Conformational modulation and hydrodynamic radii of CP12 protein and its complexes probed by fluorescence correlation spectroscopy Rigneault1, Satish Babu Moparthi1, Gabriel Thieulin-Pardo2, Pascal Mansuelle3, Herve 2 1 ro ^ me Wenger Brigitte Gontero and Je , France 1 Centrale Marseille, Institut Fresnel, Aix Marseille Universite nerge tique et Inge nierie des Prote ines, Aix Marseille Universite , France 2 Laboratoire de Bioe omique, Marseille Prote omique, Institut de Microbiologie de la Me diterrane e, France 3 Plate-forme Prote
Keywords CP12; fluorescence correlation spectroscopy; GAPDH; hydrodynamic radius; intrinsic disorder proteins; PRK Correspondence S. B. Moparthi, CNRS, Centrale Marseille, , Institut Fresnel, Aix Marseille Universite UMR 7249, 13013 Marseille, France Fax: +(33) 4 9 128 80 67 Tel: +(33) 4 9 128 84 94 E-mail: [email protected] (Received 7 February 2014, revised 30 April 2014, accepted 16 May 2014) doi:10.1111/febs.12854
Light/dark regulation of the Calvin cycle in oxygenic photosynthetic organisms involves the formation and dissociation of supramolecular complexes between CP12, a nuclear-encoded chloroplast protein, and the two enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.13) and phosphoribulokinase (PRK) (EC 2.7.1.19). Despite the high importance of understanding the structural basis of the interaction of CP12 with GAPDH and PRK to investigate the regulation of the Calvin cycle, information is still lacking about the structural remodulation of CP12 and its complex formation. Here, we characterize the diffusion dynamics and hydrodynamic radii of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH and PRK using fluorescence correlation spectroscopy experiments. We quantify a hydrodynamic radius of 3.4 0.2 nm for the CP12 protein with an increase up to 5.2 0.3 nm upon complex formation with GAPDH and PRK. In addition, unfolding experiments reveal a 1.6- and 2.0-fold increase respectively of the hydrodynamic radii for the N-terminal and C-terminal cysteine CP12 mutant proteins compared with their native folded structures. The different behavior of the CP12 mutant proteins during hydrophobic collapse transition is a direct clue to different structural orientations of the CP12 mutant proteins. These different structures are expected to facilitate the binding of either GAPDH or PRK during binary complex and ternary complex formation. Structured digital abstract GAPDH, CP12 and PRK physically interact by fluorescence correlation spectroscopy (View interaction) CP12 and PRK bind by fluorescence correlation spectroscopy (View interaction) GAPDH and CP12 bind by fluorescence correlation spectroscopy(View interaction)
Introduction CP12 is a small chloroplast protein present in many photosynthetic organisms including the eukaryotic unicellular green alga Chlamydomonas reinhardtii, where it is composed of 80 amino acids and has a molecular mass of 8.5 kDa [1–6]. CP12 plays a key
role in regulating the Calvin cycle by forming supramolecular complexes with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) downregulating their activity [3,7–13]. In the dark, oxidized CP12 forms a supramolecular complex
Abbreviations DTT, dithiothreitol; FCS, fluorescence correlation spectroscopy; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GuHCl, guanidinium hydrochloride; IDP, intrinsically disordered protein; PRK, phosphoribulokinase.
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with GAPDH and PRK, while in the light, due to the action of reduced thioredoxins and consequent disruption of disulfide bridges, the supramolecular complex dissociates [5,14]. Understanding the structural basis of the interaction of CP12 with GAPDH and PRK is thus of high importance to investigate the regulation of the Calvin cycle. CP12 is an intrinsically disordered protein (IDP) [2,5,6,15]. IDPs are proteins that under physiological conditions lack a rigid well-folded structure and exhibit low compactness and high flexibility. Their sequences are enriched in polar and charged residues and they have low content of hydrophobic amino acids that normally form the core of folded globular proteins [16–18]. IDPs are mainly found in eukaryotes (30–50% of eukaryotic proteins) and only a small proportion of these proteins are of eubacterial or archaeal origin (less than 5%) [19–21]. In plants only a few examples of IDPs have been described beside CP12, including dehydrins [22,23] and an Mn stabilizing protein belonging to photosystem II [24]. Several ensemble-based methods have been used to investigate the role and above all the interaction of CP12 with GAPDH and PRK, including size exclusion chromatography, immunoprecipitation assays, nuclear magnetic resonance or electron paramagnetic resonance spectroscopy [2,10,25,26]. These indicate that GAPDH and PRK proteins bind to different sites on CP12 [7,8,10]. CP12 from C. reinhardtii bears four cysteine residues at positions 23, 31, 66 and 75, which form two consecutive disulfide bridges at both N-terminal (Cys23-Cys31) and C-terminal (Cys66-Cys75) extremities of the protein (Fig. 1A) [1,2,4–6,27]. The N-terminal disulfide bridge is expected to be involved in the binding of PRK homo-dimer, while the C-terminal disulfide bridge appears necessary for redox regulation of the GAPDH homo-tetramer [1,2,4–6,27] (Fig. 1B). Here, we take advantage of fluorescence correlation spectroscopy (FCS) experiments to probe the diffusion dynamics of CP12 molecules from C. reinhardtii upon binding to GAPDH and PRK. FCS is a powerful and versatile method based on the statistical analysis of the temporal fluctuations affecting the fluorescence intensity from a few diffusing molecules [28–37]. It allows the investigation of a large variety of dynamic processes and photophysical properties, including translational diffusion, molecular concentrations, fluorescence brightness, chemical kinetics and binding reactions. The aim of this work was to determine the hydrodynamic radii of the wild-type CP12 (CP12wt) and its two site-specific mutant proteins altered both at the Nterminus (cysteine residue at position 31 replaced by a FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
CP12 structural modulation
serine, referred to as CP12C31S) and at the C-terminus (cysteine residue at position 75 replaced by a serine, referred to as CP12C75S). To investigate the binding properties between CP12, GAPDH and PRK, we monitor the changes of the hydrodynamic radii upon interaction with PRK and GAPDH. Our results clearly point out the crucial influence of the mutations affecting the disulfide bridges for the complex formation between CP12, GAPDH and PRK. Moreover, the change in hydrodynamic radii of the CP12 mutant proteins upon denaturation with increasing concentration of guanidinium hydrochloride (GuHCl) is analyzed using FCS. The different behavior of the CP12 mutant proteins during hydrophobic collapse transition provides a direct clue to different structural orientations resulting from the mutation of a single disulfide bridge.
Results CP12wt interaction with GAPDH and PRK FCS results for CP12wt are summarized in Fig. 2 and Table 1. The measured hydrodynamic radius of at pH 8.0, and is similar to CP12wt is around 34 A the hydrodynamic radii found for the two mutants CP12C31S and CP12C75S. In addition, longer diffusion times were obtained with CP12wt both in the presence of GAPDH or PRK alone and also in the presence of the two enzymes simultaneously. The presence of GAPDH significantly increased the hydro (Fig. 2B). Similarly, dynamic radius from 34 to 47 A the addition of PRK alone increased the apparent Forhydrodynamic radius of CP12wt from 34 to 43 A. mation of the whole complex CP12wt–GAPDH–PRK showed further significant increase of the apparent Upon treatment with hydrodynamic radius up to 52 A. dithiothreitol (DTT) as reducing agent, all hydrodynamic radii were similar to that of CP12wt alone and in This set of experiments indicates the range 34–36 A. that no complex was formed between CP12wt, GAPDH and/or PRK in the presence of DTT (Fig. 2C). CP12 self-association We checked that under the experimental conditions the CP12 mutant proteins were in the form of monomers and did not tend to form aggregates. To this end, the fluorescence correlation functions were measured by keeping the labeled CP12 concentration constant at 40 nM and adding increasing concentrations of unlabeled CP12 from 500 nM up to 50 lM. Regardless of the CP12 concentration, the hydrody-
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A C
C23 C31
kDa 130 100 70 50
1
MW
2
3
4
5
35 25
C75
15
C66 N
1) 2) 3) 4) 5)
C
B
GAPDH PRK CP12 wild-type CP12 C31S CP12 C75S
N
C N
C PRK-CP12C75S
GAPDH-CP12C31S
CP12 C N
PRK
GAPDH
C
N GAPDH-CP12-PRK
(Fig. 3A). These results namic radius was about 32 A indicate that in vitro there is no substantial interaction between the labeled and non-labeled CP12 protein even at concentrations up to 50 lM and that, very likely, the CP12 exists as a monomer in the absence of GAPDH and PRK. GAPDH–PRK–CP12 mutant interactions The role of the disulfide bridges of CP12 in the interaction with GAPDH and PRK can be investigated through the use of CP12 mutant proteins affected 3208
Fig. 1. Structure and interaction of CP12 with GAPDH and PRK. (A) 3D structural model of Chlamydomonas reinhardtii CP12. (B) Schematic representation of the association–dissociation of GAPDH, CP12 and PRK proteins. Both in vivo or in vitro CP12 forms two disulfide bridges, one at the N-terminus and one at the C-terminus, which then act as linkers for the formation of non-covalent complexes GAPDH–CP12, PRK–CP12 and GAPDH–CP12–PRK. (C) SDS gel (4–10%) stained with Coomassie Blue to test the homogeneity of GAPDH (lane 1), PRK (lane 2), CP12wt (lane 3), CP12C31S (lane 4) and CP12C75S (lane 5).
