UV range shifted upon titration with increasing concentrations of NADP (Figure 3A) and NAD (not shown) and allowed to quantify ligand binding by a method independent of tryptophan fluorescence

quenching. In the titration experiment with NHGal80 the isodichroic point (cross point between the different spectra) observed at 335 nm is indicative of different characteristics of the dinucleotide in the bound and unbound state. There was no evidence for major conformational transitions in the protein upon ligand binding as judged from the comparison of CD-spectra in the far-UV range (Figure S1). For NHGal80-W31G the spectra were hardly influenced by dinucleotides (Figure 3B). Similarly, replacing tryptophan 31 by alanine (NHGal80-W31A) (data not shown) or phenylalanine (NHGal80-W31F) resulted in an NAD(P) binding deficient KlGal80 variant (Figure S2, Table 1). Wild type and NHGal80-W31G proteins were also compared by analytical ultracentrifugation in the absence and presence of NADP (Figure 4). For NHGal80, the amplitude of the sedimenting absorption signal increased with increasing NADP concentrations, indicating co-sedimentation of protein and cofactor, whereas no influence of NADP on the amplitude of sedimenting NHGal80W31G was observed. The apparent dissociation constant of NHGal80-NADP calculated from these experiments was 170 μm, which is in good agreement with the values reported above (Table 1). Independent of the ligand concentrations, sedimentation profiles were consistent with dimers for both NHGal80 and NHGal80-W31G. Taken together, we conclude that in KlGal80, as in ScGal80 tryptophan, W31 is essential for binding of dinucleotides.



B

5 × 103

-5 × 103

Θmolar (deg · cm2 · dmol-1)

0 Θmolar (deg · cm2 · dmol-1) (310 mM)

Θmolar (deg · cm2 · dmol-1)

A

-5 × 102 -1 × 103

-1.5 × 103

-1 × 104

-2 × 103

-2.5 × 103

0

0.5 1 [NADP] (mM)

-1.5 × 104 300

350

400

450

5 × 103

0

-5 × 103

-1 × 104

1.5

-1.5 × 104 500

λ (nm)

300

350

400

450

500

λ (nm)

Figure 3 Near-UV CD spectra of NHGal80 in the presence of increasing amounts NADP. CD-spectra of 120 μm NHGal80 and mutated derivatives were recorded in the absence (black line) and presence of increasing concentrations of dinucleotides (dashed lines for 0.06–1.2 mm, gray line for 1.5 mm). (A) NHGal80 titrated with NADP; (B) NHGal80-W31G titrated with NADP. The insets show the changes of the molar ellipticity at 310 nm as a function of the ligand concentration. Dissociation constants were derived from curve fitting as described in Material and methods and are listed in Table 1.

Figure 4 NADP-binding to NHGal80 and NHGal80-W31G analyzed by analytical ultracentrifugation. Five μm protein solutions of NHGal80 (open circles) or NHGal80W31G (closed triangles) in 20 mm Tris/HCl, 60 mm NaCl, 60 mm EDTA (pH 8.2) were sedimented with NADP at concentrations of 0–600 μm. The sedimentation of the protein-associated absorption amplitude (280 nm) was measured at 40,000 rpm, 20°C. Dissociation constants were derived from curve fitting as described in Material and methods and are listed in Table 1.

and B). Positions 21 to 30 contain a GX2GX5G motif that is in contact with NAD in the ScGal80 co-crystal structure (Figure 5A and C). This conserved motif deviates from the GX2GXG motif found in the classic Rossmann-fold (Aravind and Koonin, 1998). Two Gal80 homologs (TBLA0D03760 and Sklu_YGOB_Anc-1.500) have a fourth glycine at position 28. In KlGal80 G28 is also present but G30 is replaced by a serine residue. There is also a highly conserved proline at position 38, which is replaced by a leucine in KlGal80. We have speculated that the sequence deviations in this region might be responsible for the difference in affinity for NAD(P) between KlGal80 and ScGal80. To test this hypothesis, residues 26 to 38 of KlGal80 (TSGKSWVAKTHFL) were replaced with NAAKGWAIKTHYP as found in the equivalent positions of ScGal80 (Figure 5A). The swapped variant (KlGal80-SW) was recombinantly expressed in E. coli and purified as described for the other KlGal80 variants, and NADP-binding was measured using fluorescence quenching. We found that KlGal80-SW had much higher affinity for NADP than KlGal80 (KD,app. of 3.0 μm vs. ≈200 μm, Table 1) confirming that residues around position 30 play a crucial role in determining the affinity for the dinucleotide.

