research communications\(\def\hfill{\hskip 5em}\def\hfil{\hskip 3em}\def\eqno#1{\hfil {#1}}\)

Journal logoSTRUCTURAL BIOLOGY
COMMUNICATIONS
ISSN: 2053-230X

Structure of the mouse acidic amino acid de­carboxylase GADL1

aDepartment of Biomedicine, University of Bergen, Jonas Lies Vei 91, 5009 Bergen, Norway, bK. G. Jebsen Centre for Research on Neuropsychiatric Disorders, University of Bergen, Jonas Lies Vei 91, 5009 Bergen, Norway, cFaculty of Biochemistry and Molecular Medicine, University of Oulu, PO Box 5400, 90014 Oulu, Finland, and dDivision of Psychiatry, Haukeland University Hospital, Bergen, Norway
*Correspondence e-mail: petri.kursula@uib.no, jan.haavik@uib.no

Edited by I. Tanaka, Hokkaido University, Japan (Received 9 October 2017; accepted 13 December 2017)

Pyridoxal 5′-phosphate (PLP) is a ubiquitous cofactor in various enzyme classes, including PLP-dependent decarboxylases. A recently discovered member of this class is glutamic acid decarboxylase-like protein 1 (GADL1), which lacks the activity to decarboxylate glutamate to γ-aminobutyrate, despite its homology to glutamic acid decarboxylase. Among the acidic amino acid decarboxylases, GADL1 is most similar to cysteine sulfinic acid decarboxylase (CSAD), but the physiological function of GADL1 is unclear, although its expression pattern and activity suggest a role in neurotransmitter and neuroprotectant metabolism. The crystal structure of mouse GADL1 is described, together with a solution model based on small-angle X-ray scattering data. While the overall fold and the conformation of the bound PLP are similar to those in other PLP-dependent decarboxylases, GADL1 adopts a more loose conformation in solution, which might have functional relevance in ligand binding and catalysis. The structural data raise new questions about the compactness, flexibility and conformational dynamics of PLP-dependent decarboxylases, including GADL1.

1. Introduction

Pyridoxal 5′-phosphate (PLP), or vitamin B6, is a versatile cofactor that is involved in many enzymatic reactions spanning multiple enzyme classes and chemical reactions (Percudani & Peracchi, 2003[Percudani, R. & Peracchi, A. (2003). EMBO Rep. 4, 850-854.]). PLP-dependent decarboxylases constitute a large family of enzymes that catalyze a range of metabolic reactions. Many of these enzymes catalyze biologically well defined processes; inactivating mutations affecting them are associated with severe phenotypes, and some of them are treatment targets in human disease (Baekkeskov et al., 1990[Baekkeskov, S., Aanstoot, H.-J., Christgau, S., Reetz, A., Solimena, M., Cascalho, M., Folli, F., Richter-Olesen, H. & Camilli, P.-D. (1990). Nature (London), 347, 151-156.]; Eliot & Kirsch, 2004[Eliot, A. & Kirsch, J. (2004). Annu. Rev. Biochem. 73, 383-415.]; El-Sayed & Shindia, 2011[El-Sayed, A. S. & Shindia, A. A. (2011). Targets in Gene Therapy, edited by Y. You, ch. 7. Rijeka: Intech. https://doi.org/10.5772/17449.]; Paiardini et al., 2014[Paiardini, A., Contestabile, R., Buckle, A. M. & Cellini, B. (2014). Biomed. Res. Int. 2014, 856076.]; Sköldberg et al., 2004[Sköldberg, F., Rorsman, F., Perheentupa, J., Landin-Olsson, M., Husebye, E., Gustafsson, J. & Kämpe, O. (2004). J. Clin. Endocrinol. Metab. 89, 1636-1640.]).