either at the N-terminal disulfide bridge (CP12C31S) or at the C-terminal disulfide bridge (CP12C75S). Figure 3B displays normalized FCS correlation traces obtained on the N-terminal disulfide bridge mutant CP12C31S in the absence and presence of GAPDH or PRK. In the presence of GAPDH, the correlation traces shift towards longer diffusion times indicating the formation of a sub-complex CP12C31S–GAPDH (Fig. 3B). Meanwhile, the presence of PRK did not induce any noticeable change in the correlation traces of CP12C31S compared with the CP12 mutant alone. The hydrodynamic radii deduced from the FCS data FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
Normalised auto correlation g (t)
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Table 1. Diffusion time and hydrodynamic radii for CP12wt protein in the presence and absence of GAPDH, PRK proteins and DTT.
A 1.0 CP12 wild-type
Sample
0.8
sd (ls)
RH ( A)
352 479 357 422 373 543 353
33.8 46.1 34.3 40.6 35.8 52.2 33.9
+ GAPDH
CP12wt CP12wt CP12wt CP12wt CP12wt CP12wt CP12wt
+ PRK
0.6
+ GAPDH + PRK
0.4 0.2 0.0 0.01
0.1
1
10
+ + + + + +
GAPDH GAPDH + DTT PRK PRK + DTT GAPDH + PRK GAPDH + PRK + DTT
1.7 2.3 1.7 2.0 1.7 2.6 1.6
100
Time (ms)
B Hydrodynamic radii (nm)
5.5 5.0 4.5 4.0 3.5 3.0 2.5 CP12Wt
C
+ GAPDH + PRK
Normalised auto correlation g (t)
1.0
5.5 Hydrodynamic radii (nm)
+ PRK
+ GAPDH
5.0 4.5
0.8 0.6 0.4 0.2 0.0 0.01
0.1
4.0
1
10
100
Time (ms)
3.5
GAPDH binding site on CP12 unaffected. Furthermore, successive additions of PRK to the CP12C31S–GAPDH complex did not modify the apparent hydrodynamic radius of CP12. This result indicates that PRK is not able to bind to the CP12C31S mutant even in the presence of GAPDH. Results for the other CP12C75S mutant affected at the C-terminal disulfide bridge are summarized in Fig. 4A,B and Table 2. Both CP12 mutant proteins CP12C31S and CP12C75S have a comparable hydro at pH 8.0. Unlike the correladynamic radius of 32 A tion traces of CP12C31S, the traces obtained with CP12C75S showed a shift towards longer diffusion times in the presence of PRK, while the presence of GAPDH did not significantly increase the diffusion dynamics. Here, the addition of PRK increased the apparent hydrodynamic radius of CP12C75S from 32 This indicates that the C-terminal C75S mutato 38 A. tion on CP12 does not affect the binding site for PRK while it significantly hampers the subsequent formation of a complex with GAPDH.
3.0 2.5 CP12Wt
+ GAPDH + DTT
+ PRK + DTT
+ GAPDH + PRK + DTT
Fig. 2. FCS results on the wild-type CP12 protein. (A) Normalized FCS correlation traces and (B) apparent hydrodynamic radii obtained in the presence of GAPDH or PRK or both. Five-fold molar excess proportions of GAPDH and PRK were used with CP12wt, by keeping CP12wt constant at 10 nM. (C) Reduction effect of the disulfide bridges by 1 mM DTT on the values of the hydrodynamic radii of all samples.
in Fig. 3B are summarized in Fig. 3C and Table 2 for the N-terminal mutant CP12C31S. The addition of GAPDH increased the apparent hydrodynamic radius while the addition of of CP12C31S from 32 to 47 A PRK did not affect the hydrodynamic radius. This result indicates that the N-terminal C31S mutation on CP12 severely affects PRK binding while leaving the FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
Effect of PRK concentration on CP12–PRK complex formation To investigate the effect of PRK concentration on the formation of the CP12C75S–PRK complex, the apparent hydrodynamic radius of CP12C75S at a constant concentration of 100 nM was analyzed when increased concentrations of unlabeled PRK protein up to a molar excess of 1 : 20 were added (Fig. 4C). Concentrations of PRK in a ratio less than 1 : 1 did not induce any significant change in the hydrodynamic radius. At concentration ratios higher than 1 : 1 the CP12C75S–PRK complex was formed affecting the apparent hydrodynamic radius until saturation was reached at a molar excess of 1 : 4 of PRK compared with CP12. Our observations tend to indicate a cooperative binding, involving at least two conformers of CP12 in equilibrium with different affinities for PRK. Binding of PRK will displace the equilibrium towards
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1.0
CP12C31S
Table 2. Diffusion time, hydrodynamic radii and molecular brightness CRM for CP12 mutant proteins in the presence and absence of GAPDH and PRK proteins.
0.8
+ GAPDH
Sample
sd (ls)
RH ( A)
0.6
+ PRK
CP12C31S CP12C31S + GAPDH CP12C31S + PRK CP12C31S + GAPDH + PRK CPCP12C75S CP12C75S + GAPDH CP12C75S + PRK CP12C75S + GAPDH + PRK
290 423 286 407 285 280 342 365
32.4 47.2 31.9 45.4 31.8 31.2 38.2 40.7
Normalised auto correlation g (t)
A
+ GAPDH + PRK
0.4 0.2 0.0 0.01
0.1
1 Time (ms)
10
100
B Hydrodynamic radii (nm)
5.0 4.5 4.0 3.5 3.0 2.5 CP12C31S
+ GAPDH
+ PRK
+ GAPDH + PRK
CP12C31S
+ 500 nM
+ 5 µM
+ 50 µM
Hydrodynamic radii (nm)
C 3.4
3.2
3.0
2.8
Fig. 3. FCS data on the N-terminal disulfide bridge mutant CP12C31S. (A) CP12C31S diffusion time independence of increasing concentrations of unlabeled CP12C31S. (B) Normalized FCS correlation traces and (C) deduced hydrodynamic radii obtained on CP12C31S in the absence and presence of GAPDH or PRK. The molar ratio was set to 1 : 1 with each species at a fixed concentration of 100 nM.
the higher affinity conformer giving rise to the sigmoid curve obtained (Fig. 4C). Unfolding of CP12 mutant proteins analyzed by FCS To determine the structural transitional dimensions of CP12 mutant proteins, increased concentrations of 3210
1.6 2.3 1.5 2.2 1.3 1.4 1.9 2.0
GuHCl up to 5 M were added and the hydrodynamic radii of the partially denatured states were determined by FCS. In the presence of high concentrations of GuHCl, both the solvent’s refractive index and viscosity are changed. Both effects were taken into account by calibrating the ratio of the transversal waist to the solvent’s viscosity for each GuHCl concentration (Fig. 5). With that calibration, the measured hydrodynamic radii of both Alexa Fluor 647 and Atto647N do not vary upon addition of GuHCl, confirming our experimental method. Figure 6A represents the correlation functions of both CP12 mutant proteins in the absence or presence of GuHCl; all the data were fitted to a free single-component diffusion model with a diffusion time of sd and an average number N of molecules by using Eqn (1) (later). The accuracy of the fits was established using residual distribution analysis. Figure 6B depicts the hydrodynamic radii of both CP12 mutant proteins in a series of increasing GuHCl concentrations. While under native conditions both CP12C31S and CP12C75S had similar hydrodynamic radii, the hydrodynamic radius values obtained at 5 M GuHCl for strongly denatured CP12C31S and CP12C75S revealed an increase of 1.6- and 2.0-fold respectively compared with their corresponding native structures. Moreover, difference in hydrodynamic radius the significant 10 A between the two mutant proteins under denatured conditions indicates that the N- and C-terminal mutant proteins differ in structure.