The affinity of KlGal80 for NAD(P) can be strongly increased by exchange of amino acids around W31

The negative influence of NADP on Gal80Gal4 interaction is conserved between ScGal80 and KlGal80

Gal80 variants in different yeasts show high sequence and structural similarity in the region around W31 (Figure 5A

To test whether binding of the dinucleotide ligands to KlGal80 affected interaction with its binding partners – KlGal1


860 D. Blüher et al.: NAD(P) binding of Gal80

Figure 5 Structures of Gal80. (A) Multiple sequence alignment of the N-terminal Rossmann-fold (amino acids 14 ff.) of Gal80 orthologues. Alignment was performed by MUSCLE (3.8) via http://ygob.ucd.ie/cgi/ygob.pl? gene = YML051W (February 2014) and includes ORF translations from the following genomes (top to bottom): Kazachstania naganishii, Kluyveromyces lactis, Lachancea kluyveri, Lachancea thermo-tolerans, Tetrapisispora blattae, Zygosaccharomyces rouxii, Saccharomyces bayanus var. uramum, Tetrapisispora phaffii, Kazachstania africana, Naumovozyma dairenensis, Naumovozyma castellii, Vanderwaltozyma polyspora, Kazachstania naganishii, Tetrapisispora blattae, Saccharomyces bayanus var. uramum, Saccharomyces mikatae, Saccharomyces cerevisiae, Naumovozyma dairenensis, Naumovozyma castellii. The conserved glycine residues as mentioned in the text are marked and numbered in the top line. (B) Alignment of ScGal80 [pdb: 3BTV (Kumar et al., 2008)] and KlGal80p [pdb: 2NVW from (Thoden et al., 2007)]. Alignment was created with PyMOL (The PyMOL Molecular Graphics System, Version 1.3 Schrödinger, LLC.) ScGal80 is depicted in pale green, KlGal80p in pale blue. The swap domain (amino acid 26–38) is shown in pale gray (ScGal80) and dark gray (KlGal80). Conserved glycine residues are marked in orange, proline 38 in black, serine 30 in yellow and tryptophan 31 in rose (ScGal80) and pink (KlGal80). (C) The NAD-binding pocket of ScGal80 in complex with NAD [pdb: 3BTS (Kumar et al., 2008)]. The color code is identical to (5B). NAD is shown in stick presentation in blue. Tryptophan 31 (in pink) forms a stacking interaction with the nicotinamide ring of NAD.

on the one hand and KlGal4 on the other – we made use of a method that has recently been developed in our laboratory. It is based on inhibition of the galactokinase activity of KlGal1 by KlGal1-KlGal80 interaction. The strength of the enzyme inhibition is proportional to the affinity between both proteins (Anders et al., 2006). We applied this assay to compare NHGal80 and NHGal80-SW. In Table 2, galactokinase activity in the presence of these KlGal80 variants is expressed relative to the activity in the absence of KlGal80.

The inhibitory influence of NHGal80-SW was somewhat lower than wild type, indicating a moderate influence of the amino acid exchanges on KlGal80-KlGal1 interaction. However, importantly, there was no significant influence of NADP on galactokinase inhibition whether or not the variant was able to bind NADP (see below). This result indicates that KlGal80-KlGal1 interaction is not affected by the ligand. The interaction between NHGal80 and Gal4 was analyzed by the same assay, including a 22 amino acid peptide



Table 2 Influence of NADP and AD-22 on KlGal80-mediated KlGal1 inhibition. KlGal80 variant