Despite extensive research, the biological functions of many PLP-dependent enzymes are still unclear. One such enzyme is glutamic acid decarboxylase-like protein 1 (GADL1), which was originally named based on its sequence homology to glutamic acid decarboxylase (GAD), which synthesizes the inhibitory neurotransmitter γ-aminobutyric acid (GABA; Fenalti et al., 2007[Fenalti, G. et al. (2007). Nature Struct. Mol. Biol. 14, 280-286.]). However, GADL1 has no reactivity towards glutamic acid and therefore is unlikely to be involved in GABA signalling (Liu et al., 2012[Liu, P., Ge, X., Ding, H., Jiang, H., Christensen, B. M. & Li, J. (2012). J. Biol. Chem. 287, 40898-40906.]; Winge et al., 2015[Winge, I., Teigen, K., Fossbakk, A., Mahootchi, E., Kleppe, R., Sköldberg, F., Kämpe, O. & Haavik, J. (2015). Neurochem. Int. 90, 173-184.]). It has been suggested that GADL1 is involved in taurine production (Liu et al., 2012[Liu, P., Ge, X., Ding, H., Jiang, H., Christensen, B. M. & Li, J. (2012). J. Biol. Chem. 287, 40898-40906.]). On the other hand, in our recent comparative study of GADL1 and cysteine sulfinic acid decarboxylase (CSAD), an enzyme homologous to GADL1 that is involved in taurine biosynthesis, we showed that these enzymes act differently. Compared with CSAD, the activity of GADL1 towards cysteine sulfinic acid (CSA) as a substrate is much lower, and GADL1 has a stronger preference for aspartate, suggesting that GADL1 may be involved in the biosynthesis of β-alanine and its peptide derivatives, such as the neuro­protectant carnosine (Min et al., 2008[Min, J., Senut, M., Rajanikant, K., Greenberg, E., Bandagi, R., Zemke, D., Mousa, A., Kassab, M., Farooq, M. U., Gupta, R. & Majid, A. (2008). J. Neurosci. Res. 86, 2984-2991.]; Park et al., 2014[Park, H., Han, K., Shin, J., Park, J., Song, K. & Kim, D. (2014). J. Korean. Neurosurg. Soc. 55, 125-130.]; Winge et al., 2015[Winge, I., Teigen, K., Fossbakk, A., Mahootchi, E., Kleppe, R., Sköldberg, F., Kämpe, O. & Haavik, J. (2015). Neurochem. Int. 90, 173-184.]).

We showed in our earlier study that mouse and human brain have distinct patterns of expression of CSAD and GADL1 (Winge et al., 2015[Winge, I., Teigen, K., Fossbakk, A., Mahootchi, E., Kleppe, R., Sköldberg, F., Kämpe, O. & Haavik, J. (2015). Neurochem. Int. 90, 173-184.]). Whereas both CSAD and GADL1 were expressed in neurons, only the CSAD mRNA was detected in astrocytes. Using a homology model of GADL1 based on the crystal structure of human CSAD (HsCSAD), we performed in silico screening of potential substrate analogues and discovered the first generation of inhibitors with partial selectivity against either GADL1 or CSAD. However, detailed mechanistic studies have been hampered by the lack of structural information.

In this study, we describe the crystal structure of mouse GADL1 (MmGADL1). Additionally, we used small-angle X-ray scattering (SAXS) to elucidate the solution shape of MmGADL1. The overall fold of MmGADL1 is similar to those of CSAD and other close homologues, with a flexible loop, not defined in electron density, from the apposing monomer covering the active site, which is possibly relevant in catalysis. SAXS data demonstrate that MmGADL1 adopts a loosened state in solution, which might correspond to an open conformation significant for cofactor or substrate binding.

2. Materials and methods

2.1. Macromolecule production

2.1.1. Construct preparation, protein expression and purification

The expression vector for MmGADL1 was prepared in the Gateway system using pTH27 (Hammarström et al., 2006[Hammarström, M., Woestenenk, E., Hellgren, N., Härd, T. & Berglund, H. (2006). J. Struct. Funct. Genomics, 7, 1-14.]) as the destination vector. Cloning involved a two-step PCR protocol and homologous recombination into Gateway vectors using standard procedures. The resulting construct codes for an N-terminal His6 tag, a Tobacco etch virus (TEV) protease cleavage site and MmGADL1 residues 1–502 (UniProt entry E9QP13). A longer isoform of MmGADL1 also exists (UniProtKB entry Q80WP8), and the construct corresponds to residues 49–550 of this isoform.