Discussion CP12 is very flexible and largely devoid of secondary structure; however, when disulfide bonds are present, a-helix content and the overall degree of order of CP12 is increased [2]. This protein is able to bind metal ions such as copper and nickel in C. reinhardtii [38,39] and calcium in higher plant [40]. CP12 also FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
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A
1.0
+ GAPDH 0.6
+ PRK
0.4
+ GAPDH + PRK
150 140 130 120 0
1
2
3
4
5
4
5
[GuHCl] (M)
0.0 0.1
1
10
100
B
Time (ms)
500
Waist (nm)
480
B 4.5
4.0
460 440 420 400 380
3.5
3.0
C
2.5 CP12C75S
+ GAPDH
+ PRK
+ GAPDH + PRK
C 4.5
Refractive Index
Hydrodynamic radii (nm)
160
0.2
0.01
Hydrodynamic radii (nm)
180 170
CP12C75S 0.8 td (µs)
Normalised auto correlation g (t)
A
0
1
0
1
2 3 [GuHCl] (M)
1.44 1.42 1.40 1.38 1.36 1.34 1.32
4.0
2
3
4
5
[GuHCl] (M)
3.5
3.0 4 6 8 10
2
4 6 8 100
2
4 6 8 1000
2
PRK concentration (nM) Fig. 4. FCS data on the C-terminal disulfide bridge mutant CP12C75S. (A) Normalized FCS correlation traces and (B) deduced hydrodynamic radii obtained on CP12C75S in the absence and presence of GAPDH or PRK. In (A) and (B), the concentrations of CP12C75S, GAPDH and PRK were 100, 500 and 500 nM respectively, corresponding to a 5-fold molar excess of GAPDH and/or PRK relative to CP12C75S. (C) Dependence of the CP12C75S hydrodynamic radius on the concentration of PRK. The labeled CP12C75S concentration was kept constant at 100 nM.
plays a role in oxidative stress as shown by antisense CP12 mutant plants [41]. In early studies, other proteins such as malate dehydrogenase, elongation factor FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
Fig. 5. Calibration procedure for the influence of increased concentrations of GuHCl. (A) Diffusion time measured for Alexa Fluor 647 as function of GuHCl concentration. (B) Effective transversal waist deduced from the measurements in (A) and the calibrated 0.7 nm hydrodynamic radius of Alexa Fluor 647 at 22 °C. (C) The evolution of the transversal waist in (B) is found to correlate with increase of the solvent’s refractive index upon addition of GuHCl.
1 alpha 2 and 38 kDa-ribosome-associated protein were able to interact with CP12 but to a lesser extent than PRK, GAPDH and the aldolase, enzymes from the Calvin cycle [9]. CP12 thus belongs to the IDP family and it seems to be a jack of all trades and a master of the Calvin cycle [15]. This protein is found in most photosynthetic organisms [3,42] and was even shown to be produced by cyanophages upon infection of their hosts, thereby inhibiting their Calvin cycle and re-routing the NADPH towards phage nucleotide biosynthesis [43]. A recent analysis in cyanobacteria
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Auto correlation g (t)
A 1.008 CP12C31S - 0M GuHCl
1.006
CP12C31S - 5M GuHCl CP12C75S - 0M GuHCl
1.004
CP12C75S - 5M GuHCl
1.002 1.000 0.1
1
10
Time (ms)
Hydrodynamic radii (nm)
B 6
CP12C75S
5 4 CP12C31S
3 2
Control
1 0
1
2
3
4
5
GuHCL concentration (M) Fig. 6. Conformational changes of CP12 mutant proteins are highlighted by unfolding experiments under denaturation conditions. (A) Raw fluorescence correlation functions of CP12 mutant proteins in the presence and absence of GuHCl; the data were fitted to a single-component diffusion model. (B) Hydrodynamic radii of CP12 mutant proteins as function of the concentration of GuHCl. A control experiment is displayed for Atto647N free dye to indicate the absence of experimental artifacts.
showed that about eight types of CP12 are present and among them are three CP12-CBS containing a cystathionine-b-synthase (CBS) domain that is fused to CP12. The CBS domains function as regulatory modules for a wide range of cellular activities, and some bind adenine nucleotides [42]. The role of CP12 like that of many other IDPs in the cell is thus of paramount importance [44]. Although the interaction between this protein, GAPDH and PRK has been studied, the structure and dynamics of CP12 are still unknown. Crystallographic data of GAPDH/CP12 from Synechococcus elongatus and Arabidopsis thaliana showed that the C-terminal part of CP12 can fold upon binding to GAPDH but most of the CP12 remains highly flexible and almost 50 residues of about 80 were not visible in the density map of either structure [10,45]. In C. reinhardtii it was recently shown by site-directed spin labeling combined with electron paramagnetic resonance spectroscopy 3212
that the GAPDH–CP12 complex is a fuzzy complex [26]. Only a modeled structure of the algal CP12 is therefore available [46]. Data on the interaction between PRK and CP12 are scarce as this interaction is weaker (lM range) than that with GAPDH and CP12 (nM range) in green alga, and again no structural data are available [2]. Hence not much is known on the conformational change of the CP12 protein either upon folding or upon interaction with GAPDH and PRK proteins. Our study focuses for the first time on structural transition parameters of CP12–GAPDH and CP12– PRK complexes. Our results indicate that the CP12wt binds to GAPDH and/or PRK independently and also to both enzymes to form a ternary complex. To understand the molecular background of the function of CP12wt it is crucial, due to the high flexibility and the lack of rigid structure of CP12, to investigate its structural properties. Since there are no structural data for the algal complex, our present results attempt to elucidate the algal CP12 structure in terms of hydrodynamic radii to decipher its mode of binding to two key regulated enzymes, namely GAPDH and PRK of the Calvin cycle. These observations are a first step towards an understanding of protein–protein interactions in the GAPDH–CP12–PRK complex and the nature of the physicochemical forces involved during ternary complex formation. Our results showed that the disruption of the N-terminal disulfide bridge on CP12 affects the formation of the CP12C31S–PRK complex, while it has no effect on the CP12C31S–GAPDH complex. On the other hand, we observed the opposite effect with the mutant CP12C75S affected at the C-terminal disulfide bridge that forms a complex with PRK but not with GAPDH. Further addition of PRK or GAPDH to the corresponding CP12C31S–GAPDH or CP12C75S– PRK complexes showed no signs of the formation of GAPDH–CP12(C31S/C75S)–PRK supramolecular complex suggesting that both C-terminal and N-terminal disulfide bridges are crucial for formation of the ternary structure complex. In addition, the binding of both GAPDH and PRK proteins to CP12 was suppressed by DTT that results in the disruption of the disulfide bridges of CP12. This study also showed that GAPDH and PRK bind at specific sites on CP12. Based on the similar fluorescence brightness found for CP12 mutant proteins alone and for the sub-complexes, only one monomer of CP12C31S or CP12C75S bound to GAPDH and PRK respectively. The mutant CP12C31S behaves as CP12 locked in a reduced state and therefore this result is in agreement with previous results where only one moleFEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
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cule of reduced CP12 was bound to GAPDH while two molecules of oxidized CP12 were bound to tetrameric GAPDH [47,48]. The present study also indicates that the ratio 1 : 4 of CP12 to PRK is sufficient to form the CP12C75S–PRK complex; with a KD of 1.6 lM [2] determined by surface plasmon resonance (SPR) and the experimental conditions used in this report, one would expect about 20% of complex formation between CP12 and PRK. SPR and fluorescence spectroscopy are complementary techniques although in SPR one partner is immobilized on a chip while in FCS the interaction between the two partners occurs in solution, explaining the apparent discrepancy in the results obtained. A set of equations correlating protein density in several conformational states (native, unfolded, pre-molten globular and molten globular) in a wide range of molecular masses has been reported [49]. Analysis of similar dependences for various IDPs was added later [18,50]. The measured hydrodynamic radii for both CP12 mutant proteins in their native state were around 32 A, a value that fits more with an IDP hydrodynamic radius compared with the native state hydrodynamic radius. To cite but a few examples, the hydrodynamic radius for a globular protein like cytochrome c is about 17.8 A and for an IDP like TyrRS(D1) is 21 A, while these proteins have almost the same number of amino acids (104 and 107 respectively) [51]. Moreover, a clear difference was found between the extended conformation (maximal chain expansion) under denaturing conditions (Fig. 4A) and under native conditions. CP12 is highly flexible in its native state and may become somewhat ordered upon interaction with either GAPDH or PRK by forming the GAPDH–CP12 or the PRK–CP12 binary complexes as observed by the diffusion time changes upon formation of these sub-complexes. The present report also focuses on the unfolding nature of the CP12 mutation sites specifically both at the C-terminus and N-terminus to assess the degree of re-modulation and further to evaluate the specificity towards the GAPDH and PRK interactions. It is likely that CP12 like other IDPs shows gradual hydrophobic collapse during unfolding.