Relative galactokinase activity (%)a

-NADP

NHGal80 NHGal80-W31G NHGal80-W31F NHGal80-SW

44.8 ± 2.9 63.2 ± 7.3 62.1 ± 1.7 60.0 ± 4.7

KlGal80b

+NADP b

-NADP

61.9 ± 2.3 76.7 ± 3.0 73.4 ± 2.9 70.2 ± 4.3

47.6 ± 3.5 62.1 ± 5.5 66.0 ± 3.3 61.8 ± 5.2

KlGal80b + AD-22b +NADP b

55.6 ± 3.5 76.7 ± 4.0 74.6 ± 1.9 60.2 ± 0.7

a Activities are given relative to the samples without any supplement and represent mean values ± SD. b Concentrations were as follows: 380 nm NHGal80 and variants; 2.6 μm AD-22 peptide (NHGal80, -W31F, -SW) and 1.2 μm AD-22 peptide (NHGal80-W31G); 1 mm NADP.

(AD-22) derived from the KlGal4 TAD. Addition of this peptide to a KlGal1-KlGal80 mixture had been shown to reduce galactokinase inhibition because of competing AD22-KlGal80 interaction and consequent release of active KlGal1 (Anders et al., 2006). Thus, the efficiency of Gal4TAD to relieve KlGal80-mediated galactokinase inhibition is indicative of the affinity between KlGal80 and Gal4-TAD. We observed higher galactokinase activity in the presence vs. absence of the AD-22 peptide for both wild type and KlGal80-SW (Table 2, compare columns 1 and 3). Importantly, the presence of NADP in addition to AD-22 restored galactokinase inhibition by wild type NHGal80 partially and completely by NHGal80-SW (Table 2, columns 3 and 4) but not by the NAD(P) binding deficient NHGal80-W31G or NHGal80-W31F variants (Table 2, columns 3 and 4). The negative influence of NADP on KlGal80-AD-22 interaction could independently be confirmed by

fluorescence quenching measurements. Addition of the peptide reduced the apparent affinity of NHGal80 for NADP about five-fold and a similar effect was observed with NHScGal80 (Table 1). However, surprisingly, the KlGal80-AD22 interaction was also impaired by NAD, in contrast to ScGal80. Thus, a differential effect of NADP and NAD on ScGal80-Gal4 activation domain interaction as described before (Kumar et al., 2008) could be confirmed in our assay but is not conserved in KlGal80. Hence the regulatory model that depends on this differential effect of NADP and NAD cannot apply to K. lactis.

The dinucleotide binding function of KlGal80 is involved in its regulation in vivo So far, the biological function of dinucleotide binding to Gal80 for the GAL transcriptional switch is far from being clear. As in KlGal80, NAD and NADP both have a similar, negative influence on the interaction with Gal4-TAD in vitro, mutations interfering with dinucleotide binding might be expected to positively affect repression in vivo. To monitor KlGal4 controlled gene expression, the endo genous K. lactis β-galactosidase gene (LAC4) was used as a reporter and a KlGal80 deletion was replaced by wild type and mutated alleles of KlGAL80. In most mutated KlGal80 variants, β-galactosidase activity was higher than in wild type, indicating reduced Gal80 inhibitory activity. This phenotype was not caused by NADP-binding deficiency per se as binding deficient mutant KlGal80W31F showed almost wild type repression (Figure 6A). Induction by galactose in this mutant was not impaired

Figure 6 Influence of KlGAL80 alleles on LAC gene repression and induction. Kluyveromyces lactis cells with KlGAL80 wild type (JA6) or mutant GAL80 alleles as indicated were pre-grown in YEP medium containing 2% glucose and then shifted to fresh medium containing 2% glucose (A) and 2% glucose + 2% galactose (B), respectively. Samples were harvested at the indicated time points after the shift [4 hours in (A)] and β-galactosidase activity was determined as described (Zenke et al., 1993). Mean values ± SD of two biological replicates are given relative to the wild type. Significance was defined at *p G almost abolished KlGal80-mediated Gal4 inhibition (data not shown), W31A showed a marked reduction (Figure 6A) whereas W31F behaved close to wild type (Figure 6A and B). We conclude W31 is not only essential for dinucleotide binding but is also involved in Gal80-Gal4 interaction in vivo.