2.1.2. Expression and purification of MmGADL1

His6-tagged MmGADL1 was expressed in Escherichia coli BL21-CodonPlus(DE3)-RIPL cells (Stratagene) at 288 K using 150 mM IPTG induction. Cell pellets were lysed by sonication in a buffer consisting of 50 mM sodium phosphate buffer pH 7.4, 500 mM NaCl, 20 mM imidazole, 0.2 mg ml−1 lysozyme, 1 mM MgCl2, 2 mM pyridoxine hydrochloride and cOmplete EDTA-free protease inhibitors (Roche). Phenylmethyl­sulfonyl fluoride was added to 1 mM immediately following sonication. The unclarified lysate was applied directly onto an IMAC HiTrap TALON crude column (GE Healthcare). The column was washed with 50 mM sodium phosphate pH 7.4, 500 mM NaCl, 50 mM sodium phosphate pH 7.4, 500 mM NaCl, 20 mM imidazole. Elution was carried out with 100 mM imidazole in 50 mM sodium phosphate pH 7.4, 500 mM NaCl. Size-exclusion chromatography was performed using a Superdex HR 200 column (GE Healthcare) equilibrated with 20 mM HEPES, 200 mM NaCl pH 7.5.

2.2. Crystallization

Initial crystallization conditions for MmGADL1 were obtained from the JCSG-plus screen (Molecular Dimensions) using sitting-drop vapour diffusion. The crystallization conditions, which yielded crystals with a needle morphology arranged as single crystals or point-originated clusters, were comprised of 80 mM sodium cacodylate pH 6.0–7.4, 13–14%(w/v) PEG 8000, 120–160 mM calcium acetate, 15.0–17.5%(w/v) glycerol. 0.3–1.5 µl drops with different volume ratios of protein solution (6.5–7.5 mg ml−1 in 20 mM HEPES pH 7.4, 200 mM NaCl) and reservoir solution were used at 281 and 293 K, equilibrating against 40 µl reservoir solution. Crystals were briefly soaked in cryoprotectant solutions and flash-cooled in liquid N2. The detailed conditions used to obtain the crystals used for data collection are given in Table 1[link].

Table 1
Crystallization

Crystal form 1 2
Method Sitting-drop vapour diffusion Sitting-drop vapour diffusion
Plate type Swissci 3-drop 96-well plate Swissci 3-drop 96-well plate
Temperature (K) 293 281
Protein concentration (mg ml−1) 7.5 7
Buffer composition of protein solution 20 mM HEPES pH 7.4, 200 mM NaCl 20 mM HEPES pH 7.4, 200 mM NaCl
Composition of reservoir solution 80 mM sodium cacodylate pH 6.0, 14% PEG 8000, 160 mM calcium acetate, 15% glycerol 80 mM sodium cacodylate pH 7.0, 13% PEG 8000, 160 mM calcium acetate, 15% glycerol
Volume and ratio (protein:well solution) of drop 0.3 µl (1:1) 1.5 µl (1:2)
Volume of reservoir (µl) 40 40
Cryoprotection solution 75%(v/v) reservoir + 25%(v/v) PEG 200 80%(v/v) reservoir + 20%(v/v) glycerol