Experimental procedures Recombinant protein expression and purification C. reinhardtii CP12, GAPDH and PRK were obtained as described earlier [7]. The concentrations of all proteins were calculated using Bradford assays [52]. The purity of recombinant proteins was checked by SDS/PAGE [53] followed by Coomassie Blue R-250 staining and mass spectrometry (Fig. 1C). For all experiments, His-tagged CP12 proteins
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were used as it was shown that wild-type His-tagged CP12 behaves like the native protein, indicating that the His tag does not interfere in our experiments [48]. Numbering of residues, however, was based on the protein without the tag, with SGQPA being the first residues (http://genome.jgi.doe.gov/cgi-bin/dispGeneModel?db=Chlre4&id=148487).
Protein labeling Lysine residues in the CP12wt protein were labeled with the amine-reactive N-hydroxysuccinimide ester derivative of the Atto590 fluorophore, and in both single cysteine-modified CP12 mutant proteins were labeled with the thiol-reactive maleimide derivative of the fluorophore Atto647N by following the protocol provided by the manufacturer (AttoTec). In all cases, excess fluorophore was added to the aqueous protein solution containing 50 mM Tris/HCl at pH 8.0 and left for 8 h at 4 °C. The labeled protein was subsequently purified using size exclusion chromatography on a SephadexTM G-25 Medium (GE Healthcare, Little Chalfont, Buckinghamshire, UK) column with 50 mM Tris/HCl (pH 8.0) elution buffer. In this report, CD experiments were not carried out as it was not possible to obtain enough labeled protein (10 lM). However, previous reports showed that cysteine labeling of the proteins mutated either on the N-terminal disulfide bridge [26] or on the C-terminal disulfide bridge [54] did not alter the global behavior of the CP12 proteins. CD spectra of labeled and unlabeled proteins were similar. Sample purity and specificity was confirmed by matrixassisted laser desorption/ionization mass spectrometry.
In vitro reconstitution of the GAPDH–CP12–PRK complex All in vitro reconstitution assays to test formation of the sub-complexes CP12wt and CP12 mutants (C31S or C75S) with GAPDH or PRK, or the ternary complex GAPDH– CP12(wt or C31S or C75S)–PRK were performed as previously described [7]. In the case of GAPDH–CP12 or CP12– PRK interaction experiments, 100 nM of labeled CP12wt or CP12C31S or CP12C75S was mixed separately or in combination with 500 nM of GAPDH and 500 nM of PRK in 50 mM Tris/HCl, 4 mM EDTA, 0.1 mM NAD and 5 mM cysteine at pH 8.0 for 12 h at 22 °C. In the case of CP12wt, the mixture was diluted to 10 nM of CP12wt and 50 nM of GAPDH or PRK respectively. In corresponding samples, 1 mM DTT was used as a reducing agent and samples were incubated for 2 h at 22 °C. In the case of the CP12 self-association experiments, 40 nM of labeled CP12C31S mutant was mixed with ratios 1 : 12.5 nM, 1 : 125 nM and 1 : 1250 nM of unlabeled CP12C31S mutant in 50 mM Tris/HCl, 4 mM EDTA at pH 8.0 for 12 h at 22 °C. To test the effect of PRK concentration on CP12– PRK complex formation, 100 nM labeled CP12C75S was mixed with concentration ratios from 1 : 0.04 nM to
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1 : 20 nM of unlabeled PRK for 12 h at 22 °C in 50 mM Tris/HCl, 4 mM EDTA, 0.1 mM NAD at pH 8.0. All the mixtures were tested for complex formation using in vitro reconstitution assays by electrophoresis under non-denaturing conditions (native PAGE) followed by Coomassie Blue R-250 staining (data not shown).
Fluorescence correlation spectroscopy FCS experiments were carried out with a custom-built confocal fluorescence microscope with a Zeiss C-Apochromat 40 9 1.2NA water-immersion objective [55]. The excitation source was a CW He-Ne laser operating at 633 nm. For FCS measurements on CP12 with either GAPDH or PRK proteins the laser beam was set to fill the microscope objective back-aperture so as to use the maximum numerical aperture available for the microscope objective. A 30 lm confocal pinhole conjugated to the sample plane defined the confocal volume whose transversal waist wxy was calibrated to 285 nm using the known diffusion coefficient of Alexa 647 in pure water (3.1 9 106 cm2s1 at 22 °C). For the experiment on CP12 unfolding, the increase in the refractive index of the medium upon addition of GuHCl (Fig. 5) was imposed to limit the negative effects of spherical aberrations. This was achieved by under filling the microscope objective back-aperture (4.5 mm instead of 8.9 mm) and using a 50 lm confocal pinhole. With these conditions, the transversal waist wxy was calibrated to 410 nm in the absence of GuHCl. A moderate variation of 20% on the diffusion time was found with the calibration procedure using Alexa Fluor 647 upon increasing concentrations of GuHCl. This phenomenon was due to the combination of both refractive effects and increased solvent viscosity g (Fig. 5). The effect was systematically taken into account to calibrate the quotient wxy2/g in the presence of GuHCl so as to deduce the hydrodynamic radius of the protein samples. All FCS experiments were carried out at 22 °C using 50 mM Tris/HCl, 4 mM EDTA, 0.1 mM NAD and 5 mM cysteine as a reconstitution buffer at pH 8.0. We also added 0.1% Tween-20 (Sigma) to the samples in order to diminish surface interactions with the glass coverslip. The fluorescence intensity temporal fluctuations were analyzed with a hardware correlator (Flex02-12D/C correlator.com, Bridgewater, NJ, USA, with 12.5 ns minimum channel width). All the experimental data were fitted by considering a single species and free Brownian 3D diffusion in the case of a Gaussian molecular detection efficiency:
g2 ðsÞ ¼ 1 þ
2 1 B s 1 1 þ nT exp N F sT
1 þ ssd
1 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 þ s2 ssd ð1Þ
where N is the average number of molecules in the focal volume, F is the total fluorescence signal, B is the back-
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ground noise, nT is the amplitude of the dark state population, sT is the dark state blinking time, sd is the mean diffusion time and s is the ratio of transversal to axial dimensions of the analysis volume. The background noise B originated mainly from the back-reflected laser light and from the detector dark current and remained below 1 kHz for the experiments reported here. Numerical fit of the FCS data following Eqn (1) provided the average number of molecules, N, which was used to calculate the fluorescence count rate per molecule (CRM) as CRM ¼
FB N
ð2Þ
All the data were fitted by considering that the regular distribution of the weighted residuals is around zero. The FCS measurement of the mean diffusion time sd and the separate calibration of the transversal waist wxy of the confocal detection volume allowed the molecular diffusion coefficient D to be calculated according to the relation sd ¼ w2xy =4D
ð3Þ
The hydrodynamic radius RH can then be deduced from the measured diffusion coefficient D using the Stokes–Einstein equation D¼
kB T 6 p g RH
ð4Þ
where kB is Boltzmann’s constant, T is the absolute temperature and g is the viscosity of the medium. We calibrated the parameter w2xy /g before each measurement on CP12 by recording the FCS trace for Alexa Fluor 647 dyes which have a known hydrodynamic radius of 0.7 nm in pure water. With that calibration, the measured hydrodynamic radii of both Alexa Fluor 647 and Atto647N do not vary upon addition of GuHCl, confirming our experimental method.
Conclusion We report detailed information about the hydrodynamic radii of wild-type CP12 and its site-specific mutants at their disulfide bridges at the N-terminus (CP12C31S) and the C-terminus (CP12C75S). We quantify a hydrodynamic radius of 3.4 0.2 nm for the CP12 protein with an increase up to 5.2 0.3 nm upon complex formation with GAPDH and PRK. Our results on CP12C31S and CP12C75S clearly point out the crucial influence of the mutations affecting the disulfide bridges for complex formation between CP12, GAPDH and PRK. Using denaturation conditions, we monitor the change in hydrodynamic radii of the CP12 mutant proteins upon unfolding. The different behavior between CP12 mutant proteins indicates FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
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different structural conformations that favor the binding of either the GAPDH tetramer or the PRK dimer.
Acknowledgements This work was funded by a contract from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ ERC Grant agreement no. 278242. GP was in receipt of a Fellowship from the Ecole Normale Superieure, Cachan. This work (BG) was in part supported by the Agence Nationale de la Recherche ANR SPINFOLD no. 09-BLAN-0100, La Region PACA, APO projet 063 506, the Centre National de la Recherche Scientifique (CNRS) and Aix-Marseille Universite.