A regulatory role for the NAD(P)/NAD(P)H ligand Li et al. reported that in S. cerevisiae NADP depletion by deletion of NAD kinase genes impaired GAL gene activation (Li et al., 2010). Such phenotypes can be expected



if NADP but not NAD had a negative influence on Gal80Gal4 interaction. However, given the relatively weak effect of ScGal80 dinucleotide binding deficiency reported by Kumar et al. (2008) it is very likely that pleiotropic effects of NADP(H) depletion in the cell contribute to the strong phenotype of NAD kinase mutants. In K. lactis, both NAD and NADP have a negative influence on KlGal80-AD22 interaction in vitro, making mechanisms based on their differential effects unlikely. Virtually all NAD(P) binding deficient KlGal80 mutants in our hands seem to have at least subtle defects in KlGal1 and/or KlGal4 binding per se. For this reason, it has been problematic to unambiguously assign a role for dinucleotide binding in vivo by comparing binding deficient variants to wild type. However, by varying the metabolic (and likely directly the NAD(P)(H)) state of the cells by extracellular NA we were able to demonstrate such a role: while the strain expressing KlGal80 wild type protein was significantly influenced by NA this dependence was abolished in a strain with dinucleotide binding deficient KlGal80. This result clearly shows that the NAD(P) binding activity of KlGal80 is involved in its regulation. Whether NAD(P)(H) or other related metabolites are responsible for the observed regulation remains to be shown. KlGal4 activation in KlGAL80 wild type cells was increased at increasing NA concentrations in the medium. Our in vivo and in vitro observations for wild type KlGal80 are in agreement with a simple model in which NAD(P) reduces its affinity for Gal4 and thereby supports induction (and counteracts repression). We favor the hypothesis that the low affinity of KlGal80 for the ligands reflects the evolutionary origin of Gal80 from an oxidoreductase, which now might be used to sense depletion of NAD(H) in K. lactis, resulting in faster repression or impaired induction of KlGal4 at low levels. So far, the strongest effects

of NA variation were observed in glucose plus galactose medium, the two opposing hexoses generated by lactose hydrolysis. In K. lactis, NAD(P) sensing of Gal80 may couple lactose metabolism to the cellular need. With the loss of the lactose metabolic genes in S. cerevisiae, ScGal80 would have been released from this selective pressure and would have adapted otherwise. As the higher affinity for the ligand assures that ScGal80 is saturated with dinucleotides most of the time, but NAD and NADP have differential effects, it may now sense NAD(H)/NADP(H) ratios. The KlGal80-SW variant may reflect this changed mode of dinucleotide regulation. The KlGAL80-SW strain showed the opposite phenotype of KlGAL80 wild type regarding its response to different NA concentrations in the medium. At first glance, our in vitro data showing negative effects of both NAD and NADP on the interaction of this Gal80 variant with Gal4-AD are not compatible with the proposed differential effects of dinucleotides. However, such mode of regulation could also apply if the effects of different dinucleotides on Gal4 interaction would be quantitatively different (and not only if they were qualitatively different as in the case of ScGal80). In any case, the evolutionary conservation of the ligand binding fold, particularly in the light of a fast evolving genetic locus, is a strong indication that it is under positive selection.

Materials and methods Yeast strains Kluyveromyces lactis strains are listed in Table 3. All K. lactis strains were congenic to JA6 (MATα ade1-600 adeT-600 ura3-12 trp1-11) (Breunig and Kuger, 1987). The various KlGAL80 alleles were generated by site-directed mutagenesis using the QuikChange Kit (Agilent

Table 3 Kluyveromyces lactis strains. Strain

Relevant genotype

JA6 JA6/D802 JA6/DL4 JA6/G80-W31G JA6/G80-W31A JA6/G80-W31F JA6/G80-SW

KlGAL80 Klgal80-Δ2::ScURA3 Kllac4::ScURA3 KlGAL80-W31G KlGAL80-W31A KlGAL80-W31F KlGAL80-SW (domain swap: replacement of codons 26–38 by corresponding sequence of ScGAL80) KlGAL80-SW31(KlGAL80-SW-W31A) Derivative of JA6; PLAC4::Y-GFP Derivative of JA6/G80-W31A; PLAC4::Y-GFP Derivative of JA6/G80-W31F; PLAC4::Y-GFP Derivative of JA6/G80-SW; PLAC4::Y-GFP