2.3. Data collection, structure solution and refinement

The MmGADL1 structure was solved from the two crystal forms by molecular replacement in Phaser (McCoy et al., 2007[McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. (2007). J. Appl. Cryst. 40, 658-674.]) using the structure of HsCSAD (PDB entry 2jis; Structural Genomics Consortium, unpublished work) as the search model. Crystal form 1 diffracted to 3 Å resolution, while crystal form 2, which was used for initial structure solution, diffracted to 2.4 Å resolution; the latter suffered from near-perfect pseudomerohedral twinning and high translational pseudosymmetry. Owing to these observations, both crystal forms were solved and initially refined, and the lower resolution structure, which completely lacked twinning and pseudotranslation, was considered to be better for final refinement. In essence, despite the higher nominal resolution, the twinned crystal suffering from pseudotranslation gave lower-quality electron-density maps. The twinning and pseudotranslation operations are given in Table 2[link]. NCS restraints were employed throughout refinement. Refinement was performed with phenix.refine (Afonine et al., 2012[Afonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J., Moriarty, N. W., Mustyakimov, M., Terwilliger, T. C., Urzhumtsev, A., Zwart, P. H. & Adams, P. D. (2012). Acta Cryst. D68, 352-367.]) and model building with Coot (Emsley & Cowtan, 2004[Emsley, P. & Cowtan, K. (2004). Acta Cryst. D60, 2126-2132.]). The structure was validated with MolProbity (Chen et al., 2010[Chen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. & Richardson, D. C. (2010). Acta Cryst. D66, 12-21.]). Data collection and refinement statistics can be found in Table 2[link]. The resolution limits used were based on recent studies (Karplus & Diederichs, 2015[Karplus, P. A. & Diederichs, K. (2015). Curr. Opin. Struct. Biol. 34, 60-68.]) showing that useful information is available for refinement even for data with a CC1/2 much lower than 50%.

Table 2
Data collection, processing and structure refinement

Values in parentheses are for the highest resolution shell.

Crystal form 1 2
Data-collection statistics
 Wavelength (Å) 1.0332 0.9763
 Space group C2 P21
a, b, c (Å) 137.4, 80.6, 128.5 80.9, 121.7, 101.1
α, β, γ (°) 90, 117.9, 90 90, 90.08, 90
 Resolution range (Å) 50–3.00 (3.08–3.00) 50–2.40 (2.46–2.40)
 Completeness (%) 99.4 (99.5) 98.7 (95.9)
 Multiplicity 6.4 (6.0) 3.6 (3.1)
 〈I/σ(I)〉 7.1 (0.7) 5.9 (0.9)
Rmeas 0.328 (3.535) 0.188 (1.466)
Rp.i.m. 0.130 (1.443) 0.099 (0.832)
 CC1/2 (%) 98.9 (28.4) 99.3 (61.4)
 Overall B factor from Wilson plot (Å2) 67 44
Refinement statistics
 Resolution range (Å) 50–3.0
 Final Rcryst 0.236
 Final Rfree 0.288
 R.m.s.d.s
  Bond lengths (Å) 0.003
  Bond angles (°) 0.7
 Average B factor (Å2) 91.0
 Ramachandran plot
  Favoured (%) 92.0
  Outliers (%) 0.6
MolProbity score [percentile] 1.98 [99th]
Twin operator/twin fraction (%) h, −k, −l/45
Pseudotranslation operator/fraction (%) 0.060, −0.500, 0.417/38
†The mean I/σ(I) in the outermost shell falls below 2.0 at 3.3 Å for crystal form 1 and 2.7 Å for crystal form 2.

2.4. Small-angle X-ray scattering

Synchrotron SAXS data were collected on the EMBL/DESY BioSAXS beamline P12 (Blanchet et al., 2015[Blanchet, C. E., Spilotros, A., Schwemmer, F., Graewert, M. A., Kikhney, A., Jeffries, C. M., Franke, D., Mark, D., Zengerle, R., Cipriani, F., Fiedler, S., Roessle, M. & Svergun, D. I. (2015). J. Appl. Cryst. 48, 431-443.]). The protein was at 1.6–6.5 mg ml−1 in 20 mM HEPES pH 7.4, 200 mM NaCl. The scattering data were processed and analyzed with programs from the ATSAS package (Konarev et al., 2006[Konarev, P. V., Petoukhov, M. V., Volkov, V. V. & Svergun, D. I. (2006). J. Appl. Cryst. 39, 277-286.]). The molecular weight was determined by comparison of the forward scattering intensity, I(0), with a fresh monomeric bovine serum albumin standard. Models of MmGADL1 were built with GASBOR (Svergun et al., 2001[Svergun, D. I., Petoukhov, M. V. & Koch, M. H. J. (2001). Biophys. J. 80, 2946-2953.]) and SREFLEX (Panjkovich & Svergun, 2016[Panjkovich, A. & Svergun, D. I. (2016). Phys. Chem. Chem. Phys. 18, 5707-5719.]), using data extrapolated to zero concentration. Theoretical scattering curves from crystal structure coordinates were calculated with CRYSOL (Svergun et al., 1995[Svergun, D., Barberato, C. & Koch, M. H. J. (1995). J. Appl. Cryst. 28, 768-773.]). Model superposition was performed using SUPCOMB (Kozin & Svergun, 2001[Kozin, M. B. & Svergun, D. I. (2001). J. Appl. Cryst. 34, 33-41.]). Details of SAXS data collection, processing and analysis are given in Table 3[link], and the raw SAXS scattering data are provided as Supporting Information.

