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ISSN: 2053-230X

A cryoprotectant induces conformational change in glyceraldehyde-3-phosphate de­hydrogenase

aDepartment of Herbal Medicine Resources, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan 54596, Republic of Korea, and bDepartment of Lifestyle, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan 54596, Republic of Korea
*Correspondence e-mail: nationface@jbnu.ac.kr

Edited by K. K. Kim, Sungkyunkwan University School of Medicine, Republic of Korea (Received 8 February 2018; accepted 19 March 2018; online 16 April 2018)

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, catalyses the conversion of D-glyceraldehyde 3-phosphate to 1,3-bisphospho­glycerate. While mammalian and yeast GAPDHs are multifunctional proteins that have additional functions beyond those involved in glycolysis, including reactions related to nuclear RNA transport, DNA replication/repair, membrane fusion and cellular apoptosis, Escherichia coli GAPDH (ecGAPDH) has only been reported to function in glycolysis. The S-loop of GAPDH is required for interaction with its cofactor and with other proteins. In this study, the three-dimensional crystal structure of GAPDH treated with trehalose is reported at 2.0 Å resolution. Trehalose was used as a cryoprotectant for the GAPDH crystals. The structure of trehalose-bound ecGAPDH was compared with the structures of both NAD+-free and NAD+-bound ecGAPDH. At the S-loop, the bound trehalose in the GAPDH structure induces a 2.4° rotation compared with the NAD+-free ecGAPDH structure and a 3.1° rotation compared with the NAD+-bound ecGAPDH structure.

1. Introduction

Many crystals of biological macromolecules are sensitive to X-rays near room temperature and frequently suffer from radiation damage, especially when X-ray experiments are carried out on highly intense synchrotron beamlines (Hope, 1990[Hope, H. (1990). Annu. Rev. Biophys. Biophys. Chem. 19, 107-126.]). To prevent radiation damage from X-rays (Watenpaugh, 1991[Watenpaugh, K. D. (1991). Curr. Opin. Struct. Biol. 1, 1012-1015.]; Rodgers, 1994[Rodgers, D. W. (1994). Structure, 2, 1135-1140.]; Low et al., 1966[Low, B. W., Chen, C. C. H., Berger, J. E., Singman, L. & Pletcher, J. F. (1966). Proc. Natl Acad. Sci. USA, 56, 1746-1750.]) and to facilitate the transport and simple storage of protein crystals, many cryoprotectants have been developed (Garman, 1999[Garman, E. (1999). Acta Cryst. D55, 1641-1653.], 2003[Garman, E. (2003). Curr. Opin. Struct. Biol. 13, 545-551.]) and many cryoprotectant products are commercially available, such as CryoPro from Hampton Research.

In general, cryoprotectants are small polyols and organics, such as glycerol, ethylene glycol, 1,2-propanediol, diethylene glycol, 2-methyl-2,4-pentanediol, dimethyl sulfoxide or other nonvolatile alcohols (Pflugrath, 2015[Pflugrath, J. W. (2015). Acta Cryst. F71, 622-642.]). Low-molecular-weight polyethylene glycols (PEGs), such as PEG 200 or PEG 400, are suitable cryoprotectants in many cases. Higher molecular-weight PEGs can also be used, albeit with more difficulty than lower molecular-weight PEGs. High concentrations of salts, such as lithium formate (Rubinson et al., 2000[Rubinson, K. A., Ladner, J. E., Tordova, M. & Gilliland, G. L. (2000). Acta Cryst. D56, 996-1001.]), or carboxylic acids, such as malonate, can also be used (Holyoak et al., 2003[Holyoak, T., Fenn, T. D., Wilson, M. A., Moulin, A. G., Ringe, D. & Petsko, G. A. (2003). Acta Cryst. D59, 2356-2358.]), as can sugars such as sucrose, trehalose, sorbitol, xylitol or glucose (Pflugrath, 2015[Pflugrath, J. W. (2015). Acta Cryst. F71, 622-642.]).