JA6/G80-SW31 JA6/Y-GFP JA6/ W31A-Y-GFP JA6/ W31F-Y-GFP JA6/G80-SW-Y-GFP


Reference Breunig and Kuger, 1987 Zenke et al., 1999 Gödecke et al., 1991 This work This work This work This work This work This work This work This work This work

866 D. Blüher et al.: NAD(P) binding of Gal80 Technologies, Santa Clara, CA, USA), verified by DNA sequencing and introduced into JA6/D802 (Klgal80::ScURA3) (Zenke et al., 1993) as PCR fragment replacing Klgal80::ScURA3 (details are available on request). Gal4 inhibition function of KlGal80 variants was monitored as β-galactosidase activity encoded by the Gal4 controlled LAC4 gene. For time-lapse microscopy a destabilized GFP reporter gene (Y-GFP) (Houser et al., 2012) was integrated between the LAC4 promoter and the LAC4 coding region of the Klgal80 mutant stains using the lactase based selection strategy described previously (Krijger et al., 2012).

Protein expression and purification Protein variants of KlGal80 with an N-terminal His6-tag (NHGal80, NHGal80-W31G, NHGal80-W31A, NHGal80-W31F and NHGal80-SW) or an internal His6-tag (IHGal80), N-terminally His6-tagged ScGal80 (NHScGal80) and KlGal1 (NHGal1) were expressed in E. coli strain Rosetta (DE3)-pLys from pET-derived (Merck KGaA, Darmstadt, Germany) expression vectors (pETNHG80, its derivatives pETNHG80W31 Getc., pETIHG80, pETNHScG80 and pETNHG1). After induction, proteins were purified via nickel-nitrilotriacetic acid-Sepharose affinity chromatography (Qiagen, Venlo, Limburg, Netherlands) essentially as described (Anders et al., 2006). The affinity purified proteins were subjected to gel filtration chromatography (running buffer: 20 mm Tris/ HCl, 60 mm NaCl, 60 mm EDTA, pH 8.2). Fractions with highly purified protein were concentrated using Amicon Ultra Centrifugal Filter Units (Merck KGaA, Darmstadt, Germany; MWCO 30.000). Glycerol was added to a final concentration of 10% and the protein solutions were stored at -70°C after shock freezing in liquid nitrogen. Prior to the experiments, glycerol was removed using Amicon Ultra Centrifugal Filter Units.

Gal4-activation domain peptide AD-22 A peptide (AD-22) consisting of the 22 C-terminal amino acids of KlGal4 (amino acid sequence: TQQLFNTTTMDDVYNYIFDNDE) (Genosphere Biotechnologies, Paris, France). This peptide was capable of competing with KlGal1 for the binding to KlGal80 (Anders et al., 2006).

Fluorescence spectroscopy Fluorescence spectra were measured upon excitation at 295 nm and recorded from 350 to 380 nm on a FluoroMax-2 spectrometer (HORIBA Jobin Yvon S.A.S., Longjumeau Cedex, France). Excitation and emission band passes were set at 5 nm. All experiments were performed at 20°C in 20 mm Tris/HCl, 60 mm NaCl, 60 mm EDTA (pH 8.2) using a cuvette with a 1 cm optical path length. NAD(P) was dissolved in buffer and titrated to 1 μm NHGal80 (2 μm for NHGal80-W31G) or 1.5 μm NHScGal80 solution. After 5 min equilibration, the wavelength scans were performed with a data pitch of 0.5 nm and an increment of 0.5 s (three scans averaged). NAD(P) binding experiments in the presence of 10 μm Gal4 peptide (AD-22) were performed after preincubation of NHGal80 or NHScGal80 with AD-22 for 5 min. All spectra were corrected for dilution, buffer and NAD(P) spectra. For correction of the inner-filter effect and dynamic quenching the same experiment was carried out using N-acetyltryptophanamide (NATA) instead of NHGal80. The quench constant (k) was determined by the Stern-Volmer equation F0/F = 1+k[Q], where F0 is the fluorescence signal in absence of the quencher, F is the fluorescence signal in presence of the quencher, and [Q] is the concentration of the quencher in mm (Eftink and Ghiron, 1981).