Table 3
Small-angle X-ray scattering

Data-collection parameters
 Protein MmGADL1, His-tagged
 Instrument P12, PETRA III, EMBL/DESY with Dectris PILATUS 2M detector (Blanchet et al., 2015[Blanchet, C. E., Spilotros, A., Schwemmer, F., Graewert, M. A., Kikhney, A., Jeffries, C. M., Franke, D., Mark, D., Zengerle, R., Cipriani, F., Fiedler, S., Roessle, M. & Svergun, D. I. (2015). J. Appl. Cryst. 48, 431-443.])
 Wavelength (nm) 0.124
 Beam size (µm) 200 × 120
 Detector distance (m) 3.1
 Angular range (nm−1) 0.018–4.607
 Exposure time per frame (s) 0.045
 No. of frames per sample 20
 Monitoring for radiation damage Data frame-by-frame comparison
 Scaling method Buffer-subtracted data normalized to 1 mg ml−1
 Normalization To transmitted intensity by beamstop counter (Blanchet et al., 2015[Blanchet, C. E., Spilotros, A., Schwemmer, F., Graewert, M. A., Kikhney, A., Jeffries, C. M., Franke, D., Mark, D., Zengerle, R., Cipriani, F., Fiedler, S., Roessle, M. & Svergun, D. I. (2015). J. Appl. Cryst. 48, 431-443.])
 Concentration range (mg ml−1) 1.6, 3.3, 6.5
 Temperature (K) 293
Structural parameters  
Rg from crystal structure (nm) 2.95
Dmax from crystal structure (nm) 10.67
 Guinier analysis
  I(0) from Guinier (relative) 2519
  Rg from Guinier (nm) 3.62
  smin (nm−1) 0.143
sRg range 0.52–1.30
 Fidelity 0.92
p(r) analysis
  I(0) from p(r) (relative) 2557
  Rg from p(r) (nm) 3.80
  Dmax (nm) 13.67
s range (nm−1) 0.143–1.98
 Quality of fit (total estimate from GNOM) 0.83
 Porod volume (nm3) (ratio to calculated molecular mass in kDa) 196 (1.6)
Molecular-mass determination
 Molecular mass from I(0) using p(r) (kDa) (ratio to theoretical monomer) 121.0 (2.0)
 Molecular mass from I(0) using Guinier (kDa) (ratio to theoretical monomer) 120.6 (2.0)
 Theoretical monomeric molecular mass from sequence (kDa) 60.4
Shape and atomistic modelling
CRYSOL (comparison to crystal structure)
  χ2 value versus crystal structure 41.9
  s range (nm−1) 0.018–4.607
GASBOR (ab initio chain-like modelling)
  χ2 value 1.7
  s range (nm−1) 0.143–4.607
  Symmetry P2
SREFLEX (modelling of flexibility based on crystal structure)
  χ2 value 5.7
  s range (nm−1) 0.018–4.607
Software
 Data processing and basic analyses SASFLOW (Franke et al., 2012[Franke, D., Kikhney, A. G. & Svergun, D. I. (2012). Nucl. Instrum. Methods Phys. Res. A, 689, 52-59.]; Blanchet et al., 2015[Blanchet, C. E., Spilotros, A., Schwemmer, F., Graewert, M. A., Kikhney, A., Jeffries, C. M., Franke, D., Mark, D., Zengerle, R., Cipriani, F., Fiedler, S., Roessle, M. & Svergun, D. I. (2015). J. Appl. Cryst. 48, 431-443.]) and PRIMUSqt (Petoukhov et al., 2012[Petoukhov, M. V., Franke, D., Shkumatov, A. V., Tria, G., Kikhney, A. G., Gajda, M., Gorba, C., Mertens, H. D. T., Konarev, P. V. & Svergun, D. I. (2012). J. Appl. Cryst. 45, 342-350.])
 Distance distribution analysis GNOM (Svergun, 1992[Svergun, D. I. (1992). J. Appl. Cryst. 25, 495-503.]) through PRIMUSqt (Petoukhov et al., 2012[Petoukhov, M. V., Franke, D., Shkumatov, A. V., Tria, G., Kikhney, A. G., Gajda, M., Gorba, C., Mertens, H. D. T., Konarev, P. V. & Svergun, D. I. (2012). J. Appl. Cryst. 45, 342-350.])
Ab initio analysis GASBOR (Svergun et al., 2001[Svergun, D. I., Petoukhov, M. V. & Koch, M. H. J. (2001). Biophys. J. 80, 2946-2953.]) via ATSAS online (https://www.embl-hamburg.de/biosaxs/atsas-online/)
 Conformational analysis SREFLEX (Panjkovich & Svergun, 2016[Panjkovich, A. & Svergun, D. I. (2016). Phys. Chem. Chem. Phys. 18, 5707-5719.]) via ATSAS online (https://www.embl-hamburg.de/biosaxs/atsas-online/)
 Comparison to crystal structure CRYSOL (Svergun et al., 1995[Svergun, D., Barberato, C. & Koch, M. H. J. (1995). J. Appl. Cryst. 28, 768-773.])
 Graphics representation PyMOL (http://www.pymol.org)
 Extinction coefficient estimate ProtParam (Gasteiger et al., 2005[Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D. & Bairoch, A. (2005). The Proteomics Protocols Handbook, edited by J. M. Walker, pp. 571-607. Totowa: Humana Press.])