Author contributions Design of the experiments: SBM, HR, BG and JW. Involved in protein purification and labeling: SBM and GPT. Involved in FCS experiments: SBM and JW. Helped in protein quantification: PM. Analyzed the data: SBM, GPT, BG and JW. Wrote the paper: SBM, BG and JW. All authors read and approved the final manuscript.
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Keywords CP12; fluorescence correlation spectroscopy; GAPDH; hydrodynamic radius; intrinsic disorder proteins; PRK Correspondence S. B. Moparthi, CNRS, Centrale Marseille, , Institut Fresnel, Aix Marseille Universite UMR 7249, 13013 Marseille, France Fax: +(33) 4 9 128 80 67 Tel: +(33) 4 9 128 84 94 E-mail: [email protected] (Received 7 February 2014, revised 30 April 2014, accepted 16 May 2014) doi:10.1111/febs.12854
Light/dark regulation of the Calvin cycle in oxygenic photosynthetic organisms involves the formation and dissociation of supramolecular complexes between CP12, a nuclear-encoded chloroplast protein, and the two enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (EC 1.2.1.13) and phosphoribulokinase (PRK) (EC 2.7.1.19). Despite the high importance of understanding the structural basis of the interaction of CP12 with GAPDH and PRK to investigate the regulation of the Calvin cycle, information is still lacking about the structural remodulation of CP12 and its complex formation. Here, we characterize the diffusion dynamics and hydrodynamic radii of CP12 from Chlamydomonas reinhardtii upon binding to GAPDH and PRK using fluorescence correlation spectroscopy experiments. We quantify a hydrodynamic radius of 3.4 0.2 nm for the CP12 protein with an increase up to 5.2 0.3 nm upon complex formation with GAPDH and PRK. In addition, unfolding experiments reveal a 1.6- and 2.0-fold increase respectively of the hydrodynamic radii for the N-terminal and C-terminal cysteine CP12 mutant proteins compared with their native folded structures. The different behavior of the CP12 mutant proteins during hydrophobic collapse transition is a direct clue to different structural orientations of the CP12 mutant proteins. These different structures are expected to facilitate the binding of either GAPDH or PRK during binary complex and ternary complex formation. Structured digital abstract GAPDH, CP12 and PRK physically interact by fluorescence correlation spectroscopy (View interaction) CP12 and PRK bind by fluorescence correlation spectroscopy (View interaction) GAPDH and CP12 bind by fluorescence correlation spectroscopy(View interaction)
Introduction CP12 is a small chloroplast protein present in many photosynthetic organisms including the eukaryotic unicellular green alga Chlamydomonas reinhardtii, where it is composed of 80 amino acids and has a molecular mass of 8.5 kDa [1–6]. CP12 plays a key
role in regulating the Calvin cycle by forming supramolecular complexes with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase (PRK) downregulating their activity [3,7–13]. In the dark, oxidized CP12 forms a supramolecular complex
Abbreviations DTT, dithiothreitol; FCS, fluorescence correlation spectroscopy; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GuHCl, guanidinium hydrochloride; IDP, intrinsically disordered protein; PRK, phosphoribulokinase.
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with GAPDH and PRK, while in the light, due to the action of reduced thioredoxins and consequent disruption of disulfide bridges, the supramolecular complex dissociates [5,14]. Understanding the structural basis of the interaction of CP12 with GAPDH and PRK is thus of high importance to investigate the regulation of the Calvin cycle. CP12 is an intrinsically disordered protein (IDP) [2,5,6,15]. IDPs are proteins that under physiological conditions lack a rigid well-folded structure and exhibit low compactness and high flexibility. Their sequences are enriched in polar and charged residues and they have low content of hydrophobic amino acids that normally form the core of folded globular proteins [16–18]. IDPs are mainly found in eukaryotes (30–50% of eukaryotic proteins) and only a small proportion of these proteins are of eubacterial or archaeal origin (less than 5%) [19–21]. In plants only a few examples of IDPs have been described beside CP12, including dehydrins [22,23] and an Mn stabilizing protein belonging to photosystem II [24]. Several ensemble-based methods have been used to investigate the role and above all the interaction of CP12 with GAPDH and PRK, including size exclusion chromatography, immunoprecipitation assays, nuclear magnetic resonance or electron paramagnetic resonance spectroscopy [2,10,25,26]. These indicate that GAPDH and PRK proteins bind to different sites on CP12 [7,8,10]. CP12 from C. reinhardtii bears four cysteine residues at positions 23, 31, 66 and 75, which form two consecutive disulfide bridges at both N-terminal (Cys23-Cys31) and C-terminal (Cys66-Cys75) extremities of the protein (Fig. 1A) [1,2,4–6,27]. The N-terminal disulfide bridge is expected to be involved in the binding of PRK homo-dimer, while the C-terminal disulfide bridge appears necessary for redox regulation of the GAPDH homo-tetramer [1,2,4–6,27] (Fig. 1B). Here, we take advantage of fluorescence correlation spectroscopy (FCS) experiments to probe the diffusion dynamics of CP12 molecules from C. reinhardtii upon binding to GAPDH and PRK. FCS is a powerful and versatile method based on the statistical analysis of the temporal fluctuations affecting the fluorescence intensity from a few diffusing molecules [28–37]. It allows the investigation of a large variety of dynamic processes and photophysical properties, including translational diffusion, molecular concentrations, fluorescence brightness, chemical kinetics and binding reactions. The aim of this work was to determine the hydrodynamic radii of the wild-type CP12 (CP12wt) and its two site-specific mutant proteins altered both at the Nterminus (cysteine residue at position 31 replaced by a FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
CP12 structural modulation
serine, referred to as CP12C31S) and at the C-terminus (cysteine residue at position 75 replaced by a serine, referred to as CP12C75S). To investigate the binding properties between CP12, GAPDH and PRK, we monitor the changes of the hydrodynamic radii upon interaction with PRK and GAPDH. Our results clearly point out the crucial influence of the mutations affecting the disulfide bridges for the complex formation between CP12, GAPDH and PRK. Moreover, the change in hydrodynamic radii of the CP12 mutant proteins upon denaturation with increasing concentration of guanidinium hydrochloride (GuHCl) is analyzed using FCS. The different behavior of the CP12 mutant proteins during hydrophobic collapse transition provides a direct clue to different structural orientations resulting from the mutation of a single disulfide bridge.
Results CP12wt interaction with GAPDH and PRK FCS results for CP12wt are summarized in Fig. 2 and Table 1. The measured hydrodynamic radius of at pH 8.0, and is similar to CP12wt is around 34 A the hydrodynamic radii found for the two mutants CP12C31S and CP12C75S. In addition, longer diffusion times were obtained with CP12wt both in the presence of GAPDH or PRK alone and also in the presence of the two enzymes simultaneously. The presence of GAPDH significantly increased the hydro (Fig. 2B). Similarly, dynamic radius from 34 to 47 A the addition of PRK alone increased the apparent Forhydrodynamic radius of CP12wt from 34 to 43 A. mation of the whole complex CP12wt–GAPDH–PRK showed further significant increase of the apparent Upon treatment with hydrodynamic radius up to 52 A. dithiothreitol (DTT) as reducing agent, all hydrodynamic radii were similar to that of CP12wt alone and in This set of experiments indicates the range 34–36 A. that no complex was formed between CP12wt, GAPDH and/or PRK in the presence of DTT (Fig. 2C). CP12 self-association We checked that under the experimental conditions the CP12 mutant proteins were in the form of monomers and did not tend to form aggregates. To this end, the fluorescence correlation functions were measured by keeping the labeled CP12 concentration constant at 40 nM and adding increasing concentrations of unlabeled CP12 from 500 nM up to 50 lM. Regardless of the CP12 concentration, the hydrody-
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A C
C23 C31
kDa 130 100 70 50
1
MW
2
3
4
5
35 25
C75
15
C66 N
1) 2) 3) 4) 5)
C
B
GAPDH PRK CP12 wild-type CP12 C31S CP12 C75S
N
C N
C PRK-CP12C75S
GAPDH-CP12C31S
CP12 C N
PRK
GAPDH
C
N GAPDH-CP12-PRK
(Fig. 3A). These results namic radius was about 32 A indicate that in vitro there is no substantial interaction between the labeled and non-labeled CP12 protein even at concentrations up to 50 lM and that, very likely, the CP12 exists as a monomer in the absence of GAPDH and PRK. GAPDH–PRK–CP12 mutant interactions The role of the disulfide bridges of CP12 in the interaction with GAPDH and PRK can be investigated through the use of CP12 mutant proteins affected 3208
Fig. 1. Structure and interaction of CP12 with GAPDH and PRK. (A) 3D structural model of Chlamydomonas reinhardtii CP12. (B) Schematic representation of the association–dissociation of GAPDH, CP12 and PRK proteins. Both in vivo or in vitro CP12 forms two disulfide bridges, one at the N-terminus and one at the C-terminus, which then act as linkers for the formation of non-covalent complexes GAPDH–CP12, PRK–CP12 and GAPDH–CP12–PRK. (C) SDS gel (4–10%) stained with Coomassie Blue to test the homogeneity of GAPDH (lane 1), PRK (lane 2), CP12wt (lane 3), CP12C31S (lane 4) and CP12C75S (lane 5).
either at the N-terminal disulfide bridge (CP12C31S) or at the C-terminal disulfide bridge (CP12C75S). Figure 3B displays normalized FCS correlation traces obtained on the N-terminal disulfide bridge mutant CP12C31S in the absence and presence of GAPDH or PRK. In the presence of GAPDH, the correlation traces shift towards longer diffusion times indicating the formation of a sub-complex CP12C31S–GAPDH (Fig. 3B). Meanwhile, the presence of PRK did not induce any noticeable change in the correlation traces of CP12C31S compared with the CP12 mutant alone. The hydrodynamic radii deduced from the FCS data FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
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Table 1. Diffusion time and hydrodynamic radii for CP12wt protein in the presence and absence of GAPDH, PRK proteins and DTT.