Isothermal titration calorimetry Isothermal titration calorimetry was performed at 20°C in 20 mm Tris/ HCl, 60 mm NaCl, 60 mm EDTA (pH 8.2) on a VP-ITC Micro Calorimeter (MicroCal, Northampton, MA, USA) after vacuum degassing of the samples. For binding studies, 15 mm NAD(H) or 8 mm NADP(H) in buffer solution were injected to a 150 μm KlGal80 solution in a stirred sample cell. A total of 36 injections (1 μl initial injection, 10 × 2.5 μl and 25 × 10 μl) were made at 320 s intervals. Blank titrations were made in the absence of the protein to control for heat of dilution and mixing. The control titrations were subtracted from the experimental titrations prior to data analysis. The data were analyzed by the Origin software (MicroCal, Inc., Northampton, MA, USA) and fitted to a single-site model.

Protein concentration determination

CD spectroscopy CD-spectra of NHGal80 and its derivatives were measured on a Jasco810 spectropolarimeter equipped with Peltier temperature control (Labor- und Datentechnik, Groß-Umstadt, Germany). All experiments were performed at 20°C in 20 mm Tris/HCl, 60 mm NaCl (pH 8.2) in the presence (Near-UV) or absence of 60 mm EDTA (Far-UV) using a cuvette with a 1-cm path length. NAD(P) was dissolved in buffer and titrated to 120 μm or 50 μm NHGa80 solution. After 5 min preincubation the wavelength scans were performed for near-UV CD-spectra from 290 to 500 nm at a scan speed of 500 nm/min, time response of 1 s, data pitch of 0.2 nm and for far-UV CD-spectra from 190 to 260 nm at a scan speed of 50 nm/min, time response of 1 s, data pitch of 1 nm. All spectra (averages) were corrected for dilution, buffer and NAD(P) spectra. From the observed ellipticity Θobs, the molar ellipticity Θmolar

( Θobs ⋅100⋅ M r ) , where Mr is ( c⋅l ) the molecular weight of NHGal80 in Da, l is the optical path length in [deg/cm2/dmol] was calculated as Θmolar =

cm, and c is the protein concentration in milligram per ml.

Protein concentrations were determined using the Coomassie Plus Assay Kit (Pierce, Biotechnology, Rockford, IL, USA) and are considered for monomeric protein.

Analytical ultracentrifugation Gal80 was analyzed using a Beckman Optima XL-A (Beckman Coulter, Brea, CA, USA) centrifuge and a 50Ti rotor at initial protein concentrations of 30–300 μg/ml in 20 mm Tris/HCl, 60 mm NaCl, 60 mm EDTA (pH 8.2) in the presence of varying NADP concentrations. Sedimentation equilibrium measurements (absorbance at 230 and 280 nm) were carried out in double sector cells at 6000 and 8000 rpm and 20°C. Sedimentation velocity was monitored at 40,000 rpm, 20°C. To analyze NADP-binding to Gal80 during the sedimentation velocity experiment, the amplitude of the sedimenting absorption signal was quantified in dependence of the NADP concentration. All data were analyzed using the software provided by Beckman Coulter (Beckman Coulter, Brea, CA, USA).



Calculation of dissociaton constants For the determination of apparent dissociation constants (KD,app) of NAD(P)-binding to NHGal80 by analytical ultracentrifugation, fluorescence and CD spectroscopy, the reported signal at given wavelength was plotted as a function of the dinucleotide concentration and calculated according to the following equation (Agashe et al., 1997):  ([ R ] + [ L ] + K ) − ([ R ] + [ L ] + K ) 2 − 4⋅[ R ] ⋅[ L ]  0 0 D 0 0 D 0 0  ⋅Smax S =  2⋅[ R ]0   where S represents the reported signal and Smax is the maximum amplitude. [R]0 corresponds to the concentration of the KlGal80 and [L]0 to the cofactor concentration. The apparent KD-values of ScGal80 determined by fluorescence spectroscopy were calculated using the modified Stern-Volmer equation (Lehrer, 1971). For NHGal80-SW the apparent KD-value of NAD-binding was calculated according to NHGal80 and for NADP according to ScGal80.