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme that catalyses the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate using NAD+ as a cofactor (Baker et al., 2014[Baker, B. Y., Shi, W., Wang, B. & Palczewski, K. (2014). Protein Sci. 23, 1629-1639.]) and is known to be a moonlighting protein (Savreux-Lenglet et al., 2015[Savreux-Lenglet, G., Depauw, S. & David-Cordonnier, M. H. (2015). Int. J. Mol. Sci. 16, 26555-26581.]). This enzyme plays multiple roles in the regulation of mRNA stability (Zhou et al., 2008[Zhou, Y., Yi, X., Stoffer, J. B., Bonafe, N., Gilmore-Hebert, M., McAlpine, J. & Chambers, S. K. (2008). Mol. Cancer Res. 6, 1375-1384.]), intracellular membrane trafficking (Sirover, 2012[Sirover, M. A. (2012). J. Cell. Biochem. 113, 2193-2200.]), iron uptake and transport (Zaid et al., 2009[Zaid, H., Talior-Volodarsky, I., Antonescu, C., Liu, Z. & Klip, A. (2009). Biochem. J. 419, 475-484.]), DNA replication and repair (Zheng et al., 2003[Zheng, L., Roeder, R. G. & Luo, Y. (2003). Cell, 114, 255-266.]), and nuclear RNA transport (Dastoor & Dreyer, 2001[Dastoor, Z. & Dreyer, J. L. (2001). J. Cell Sci. 114, 1643-1653.]). In particular, yeast GAPDH is inhibited by trehalose (Araiza-Olivera et al., 2010[Araiza-Olivera, D., Sampedro, J. G., Mújica, A., Peña, A. & Uribe-Carvajal, S. (2010). FEM. Yeast Res. 10, 282-289.]). An important region of GAPDH is the S-loop (residues 178–201), a long, winding region of the enzyme that is known to interact with some proteins (Kosova et al., 2017[Kosova, A. A., Khodyreva, S. N. & Lavrik, O. I. (2017). Biochemistry, 82, 643-654.]; Duée et al., 1996[Duée, E., Olivier-Deyris, L., Fanchon, E., Corbier, C., Branlant, G. & Dideberg, O. (1996). J. Mol. Biol. 257, 814-838.]). The S-loop region of NAD+-free GAPDH has a very flexible shape and thus does not show clear electron density (Ferreira-da-Silva et al., 2006[Ferreira-da-Silva, F., Pereira, P. J., Gales, L., Roessle, M., Svergun, D. I., Moradas-Ferreira, P. & Damas, A. M. (2006). J. Biol. Chem. 281, 33433-33440.]; Querol-García et al., 2017[Querol-García, J., Fernández, F. J., Marin, A. V., Gómez, S., Fullà, D., Melchor-Tafur, C., Franco-Hidalgo, V., Albertí, S., Juanhuix, J., Rodríguez de Córdoba, S., Regueiro, J. R. & Vega, M. C. (2017). Front. Microbiol. 8, 541.]). On the other hand, in NAD+-bound GAPDH S-loop fixation occurs by the formation of a complex with the coenzyme NAD+ (Kitatani et al., 2006[Kitatani, T., Nakamura, Y., Wada, K., Kinoshita, T., Tamoi, M., Shigeoka, S. & Tada, T. (2006). Acta Cryst. F62, 727-730.]).

GAPDHs have been isolated from bacteria, archaea, prokaryotes, eukaryotes, plants and mammals. Under normal cellular conditions, GAPDH primarily has a tetrameric conformation composed of four identical 35 kDa subunits (Nicholls et al., 2012[Nicholls, C., Li, H. & Liu, J.-P. (2012). Clin. Exp. Pharmacol. Physiol. 39, 674-679.]; Frayne et al., 2009[Frayne, J., Taylor, A., Cameron, G. & Hadfield, A. T. (2009). J. Biol. Chem. 284, 22703-22712.]). GAPDH consists of an NAD+-binding domain (residues 2–148 and 312–330), which is composed of α/β dinucleotide-binding folds or Rossmann folds, and a catalytic domain (residues 149–311), which is composed of eight antiparallel β-sheets with an α-helix and several short loops (Duée et al., 1996[Duée, E., Olivier-Deyris, L., Fanchon, E., Corbier, C., Branlant, G. & Dideberg, O. (1996). J. Mol. Biol. 257, 814-838.]; Yun et al., 2000[Yun, M., Park, C.-G., Kim, J.-Y. & Park, H.-W. (2000). Biochemistry, 39, 10702-10710.]). The activity of GAPDH requires the binding of NAD+.