A 1.0 CP12 wild-type
Sample
0.8
sd (ls)
RH ( A)
352 479 357 422 373 543 353
33.8 46.1 34.3 40.6 35.8 52.2 33.9
+ GAPDH
CP12wt CP12wt CP12wt CP12wt CP12wt CP12wt CP12wt
+ PRK
0.6
+ GAPDH + PRK
0.4 0.2 0.0 0.01
0.1
1
10
+ + + + + +
GAPDH GAPDH + DTT PRK PRK + DTT GAPDH + PRK GAPDH + PRK + DTT
1.7 2.3 1.7 2.0 1.7 2.6 1.6
100
Time (ms)
B Hydrodynamic radii (nm)
5.5 5.0 4.5 4.0 3.5 3.0 2.5 CP12Wt
C
+ GAPDH + PRK
Normalised auto correlation g (t)
1.0
5.5 Hydrodynamic radii (nm)
+ PRK
+ GAPDH
5.0 4.5
0.8 0.6 0.4 0.2 0.0 0.01
0.1
4.0
1
10
100
Time (ms)
3.5
GAPDH binding site on CP12 unaffected. Furthermore, successive additions of PRK to the CP12C31S–GAPDH complex did not modify the apparent hydrodynamic radius of CP12. This result indicates that PRK is not able to bind to the CP12C31S mutant even in the presence of GAPDH. Results for the other CP12C75S mutant affected at the C-terminal disulfide bridge are summarized in Fig. 4A,B and Table 2. Both CP12 mutant proteins CP12C31S and CP12C75S have a comparable hydro at pH 8.0. Unlike the correladynamic radius of 32 A tion traces of CP12C31S, the traces obtained with CP12C75S showed a shift towards longer diffusion times in the presence of PRK, while the presence of GAPDH did not significantly increase the diffusion dynamics. Here, the addition of PRK increased the apparent hydrodynamic radius of CP12C75S from 32 This indicates that the C-terminal C75S mutato 38 A. tion on CP12 does not affect the binding site for PRK while it significantly hampers the subsequent formation of a complex with GAPDH.
3.0 2.5 CP12Wt
+ GAPDH + DTT
+ PRK + DTT
+ GAPDH + PRK + DTT
Fig. 2. FCS results on the wild-type CP12 protein. (A) Normalized FCS correlation traces and (B) apparent hydrodynamic radii obtained in the presence of GAPDH or PRK or both. Five-fold molar excess proportions of GAPDH and PRK were used with CP12wt, by keeping CP12wt constant at 10 nM. (C) Reduction effect of the disulfide bridges by 1 mM DTT on the values of the hydrodynamic radii of all samples.
in Fig. 3B are summarized in Fig. 3C and Table 2 for the N-terminal mutant CP12C31S. The addition of GAPDH increased the apparent hydrodynamic radius while the addition of of CP12C31S from 32 to 47 A PRK did not affect the hydrodynamic radius. This result indicates that the N-terminal C31S mutation on CP12 severely affects PRK binding while leaving the FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
Effect of PRK concentration on CP12–PRK complex formation To investigate the effect of PRK concentration on the formation of the CP12C75S–PRK complex, the apparent hydrodynamic radius of CP12C75S at a constant concentration of 100 nM was analyzed when increased concentrations of unlabeled PRK protein up to a molar excess of 1 : 20 were added (Fig. 4C). Concentrations of PRK in a ratio less than 1 : 1 did not induce any significant change in the hydrodynamic radius. At concentration ratios higher than 1 : 1 the CP12C75S–PRK complex was formed affecting the apparent hydrodynamic radius until saturation was reached at a molar excess of 1 : 4 of PRK compared with CP12. Our observations tend to indicate a cooperative binding, involving at least two conformers of CP12 in equilibrium with different affinities for PRK. Binding of PRK will displace the equilibrium towards
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1.0
CP12C31S
Table 2. Diffusion time, hydrodynamic radii and molecular brightness CRM for CP12 mutant proteins in the presence and absence of GAPDH and PRK proteins.
0.8
+ GAPDH
Sample
sd (ls)
RH ( A)
0.6
+ PRK
CP12C31S CP12C31S + GAPDH CP12C31S + PRK CP12C31S + GAPDH + PRK CPCP12C75S CP12C75S + GAPDH CP12C75S + PRK CP12C75S + GAPDH + PRK
290 423 286 407 285 280 342 365
32.4 47.2 31.9 45.4 31.8 31.2 38.2 40.7
Normalised auto correlation g (t)
A
+ GAPDH + PRK
0.4 0.2 0.0 0.01
0.1
1 Time (ms)
10
100
B Hydrodynamic radii (nm)
5.0 4.5 4.0 3.5 3.0 2.5 CP12C31S
+ GAPDH
+ PRK
+ GAPDH + PRK
CP12C31S
+ 500 nM
+ 5 µM
+ 50 µM
Hydrodynamic radii (nm)
C 3.4
3.2
3.0
2.8
Fig. 3. FCS data on the N-terminal disulfide bridge mutant CP12C31S. (A) CP12C31S diffusion time independence of increasing concentrations of unlabeled CP12C31S. (B) Normalized FCS correlation traces and (C) deduced hydrodynamic radii obtained on CP12C31S in the absence and presence of GAPDH or PRK. The molar ratio was set to 1 : 1 with each species at a fixed concentration of 100 nM.
the higher affinity conformer giving rise to the sigmoid curve obtained (Fig. 4C). Unfolding of CP12 mutant proteins analyzed by FCS To determine the structural transitional dimensions of CP12 mutant proteins, increased concentrations of 3210
1.6 2.3 1.5 2.2 1.3 1.4 1.9 2.0
GuHCl up to 5 M were added and the hydrodynamic radii of the partially denatured states were determined by FCS. In the presence of high concentrations of GuHCl, both the solvent’s refractive index and viscosity are changed. Both effects were taken into account by calibrating the ratio of the transversal waist to the solvent’s viscosity for each GuHCl concentration (Fig. 5). With that calibration, the measured hydrodynamic radii of both Alexa Fluor 647 and Atto647N do not vary upon addition of GuHCl, confirming our experimental method. Figure 6A represents the correlation functions of both CP12 mutant proteins in the absence or presence of GuHCl; all the data were fitted to a free single-component diffusion model with a diffusion time of sd and an average number N of molecules by using Eqn (1) (later). The accuracy of the fits was established using residual distribution analysis. Figure 6B depicts the hydrodynamic radii of both CP12 mutant proteins in a series of increasing GuHCl concentrations. While under native conditions both CP12C31S and CP12C75S had similar hydrodynamic radii, the hydrodynamic radius values obtained at 5 M GuHCl for strongly denatured CP12C31S and CP12C75S revealed an increase of 1.6- and 2.0-fold respectively compared with their corresponding native structures. Moreover, difference in hydrodynamic radius the significant 10 A between the two mutant proteins under denatured conditions indicates that the N- and C-terminal mutant proteins differ in structure.