2.5. Sequence and structure analysis

APBS (Unni et al., 2011[Unni, S., Huang, Y., Hanson, R., Tobias, M., Krishnan, S., Li, W. W., Nielsen, J. E. & Baker, N. A. (2011). J. Comput. Chem. 32, 1488-1491.]), UCSF Chimera (Pettersen et al., 2004[Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C. & Ferrin, T. E. (2004). J. Comput. Chem. 25, 1605-1612.]), PyMOL (http://www.pymol.org), ProtParam (Gasteiger et al., 2005[Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D. & Bairoch, A. (2005). The Proteomics Protocols Handbook, edited by J. M. Walker, pp. 571-607. Totowa: Humana Press.]), PDBeFold (Krissinel & Henrick, 2004[Krissinel, E. & Henrick, K. (2004). Acta Cryst. D60, 2256-2268.]), MUSCLE (Edgar, 2004[Edgar, R. C. (2004). Nucleic Acids Res. 32, 1792-1797.]) and ESPript3.0 (Robert & Gouet, 2014[Robert, X. & Gouet, P. (2014). Nucleic Acids Res. 42, W320-W324.]) were used for bioinformatics and structure analyses.

3. Results and discussion

3.1. The crystal structure of MmGADL1

Initial screening for crystallization conditions of MmGADL1 resulted in a single condition that produced diffracting crystals. Crystals formed with a needle morphology, often growing in bunches or clusters and generally being very thin, with maximum lengths reaching 500 µm. The diffraction quality was initially poor, with diffraction limits of around 6–7 Å and highly smeared reflections. By optimizing the size, the amount of nucleation, and the general appearance of the crystals, the conditions given in Table 1[link] produced thin but nonfragile crystals that allowed structure refinement in two crystal forms to resolutions of 2.4 and 3.0 Å; the higher resolution data set was plagued by significant twinning and pseudotranslation (Table 2[link]). Thus, the structure of the nontwinned crystal form is discussed here; essentially all features can also be seen in the twinned crystal.

The crystal structure revealed a single MmGADL1 homodimer in the asymmetric unit, which was the expected oligomeric state based on other PLP-dependent decarboxylases (Fig. 1[link]a). For both chains, residues 11–502 could be built, with the flexible loop common to PLP-dependent decarboxylases (Fenalti et al., 2007[Fenalti, G. et al. (2007). Nature Struct. Mol. Biol. 14, 280-286.]) being absent from the electron density (approximately residues 340–350). The overall conformation of the two chains was nearly identical (Fig. 1[link]b).