In this study, the purification, crystallization and cryo­protection of Escherichia coli GAPDH (ecGAPDH) and the three-dimensional structure of trehalose-bound ecGAPDH are described. The GAPDH crystals were cryoprotected with a solution containing 15% of the disaccharide trehalose.

2. Materials and methods

2.1. Preparation of GAPDH

E. coli BL21 (DE3) cells were grown on LB agar medium containing 100 µM ampicillin. A single colony was cultivated in 100 ml LB medium overnight at 37°C. The next day, a main culture was cultivated in 1 l LB medium for 4 h at 37°C. The cultured cells were harvested by centrifugation (4°C, 8000 rev min−1, 5 min). The harvested cells were resuspended in buffer A (20 mM Tris, 130 mM NaCl pH 7.5). The cells were disrupted by sonication on ice and the cell lysate was then separated from the cell debris by centrifugation (4°C, 20 000 rev min−1, 20 min). The supernatant was loaded onto a HisTrap column (5 ml; GE Healthcare, Little Chalfont, England) that had been equilibrated with buffer A. The column was washed with buffer A and the proteins bound to the HisTrap column were eluted with buffer B (20 mM Tris, 130 mM NaCl, 0.2 M imidazole pH 7.5). The extracted active proteins, including ecGAPDH, were loaded onto a HiTrap heparin column (5 ml; GE Healthcare) equilibrated with buffer A (Reisz et al., 2016[Reisz, J. A., Wither, M. J., Dzieciatkowska, M., Nemkov, T., Issaian, A., Yoshida, T., Dunham, A. J., Hill, R. C., Hansen, K. C. & D'Alessandro, A. (2016). Blood, 128, e32-e42.]). After washing the column with buffer A, buffer C (20 mM Tris pH 7.5, 1 M NaCl) was gradually applied as an elution buffer. ecGAPDH was eluted at concentrations of 200–300 mM NaCl and was dialysed thoroughly against buffer A.

2.2. Crystallization

Purified ecGAPDH was concentrated to 20 mg ml−1 using Amicon filters (30 kDa cutoff; Sigma–Aldrich, St Louis, Missouri, USA). Concentrated ecGAPDH (2 µl) was mixed with reservoir solution (2 µl) consisting of 2.8 M ammonium sulfate, 0.1 M 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.5–6.5 at 4°C. GAPDH crystals grew within two weeks using the hanging-drop method. X-ray data were collected from GAPDH crystals treated with a cryoprotectant consisting of 15%(v/v) trehalose at 100 K. Crystallization information is given in Table 1[link].

Table 1
Crystallization conditions

Method Hanging-drop vapour diffusion
Plate type 24-well plates
Temperature (°C) 4
Protein concentration (mg ml−1) 20
Buffer composition of protein solution 20 mM Tris pH 7.5, 0.1 M NaCl
Composition of reservoir solution 0.1 M MES pH 5.5–6.5, 2.8 M ammonium sulfate
Volume and ratio of drop 2 µl:2 µl
Volume of reservoir (ml) 1

2.3. Data collection and processing

X-ray diffraction data were collected on beamline 7A at the Pohang Accelerator Laboratory (PAL 7A SB I; Pohang, Republic of Korea) using a CCD detector (ADSC Quantum 270) at an X-ray wavelength of 1.0 Å. All images were indexed, integrated and scaled using the XDS program package and the CCP4 program SCALA. The crystals belonged to the tetragonal space group I4122, with unit-cell parameters a = 121.29, b = 121.29, c = 156.06 Å, α = 90, β = 90, γ = 90°. Data-collection and processing statistics are given in Table 2[link].