Discussion CP12 is very flexible and largely devoid of secondary structure; however, when disulfide bonds are present, a-helix content and the overall degree of order of CP12 is increased [2]. This protein is able to bind metal ions such as copper and nickel in C. reinhardtii [38,39] and calcium in higher plant [40]. CP12 also FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
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CP12 structural modulation
A
1.0
+ GAPDH 0.6
+ PRK
0.4
+ GAPDH + PRK
150 140 130 120 0
1
2
3
4
5
4
5
[GuHCl] (M)
0.0 0.1
1
10
100
B
Time (ms)
500
Waist (nm)
480
B 4.5
4.0
460 440 420 400 380
3.5
3.0
C
2.5 CP12C75S
+ GAPDH
+ PRK
+ GAPDH + PRK
C 4.5
Refractive Index
Hydrodynamic radii (nm)
160
0.2
0.01
Hydrodynamic radii (nm)
180 170
CP12C75S 0.8 td (µs)
Normalised auto correlation g (t)
A
0
1
0
1
2 3 [GuHCl] (M)
1.44 1.42 1.40 1.38 1.36 1.34 1.32
4.0
2
3
4
5
[GuHCl] (M)
3.5
3.0 4 6 8 10
2
4 6 8 100
2
4 6 8 1000
2
PRK concentration (nM) Fig. 4. FCS data on the C-terminal disulfide bridge mutant CP12C75S. (A) Normalized FCS correlation traces and (B) deduced hydrodynamic radii obtained on CP12C75S in the absence and presence of GAPDH or PRK. In (A) and (B), the concentrations of CP12C75S, GAPDH and PRK were 100, 500 and 500 nM respectively, corresponding to a 5-fold molar excess of GAPDH and/or PRK relative to CP12C75S. (C) Dependence of the CP12C75S hydrodynamic radius on the concentration of PRK. The labeled CP12C75S concentration was kept constant at 100 nM.
plays a role in oxidative stress as shown by antisense CP12 mutant plants [41]. In early studies, other proteins such as malate dehydrogenase, elongation factor FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
Fig. 5. Calibration procedure for the influence of increased concentrations of GuHCl. (A) Diffusion time measured for Alexa Fluor 647 as function of GuHCl concentration. (B) Effective transversal waist deduced from the measurements in (A) and the calibrated 0.7 nm hydrodynamic radius of Alexa Fluor 647 at 22 °C. (C) The evolution of the transversal waist in (B) is found to correlate with increase of the solvent’s refractive index upon addition of GuHCl.
1 alpha 2 and 38 kDa-ribosome-associated protein were able to interact with CP12 but to a lesser extent than PRK, GAPDH and the aldolase, enzymes from the Calvin cycle [9]. CP12 thus belongs to the IDP family and it seems to be a jack of all trades and a master of the Calvin cycle [15]. This protein is found in most photosynthetic organisms [3,42] and was even shown to be produced by cyanophages upon infection of their hosts, thereby inhibiting their Calvin cycle and re-routing the NADPH towards phage nucleotide biosynthesis [43]. A recent analysis in cyanobacteria
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Auto correlation g (t)
A 1.008 CP12C31S - 0M GuHCl
1.006
CP12C31S - 5M GuHCl CP12C75S - 0M GuHCl
1.004
CP12C75S - 5M GuHCl
1.002 1.000 0.1
1
10
Time (ms)
Hydrodynamic radii (nm)
B 6
CP12C75S
5 4 CP12C31S
3 2
Control
1 0
1
2
3
4
5
GuHCL concentration (M) Fig. 6. Conformational changes of CP12 mutant proteins are highlighted by unfolding experiments under denaturation conditions. (A) Raw fluorescence correlation functions of CP12 mutant proteins in the presence and absence of GuHCl; the data were fitted to a single-component diffusion model. (B) Hydrodynamic radii of CP12 mutant proteins as function of the concentration of GuHCl. A control experiment is displayed for Atto647N free dye to indicate the absence of experimental artifacts.
showed that about eight types of CP12 are present and among them are three CP12-CBS containing a cystathionine-b-synthase (CBS) domain that is fused to CP12. The CBS domains function as regulatory modules for a wide range of cellular activities, and some bind adenine nucleotides [42]. The role of CP12 like that of many other IDPs in the cell is thus of paramount importance [44]. Although the interaction between this protein, GAPDH and PRK has been studied, the structure and dynamics of CP12 are still unknown. Crystallographic data of GAPDH/CP12 from Synechococcus elongatus and Arabidopsis thaliana showed that the C-terminal part of CP12 can fold upon binding to GAPDH but most of the CP12 remains highly flexible and almost 50 residues of about 80 were not visible in the density map of either structure [10,45]. In C. reinhardtii it was recently shown by site-directed spin labeling combined with electron paramagnetic resonance spectroscopy 3212
that the GAPDH–CP12 complex is a fuzzy complex [26]. Only a modeled structure of the algal CP12 is therefore available [46]. Data on the interaction between PRK and CP12 are scarce as this interaction is weaker (lM range) than that with GAPDH and CP12 (nM range) in green alga, and again no structural data are available [2]. Hence not much is known on the conformational change of the CP12 protein either upon folding or upon interaction with GAPDH and PRK proteins. Our study focuses for the first time on structural transition parameters of CP12–GAPDH and CP12– PRK complexes. Our results indicate that the CP12wt binds to GAPDH and/or PRK independently and also to both enzymes to form a ternary complex. To understand the molecular background of the function of CP12wt it is crucial, due to the high flexibility and the lack of rigid structure of CP12, to investigate its structural properties. Since there are no structural data for the algal complex, our present results attempt to elucidate the algal CP12 structure in terms of hydrodynamic radii to decipher its mode of binding to two key regulated enzymes, namely GAPDH and PRK of the Calvin cycle. These observations are a first step towards an understanding of protein–protein interactions in the GAPDH–CP12–PRK complex and the nature of the physicochemical forces involved during ternary complex formation. Our results showed that the disruption of the N-terminal disulfide bridge on CP12 affects the formation of the CP12C31S–PRK complex, while it has no effect on the CP12C31S–GAPDH complex. On the other hand, we observed the opposite effect with the mutant CP12C75S affected at the C-terminal disulfide bridge that forms a complex with PRK but not with GAPDH. Further addition of PRK or GAPDH to the corresponding CP12C31S–GAPDH or CP12C75S– PRK complexes showed no signs of the formation of GAPDH–CP12(C31S/C75S)–PRK supramolecular complex suggesting that both C-terminal and N-terminal disulfide bridges are crucial for formation of the ternary structure complex. In addition, the binding of both GAPDH and PRK proteins to CP12 was suppressed by DTT that results in the disruption of the disulfide bridges of CP12. This study also showed that GAPDH and PRK bind at specific sites on CP12. Based on the similar fluorescence brightness found for CP12 mutant proteins alone and for the sub-complexes, only one monomer of CP12C31S or CP12C75S bound to GAPDH and PRK respectively. The mutant CP12C31S behaves as CP12 locked in a reduced state and therefore this result is in agreement with previous results where only one moleFEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
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cule of reduced CP12 was bound to GAPDH while two molecules of oxidized CP12 were bound to tetrameric GAPDH [47,48]. The present study also indicates that the ratio 1 : 4 of CP12 to PRK is sufficient to form the CP12C75S–PRK complex; with a KD of 1.6 lM [2] determined by surface plasmon resonance (SPR) and the experimental conditions used in this report, one would expect about 20% of complex formation between CP12 and PRK. SPR and fluorescence spectroscopy are complementary techniques although in SPR one partner is immobilized on a chip while in FCS the interaction between the two partners occurs in solution, explaining the apparent discrepancy in the results obtained. A set of equations correlating protein density in several conformational states (native, unfolded, pre-molten globular and molten globular) in a wide range of molecular masses has been reported [49]. Analysis of similar dependences for various IDPs was added later [18,50]. The measured hydrodynamic radii for both CP12 mutant proteins in their native state were around 32 A, a value that fits more with an IDP hydrodynamic radius compared with the native state hydrodynamic radius. To cite but a few examples, the hydrodynamic radius for a globular protein like cytochrome c is about 17.8 A and for an IDP like TyrRS(D1) is 21 A, while these proteins have almost the same number of amino acids (104 and 107 respectively) [51]. Moreover, a clear difference was found between the extended conformation (maximal chain expansion) under denaturing conditions (Fig. 4A) and under native conditions. CP12 is highly flexible in its native state and may become somewhat ordered upon interaction with either GAPDH or PRK by forming the GAPDH–CP12 or the PRK–CP12 binary complexes as observed by the diffusion time changes upon formation of these sub-complexes. The present report also focuses on the unfolding nature of the CP12 mutation sites specifically both at the C-terminus and N-terminus to assess the degree of re-modulation and further to evaluate the specificity towards the GAPDH and PRK interactions. It is likely that CP12 like other IDPs shows gradual hydrophobic collapse during unfolding.