[Figure 1]
Figure 1
Overall structure of MmGADL1. (a) The MmGADL1 dimer. The PLP molecule covalently bound to Lys314 is shown in magenta. The green dashed line indicates the position of the flexible loop covering the active site. (b) Superposition of the two MmGADL1 monomers in the homodimer. The covalently bound PLP is shown in magenta. The two monomers are essentially identical. (c) Crystal lattice arrangement of MmGADL1 in two different planes. Note the uniform 11 Å cavities in the xy plane between protein layers. (d) Omit FoFc difference map (blue mesh) contoured at 2σ for the covalently bound PLP cofactor on Lys314.

In the crystal lattice, the protein dimers form layers in the xy plane, separated by a rather uniform spacing (Fig. 1[link]c). As the first ∼30 amino acids of the tagged protein were not visible, they are most likely to occupy the space between protein dimers in the crystal. This is the likely source of the high mosaicity and incomplete lattice arrangement in the current MmGADL1 crystals.

Both active sites in the dimer are occupied by the PLP cofactor, which is covalently bound to the N atom of Lys314 in each chain through a Schiff base linkage, being located at the dimer interface (Fig. 1[link]d). Closer observation of the active-site cavity reveals that only the active imine of the linked PLP is solvent-exposed, and it resides at the end of a narrow cavity, which represents the substrate-binding pocket (Fig. 2[link]a). Electrostatic surface analysis reveals the GADL1 active site to have a high positive charge potential (Fig. 2[link]b). This is logical, as the substrates of GADL1 are acidic amino acids; the basic nature of the binding cavity is involved in electrostatic interactions that attract and bind the substrate, orienting it correctly for catalysis.

[Figure 2]
Figure 2
The MmGADL1 active site. (a) Close-up view of the active-site cavity with the reactive moiety of PLP (magenta) visible (red arrow). Note how the cofactor lies at the interface between two protein monomers (blue and grey). (b) Surface electrostatics of MmGADL1. The positively charged cavity corresponds to the active site (blue).

In our earlier work, we showed that GADL1 can use aspartate and CSA as substrates, but not glutamate (Winge et al., 2015[Winge, I., Teigen, K., Fossbakk, A., Mahootchi, E., Kleppe, R., Sköldberg, F., Kämpe, O. & Haavik, J. (2015). Neurochem. Int. 90, 173-184.]). While the catalytic cavities of GAD and GADL1 are very similar, it is currently difficult to pinpoint which features of the active site might be responsible for selectivity between such similar substrates. High-resolution structures of GADL1 and its closest homologues with bound active-site ligands will clearly be required. Importantly, a large part of the active-site cavity wall will be formed by the flexible loop in the substrate-bound state; the flexible loop is invisible in most PLP-dependent decarboxylase crystal structures, but harbours a Tyr residue that is likely to be important for catalysis.

3.2. MmGADL1 adopts an open conformation in solution

We used SAXS to validate the crystal structure and to determine the conformation of MmGADL1 in solution (Fig. 3[link], Table 3[link]). As is apparent from the scattering data and the dimensionless Kratky plot, GADL1 presents a folded shape. While the molecular mass calculated from the forward scattering intensity accurately matches that of a dimer, the theoretical scattering curve calculated from the crystal structure differs significantly. The shape in solution is more elongated than in the crystal. The radii of gyration between the theoretical scattering curve from the crystal structure and the experimental value from Guinier analysis differ by nearly 1 nm, indicating a large difference in conformation. The maximum diameter is 3 nm larger in solution than in the crystal state.