Table 2
Data collection and processing

Values in parentheses are for the outer shell.

Diffraction source PAL 7A SB I
Wavelength (Å) 1.00
Temperature (K) 100
Detector ADSC Quantum 270
Crystal-to-detector distance (mm) 230
Rotation range per image (°) 1
Total rotation range (°) 90
Exposure time per image (s) 1
Space group I4122
a, b, c (Å) 121.29, 121.29, 156.06
α, β, γ (°) 90, 90, 90
Mosaicity (°) 0.126
Resolution range (Å) 47.88–2.00 (2.10–2.00)
Total No. of reflections 284143 (41653)
No. of unique reflections 39522 (5706)
Completeness (%) 99.6 (99.7)
Multiplicity 7.2 (7.3)
I/σ(I)〉 21.8 (8.1)
Rmeas 0.061 (0.257)
Overall B factor from Wilson plot (Å2) 22.9

3. Structure determination and refinement

Initial phases for GAPDH were obtained by a conventional molecular-replacement protocol (rotation, translation and rigid-body fitting) using the structure of E. coli GAPDH (PDB entry 1s7c; Berkeley Structural Genomics Center, unpublished work) as an initial search model. The model was fitted more appropriately by simulated annealing in CNS. Manual rebuilding of the GAPDH model and electron-density interpretation were performed after each refinement cycle using Coot (Emsley et al., 2010[Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. (2010). Acta Cryst. D66, 486-501.]). Restrained, individual B factors were refined and the crystal structure was finalized using the CCP4 program REFMAC5 and other programs in the CCP4 suite (Winn et al., 2011[Winn, M. D. et al. (2011). Acta Cryst. D67, 235-242.]). GAPDH was modelled with TLS refinement using anisotropic temperature factors for all atoms. The final model had Rfree and Rcryst factors of 20.5% and 18.4%, respectively (Table 3[link]). Structure validation was performed with SFCHECK (Vaguine et al., 1999[Vaguine, A. A., Richelle, J. & Wodak, S. J. (1999). Acta Cryst. D55, 191-205.]), PROCHECK (Laskowski et al., 1996[Laskowski, R. A., Rullmannn, J. A., MacArthur, M. W., Kaptein, R. & Thornton, J. M. (1996). J. Biomol. NMR, 8, 477-486.]) and ADIT (Bhat et al., 2001[Bhat, T. N., Bourne, P., Feng, Z., Gilliland, G., Jain, S., Ravichandran, V., Schneider, B., Schneider, K., Thanki, N., Weissig, H., Westbrook, J. & Berman, H. M. (2001). Nucleic Acids Res. 29, 214-218.]). DynDom, a program that determines domain movement and relative inter-domain rotation angles in proteins for which two conformations are available, was used to study domain rotation (Poornam et al., 2009[Poornam, G. P., Matsumoto, A., Ishida, H. & Hayward, S. (2009). Proteins, 76, 201-212.]; Hayward et al., 1997[Hayward, S., Kitao, A. & Berendsen, H. J. (1997). Proteins, 27, 425-437.]). Coordinates have been deposited in the Protein Data Bank with accession code 5za0.

Table 3
Structure solution and refinement

Resolution range (Å) 47.7–2.00
Completeness (%) 99.5
No. of reflections 37461
Final Rcryst (%) 18.4
Final Rfree (%) 20.5
No. of atoms/residues
 Protein 2451/330
 Others 199/173
 Sulfate 5/1
 Trehalose 23/1
 Water 171/171
 Total 2650/503
R.m.s. deviations
 Bond lengths (Å) 0.010
 Bond angles (°) 1.284
Ramachandran plot
 Most favoured (%) 97
 Allowed (%) 2.7
 Disallowed (%) 0.3