Experimental procedures Recombinant protein expression and purification C. reinhardtii CP12, GAPDH and PRK were obtained as described earlier [7]. The concentrations of all proteins were calculated using Bradford assays [52]. The purity of recombinant proteins was checked by SDS/PAGE [53] followed by Coomassie Blue R-250 staining and mass spectrometry (Fig. 1C). For all experiments, His-tagged CP12 proteins
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were used as it was shown that wild-type His-tagged CP12 behaves like the native protein, indicating that the His tag does not interfere in our experiments [48]. Numbering of residues, however, was based on the protein without the tag, with SGQPA being the first residues (http://genome.jgi.doe.gov/cgi-bin/dispGeneModel?db=Chlre4&id=148487).
Protein labeling Lysine residues in the CP12wt protein were labeled with the amine-reactive N-hydroxysuccinimide ester derivative of the Atto590 fluorophore, and in both single cysteine-modified CP12 mutant proteins were labeled with the thiol-reactive maleimide derivative of the fluorophore Atto647N by following the protocol provided by the manufacturer (AttoTec). In all cases, excess fluorophore was added to the aqueous protein solution containing 50 mM Tris/HCl at pH 8.0 and left for 8 h at 4 °C. The labeled protein was subsequently purified using size exclusion chromatography on a SephadexTM G-25 Medium (GE Healthcare, Little Chalfont, Buckinghamshire, UK) column with 50 mM Tris/HCl (pH 8.0) elution buffer. In this report, CD experiments were not carried out as it was not possible to obtain enough labeled protein (10 lM). However, previous reports showed that cysteine labeling of the proteins mutated either on the N-terminal disulfide bridge [26] or on the C-terminal disulfide bridge [54] did not alter the global behavior of the CP12 proteins. CD spectra of labeled and unlabeled proteins were similar. Sample purity and specificity was confirmed by matrixassisted laser desorption/ionization mass spectrometry.
In vitro reconstitution of the GAPDH–CP12–PRK complex All in vitro reconstitution assays to test formation of the sub-complexes CP12wt and CP12 mutants (C31S or C75S) with GAPDH or PRK, or the ternary complex GAPDH– CP12(wt or C31S or C75S)–PRK were performed as previously described [7]. In the case of GAPDH–CP12 or CP12– PRK interaction experiments, 100 nM of labeled CP12wt or CP12C31S or CP12C75S was mixed separately or in combination with 500 nM of GAPDH and 500 nM of PRK in 50 mM Tris/HCl, 4 mM EDTA, 0.1 mM NAD and 5 mM cysteine at pH 8.0 for 12 h at 22 °C. In the case of CP12wt, the mixture was diluted to 10 nM of CP12wt and 50 nM of GAPDH or PRK respectively. In corresponding samples, 1 mM DTT was used as a reducing agent and samples were incubated for 2 h at 22 °C. In the case of the CP12 self-association experiments, 40 nM of labeled CP12C31S mutant was mixed with ratios 1 : 12.5 nM, 1 : 125 nM and 1 : 1250 nM of unlabeled CP12C31S mutant in 50 mM Tris/HCl, 4 mM EDTA at pH 8.0 for 12 h at 22 °C. To test the effect of PRK concentration on CP12– PRK complex formation, 100 nM labeled CP12C75S was mixed with concentration ratios from 1 : 0.04 nM to
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1 : 20 nM of unlabeled PRK for 12 h at 22 °C in 50 mM Tris/HCl, 4 mM EDTA, 0.1 mM NAD at pH 8.0. All the mixtures were tested for complex formation using in vitro reconstitution assays by electrophoresis under non-denaturing conditions (native PAGE) followed by Coomassie Blue R-250 staining (data not shown).
Fluorescence correlation spectroscopy FCS experiments were carried out with a custom-built confocal fluorescence microscope with a Zeiss C-Apochromat 40 9 1.2NA water-immersion objective [55]. The excitation source was a CW He-Ne laser operating at 633 nm. For FCS measurements on CP12 with either GAPDH or PRK proteins the laser beam was set to fill the microscope objective back-aperture so as to use the maximum numerical aperture available for the microscope objective. A 30 lm confocal pinhole conjugated to the sample plane defined the confocal volume whose transversal waist wxy was calibrated to 285 nm using the known diffusion coefficient of Alexa 647 in pure water (3.1 9 106 cm2s1 at 22 °C). For the experiment on CP12 unfolding, the increase in the refractive index of the medium upon addition of GuHCl (Fig. 5) was imposed to limit the negative effects of spherical aberrations. This was achieved by under filling the microscope objective back-aperture (4.5 mm instead of 8.9 mm) and using a 50 lm confocal pinhole. With these conditions, the transversal waist wxy was calibrated to 410 nm in the absence of GuHCl. A moderate variation of 20% on the diffusion time was found with the calibration procedure using Alexa Fluor 647 upon increasing concentrations of GuHCl. This phenomenon was due to the combination of both refractive effects and increased solvent viscosity g (Fig. 5). The effect was systematically taken into account to calibrate the quotient wxy2/g in the presence of GuHCl so as to deduce the hydrodynamic radius of the protein samples. All FCS experiments were carried out at 22 °C using 50 mM Tris/HCl, 4 mM EDTA, 0.1 mM NAD and 5 mM cysteine as a reconstitution buffer at pH 8.0. We also added 0.1% Tween-20 (Sigma) to the samples in order to diminish surface interactions with the glass coverslip. The fluorescence intensity temporal fluctuations were analyzed with a hardware correlator (Flex02-12D/C correlator.com, Bridgewater, NJ, USA, with 12.5 ns minimum channel width). All the experimental data were fitted by considering a single species and free Brownian 3D diffusion in the case of a Gaussian molecular detection efficiency:
g2 ðsÞ ¼ 1 þ
2 1 B s 1 1 þ nT exp N F sT
1 þ ssd
1 qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 þ s2 ssd ð1Þ
where N is the average number of molecules in the focal volume, F is the total fluorescence signal, B is the back-
3214
ground noise, nT is the amplitude of the dark state population, sT is the dark state blinking time, sd is the mean diffusion time and s is the ratio of transversal to axial dimensions of the analysis volume. The background noise B originated mainly from the back-reflected laser light and from the detector dark current and remained below 1 kHz for the experiments reported here. Numerical fit of the FCS data following Eqn (1) provided the average number of molecules, N, which was used to calculate the fluorescence count rate per molecule (CRM) as CRM ¼
FB N
ð2Þ
All the data were fitted by considering that the regular distribution of the weighted residuals is around zero. The FCS measurement of the mean diffusion time sd and the separate calibration of the transversal waist wxy of the confocal detection volume allowed the molecular diffusion coefficient D to be calculated according to the relation sd ¼ w2xy =4D
ð3Þ
The hydrodynamic radius RH can then be deduced from the measured diffusion coefficient D using the Stokes–Einstein equation D¼
kB T 6 p g RH
ð4Þ
where kB is Boltzmann’s constant, T is the absolute temperature and g is the viscosity of the medium. We calibrated the parameter w2xy /g before each measurement on CP12 by recording the FCS trace for Alexa Fluor 647 dyes which have a known hydrodynamic radius of 0.7 nm in pure water. With that calibration, the measured hydrodynamic radii of both Alexa Fluor 647 and Atto647N do not vary upon addition of GuHCl, confirming our experimental method.
Conclusion We report detailed information about the hydrodynamic radii of wild-type CP12 and its site-specific mutants at their disulfide bridges at the N-terminus (CP12C31S) and the C-terminus (CP12C75S). We quantify a hydrodynamic radius of 3.4 0.2 nm for the CP12 protein with an increase up to 5.2 0.3 nm upon complex formation with GAPDH and PRK. Our results on CP12C31S and CP12C75S clearly point out the crucial influence of the mutations affecting the disulfide bridges for complex formation between CP12, GAPDH and PRK. Using denaturation conditions, we monitor the change in hydrodynamic radii of the CP12 mutant proteins upon unfolding. The different behavior between CP12 mutant proteins indicates FEBS Journal 281 (2014) 3206–3217 ª 2014 FEBS
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different structural conformations that favor the binding of either the GAPDH tetramer or the PRK dimer.
Acknowledgements This work was funded by a contract from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ ERC Grant agreement no. 278242. GP was in receipt of a Fellowship from the Ecole Normale Superieure, Cachan. This work (BG) was in part supported by the Agence Nationale de la Recherche ANR SPINFOLD no. 09-BLAN-0100, La Region PACA, APO projet 063 506, the Centre National de la Recherche Scientifique (CNRS) and Aix-Marseille Universite.
Author contributions Design of the experiments: SBM, HR, BG and JW. Involved in protein purification and labeling: SBM and GPT. Involved in FCS experiments: SBM and JW. Helped in protein quantification: PM. Analyzed the data: SBM, GPT, BG and JW. Wrote the paper: SBM, BG and JW. All authors read and approved the final manuscript.
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