Galactokinase inhibition assay Galactokinase activity (galactose + ATP → galactose-1-phosphate + ADP) was measured using a coupled enzymatic assay where the production of ADP was coupled to the consumption of NADH via the coupling enzymes pyruvate kinase (PK) and lactate dehydrogenase (LDH). One-step galactokinase inhibition experiments were performed as described previously (Anders et al., 2006) either in the absence or in the presence of 1 mm NADP. The reaction buffer was composed of 100 mm Tris/Cl pH 7.9, 5 mm MgCl2, 10 mm KCl, 100 mm potassium acetate, 300 μg/ml BSA, 1 mm ATP, 0.25 mm NADH, 2 mm phosphoenolpyruvate (PEP), 1 mm fructose-1,6-bisphosphate (FBP) and 6.6/13.5 U/ml of the coupling enzymes PK/LDH. Each reaction mixture contained NHGal1. The reaction mixture was pre-warmed to 30°C before measurement. NHGal80 (and variants, respectively) and peptide AD-22 were added as indicated. Reactions were started by the addition of 50 mm galactose and measured photometrically at 30°C by following the consumption of NADH at a wavelength of 340 nm over a period of 5 min. Samples containing the different KlGal80 variants and the AD-22 peptide were measured against a sample with KlGal1 alone giving relative galactokinase activities.

Time-lapse microscopy Growth media for microscopy were composed of low-fluorescence yeast nitrogen base (Formedium, Hunstanton, Norfolk, UK) supplemented with adenine, uracil, tryptophan and the indicated carbon source. Cells were grown at 30°C over night in 24-well plates (TPP, Trasadingen, Switzerland) in media containing 2% glucose and 2% galactose, respectively, inoculated into fresh media of the same composition and prepared for microscopy during exponential growth phase. Glucose grown cells were washed with and resuspended in medium with 0.1% glucose. Galactose grown cells were used directly from growth medium. 50 μl of the cell suspensions were transferred into 400 μl of fresh growth media with the indicated carbon sources in a 96-well glass bottom plate (Matrical Bioscience, Spokane, WA, USA). For experiments using variable NA concentrations (Figure 8), cells pregrown in either glucose or galactose containing media were washed twice with YNB without NA (YNB-NA, Formedium, Hunstanton,

Norfolk, UK) and without carbon source. Washed cells were then transferred into YNB-NA media supplemented with carbon sources and nicotinic acid as indicated. Microscopy was performed at 30°C on an automated Olympus IX81 inverted microscope (Olympus, Shinjuku, Tokyo, Japan) equipped with a 40× objective (UPlanSApo, numerical aperture 0.95) and a front illuminated EMCCD camera (Hamamatsu Photonics, Hamamatsu, SZK, Japan). GFP fluorescence images were acquired using an MT20 fluorescence lamp, a 474/23 excitation filter, a 525/45 emission filter and a 495 long-pass dichroic. For every time point, bright field and GFP fluorescence images were taken. For each condition, the wild type strain not expressing GFP (JA6) was grown in parallel to correct for background fluorescence of cells. Single cell fluorescence intensity values were extracted from the images with the freely available software CellProfiler 2.0 (Carpenter et al., 2006) by performing cell segmentation on the bright field images and using the defined masks for measuring intensities in the corresponding fluorescence images. Fluorescence intensities of the non-GFP-expressing strain (population medians of single cell medians) calculated separately for each condition and time point were subtracted from the single cell median intensities of the GFP-expressing strains, which resulted in background corrected single cell median values. Raw data extracted by CellProfiler was processed using the statistical software R (version 3.0.1) (Wickham, 2009; R Core Team, 2013).

Acknowledgments: We thank Dr. Renate Langhammer and Ursula Klokow for help in strain constructions and are grateful to all our colleagues from the DFG Doctoral Reasearch Training Group 1026 for many helpful discussions. Technical assistance by Karin Sorge is gratefully acknowledged. We are grateful to Victor Sourjik (University of Heidelberg) for being able to use the microscope. This work was supported by DFG Graduiertenkolleg GRK 1026 (Project B.1) und DFG grant BR921/4 to K.D.B.

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