4. Results and discussion

GAPDH was purified from E. coli without any genetic manipulation. Despite the use of a nonrecombinant gene, GAPDH could surprisingly be purified using a HisTrap column. Approximately 2 mg of purified ecGAPDH was obtained from a 1 l culture. For crystallization purposes, ecGAPDH was concentrated to approximately 20 mg ml−1 using an Amicon concentrator (30 kDa cutoff; Sigma–Aldrich). Although ecGAPDH was stable at room temperature, crystals suitable for X-ray analysis were grown by the hanging-drop method at 4°C (Fig. 1[link]). Tetragonal bipyramidal crystals appeared within two weeks and grew for a further week. For data collection, the crystals were cryoprotected with 15% trehalose. In the X-ray studies, a single crystal of ecGAPDH was mounted in a cryoloop. The crystals showed significant overloads. Thus, even when the resolution was reduced, X-ray diffraction was measured at 80% attenuation. Nevertheless, these crystals diffracted to 2.0 Å resolution on PAL 7A SB I (Supplementary Fig. S1).

[Figure 1]
Figure 1
Crystals of ecGAPDH.

The structure was solved by molecular replacement using the E. coli GAPDH structure (PDB entry 1s7c) as a starting model. Molecular replacement was performed using Phaser from the CCP4 suite. The final ecGAPDH model had Rfree and Rcryst factors of 20.5% and 18.4%, respectively. The solved structure had a monomer in the asymmetric unit. The monomer contained 330 residues, one sulfate ion, one trehalose molecule and 171 water molecules. The number of water molecules in the current GAPDH structure is restricted despite the high resolution and low mosaicity. To study the conformational changes induced by the binding of trehalose to ecGAPDH, a detailed structural comparison of trehalose-free and trehalose-bound GAPDH was performed.

A list of commonly used sugar cryoprotectants is shown in Supplementary Table S1. Among them, disaccharides such as sucrose, maltose and trehalose have been used to cryoprotect GAPDH crystals. Glycerol, a commonly used cryoprotectant, has also been used in a comparison with sugar-protected GAPDH. Mixtures of 30% glycerol and 15% sugars were used for cryoprotection. None of the resulting crystals exhibited any signs of cracking or melting during treatment with the cryoprotectants. The crystals obtained using glycerol as a cryoprotectant showed a resolution of 2.1 Å. The crystals obtained using sucrose and maltose showed resolutions of 2.4 and 2.2 Å, respectively. The crystals obtained using trehalose showed a resolution of 2.0 Å. Although sucrose and maltose have similar structures to that of trehalose, these sugars did not bind to ecGAPDH and glycerol was not found, even though glycerol has previously been reported to be bound in GAPDH structures (Mukherjee et al., 2010[Mukherjee, S., Dutta, D., Saha, B. & Das, A. K. (2010). J. Mol. Biol. 401, 949-968.]; Cook et al., 2009[Cook, W. J., Senkovich, O. & Chattopadhyay, D. (2009). BMC Struct. Biol. 9, 51.]; Robien et al., 2006[Robien, M. A. et al. (2006). Proteins, 62, 570-577.]; Chaikuad et al., 2011[Chaikuad, A., Shafqat, N., Al-Mokhtar, R., Cameron, G., Clarke, A. R., Brady, R. L., Oppermann, U., Frayne, J. & Yue, W. W. (2011). Biochem. J. 435, 401-409.]; Querol-García et al., 2017[Querol-García, J., Fernández, F. J., Marin, A. V., Gómez, S., Fullà, D., Melchor-Tafur, C., Franco-Hidalgo, V., Albertí, S., Juanhuix, J., Rodríguez de Córdoba, S., Regueiro, J. R. & Vega, M. C. (2017). Front. Microbiol. 8, 541.]; Moreau et al., 2017[Moreau, C., Terrasse, R., Thielens, N. M., Vernet, T., Gaboriaud, C. & Di Guilmi, A. M. (2017). J. Biol. Chem. 292, 2217-2225.]). Only the GAPDH structure with trehalose was significantly different from published structures.

An electron-density map clearly indicated ordered tre­halose molecules bound to the protein. One trehalose molecule bound to ecGAPDH in the S-loop region (Fig. 2[link]). As described above, the S-loop region of GAPDH without NAD+ bound as a cofactor is very flexible and does not exhibit definite electron density (Querol-García et al., 2017[Querol-García, J., Fernández, F. J., Marin, A. V., Gómez, S., Fullà, D., Melchor-Tafur, C., Franco-Hidalgo, V., Albertí, S., Juanhuix, J., Rodríguez de Córdoba, S., Regueiro, J. R. & Vega, M. C. (2017). Front. Microbiol. 8, 541.]). Despite the absence of NAD+, trehalose stabilized the S-loop of the enzyme. As a result, the electron-density map of the S-loop region of the protein was clear. The rotation angle and the relative rotation angle between the NAD+-binding domain and the catalytic domain were measured. The GAPDH molecule shows a rotation of approximately 2.4–3.1° when superpositioned on the NAD+-free ecGAPDH (PDB entry 1dc5) and/or the NAD+-bound ecGAPDH (PDB entry 1dc6) structures (Yun et al., 2000[Yun, M., Park, C.-G., Kim, J.-Y. & Park, H.-W. (2000). Biochemistry, 39, 10702-10710.]; Fig. 3[link]). The inhibition of yeast GAPDH is reportedly proportional to the concentration of trehalose (Araiza-Olivera et al., 2010[Araiza-Olivera, D., Sampedro, J. G., Mújica, A., Peña, A. & Uribe-Carvajal, S. (2010). FEM. Yeast Res. 10, 282-289.]). The GAPDHs from yeast and E. coli have similar structures. Although ecGAPDH has not been reported to be inhibited by trehalose, trehalose induced a conformational change in ecGAPDH in the current structure. The rotation of GAPDH also induced a conformational change in its active site. This suggests that the binding of trehalose to GAPDH induced a conformational change in its active site to prevent the binding of NAD+, although the NAD+- and trehalose-binding sites differ from one another.

[Figure 2]
Figure 2
Electron-density map of trehalose and the ecGAPDH S-loop. ecGAPDH is shown as a cartoon model with trehalose and S-loop residues shown as orange stick models. (a) 2FoFc electron-density map of trehalose contoured at 3.0σ (blue mesh). (b) 2FoFc electron-density map of the S-loop residues contoured at 3.0σ (blue mesh).
[Figure 3]
Figure 3
Rotation of domains in trehalose-bound ecGAPDH compared with NAD+-free ecGAPDH and NAD+-bound ecGAPDH. The fixed domain is represented in black. (a) NAD+-free ecGAPDH (PDB entry 1dc5; green). (b) Trehalose-bound ecGAPDH (orange). (c) NAD+-bound ecGAPDH (PDB entry 1dc6; forest). (d) The calculated relative rotation angle of trehalose-bound ecGAPDH compared with NAD+-free ecGAPDH is 2.4°. (e) The calculated relative rotation angle of trehalose-bound ecGAPDH compared with NAD+-bound ecGAPDH is 3.1°.

To examine the time dependence of the cryoprotectant effect on GAPDH crystals, the cryoprotectant soaking time was varied from 1 to 10 min and the data for all crystals from each time were evaluated systematically. The GAPDH crystals neither cracked nor melted as a result of the penetration of cryoprotectants into the solvent channels. These results suggested that the conformational changes in GAPDH owing to the binding of trehalose were independent of soaking time for up to 10 min.

Supporting information


Acknowledgements

I am grateful to Pohang Accelerator Laboratory (PAL) where the data were collected on beamline 7A. I thank Chair Professor Dr Hui-Woog Choe at Chonbuk National University and at the Department of Brain and Cognitive Sciences of DGIST, Republic of Korea for fruitful discussions and for continuous support of this work. I also thank Professor Dr Joerg Labahn at the Centre for Structural Systems Biology (CSSB), Deutsches Elektronen-Synchrotron (DESY), Hamburg, Germany for discussions during the early stage of this work.

Funding information

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), the Ministry of Science, ICT and Future Planning (2015R1C1A1A02037509), research funds for newly appointed professors of Chonbuk National University in 2014, and a grant from the Jeon-Dae Memorial Fund in 2014.

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