[Figure 3]
Figure 3
Structure of MmGADL1 in solution determined by SAXS. (a) Raw SAXS data (dots) overlaid with GASBOR (pink) and SREFLEX (green) model fits, as well as the theoretical scattering curve calculated from the crystal structure using CRYSOL (red). (b) Guinier plot. (c) The dimensionless Kratky plot indicates that MmGADL1 is folded, with limited flexibility. The red dot indicates the theoretical position of the peak in a folded globular protein. (d) Distance distribution of MmGADL1. (e) The GASBOR model (orange) superimposed with the crystal structure of MmGADL1 indicates a much more elongated conformation in solution. (f) Superposition of the SREFLEX (blue/grey) and GASBOR (orange) models suggests conformational changes relative to the crystal structure. (g) Comparison of the `open' conformation of DDC (orange/yellow; Giardina et al., 2011[Giardina, G., Montioli, R., Gianni, S., Cellini, B., Paiardini, A., Voltattorni, C. B. & Cutruzzolà, F. (2011). Proc. Natl Acad. Sci. USA, 108, 20514-20519.]) and the open conformation of MmGADL1 (blue/grey) seen here in solution.

The GASBOR chain-like dummy residue model of MmGADL1 is elongated compared with the crystal structure (Fig. 3[link]e). Attempts to model the missing N-terminal portion, while keeping the dimeric crystal structure fixed, did not fit the experimental SAXS data well (data not shown). We thus employed the recently described SREFLEX method (Panjkovich & Svergun, 2016[Panjkovich, A. & Svergun, D. I. (2016). Phys. Chem. Chem. Phys. 18, 5707-5719.]) to take advantage of normal-mode analysis of the crystal structure in SAXS modelling. The SREFLEX model fits the scattering data well and suggests a clearly loosened solution conformation (Fig. 3[link]f), in contrast to the compact globular structures observed for PLP-dependent decarboxylases in the crystalline state. The open conformation is not similar to the open conformation observed for DOPA decarboxylase in the crystalline state (Giardina et al., 2011[Giardina, G., Montioli, R., Gianni, S., Cellini, B., Paiardini, A., Voltattorni, C. B. & Cutruzzolà, F. (2011). Proc. Natl Acad. Sci. USA, 108, 20514-20519.]; Fig. 3[link]g), in which case the opening occurs in the centre of the dimer. The observed open–close movement is likely to be of functional relevance in the GADL1 catalytic cycle. Whether the different conformations are linked to the binding of ligands remains to be studied. While our GADL1 preparation is enzymatically active (Winge et al., 2015[Winge, I., Teigen, K., Fossbakk, A., Mahootchi, E., Kleppe, R., Sköldberg, F., Kämpe, O. & Haavik, J. (2015). Neurochem. Int. 90, 173-184.]), and the crystal is apparently fully occupied with covalently bound PLP, we cannot currently rule out the presence of PLP-deficient GADL1 in the purified SAXS sample, since crystallization might have enriched a cofactor-bound conformation of the protein.

3.3. Comparison to other PLP-dependent decarboxylases

While MmGADL1 and its homologues show low sequence conservation, apart from a few fully conserved core elements around the active site (Fig. 4[link]a), comparison of the structures of MmGADL1 and its homologues reveals a conserved structural fold with little variance in the arrangement of the PLP-linked Lys residue (Figs. 4[link]b and 4[link]c, Table 4[link]). The sequence conservation between MmGADL1, HsCSAD and HsGADs (Fenalti et al., 2007[Fenalti, G. et al. (2007). Nature Struct. Mol. Biol. 14, 280-286.]) is higher than those with human histidine decarboxylase (HDC; Komori et al., 2012[Komori, H., Nitta, Y., Ueno, H. & Higuchi, Y. (2012). J. Biol. Chem. 287, 29175-29183.]) and DOPA decarboxylase (DDC; Giardina et al., 2011[Giardina, G., Montioli, R., Gianni, S., Cellini, B., Paiardini, A., Voltattorni, C. B. & Cutruzzolà, F. (2011). Proc. Natl Acad. Sci. USA, 108, 20514-20519.]; Winge et al., 2015[Winge, I., Teigen, K., Fossbakk, A., Mahootchi, E., Kleppe, R., Sköldberg, F., Kämpe, O. & Haavik, J. (2015). Neurochem. Int. 90, 173-184.]). The latter present similar levels of sequence homology to GADL1 as the bacterial enzymes Sphaerobacter thermophilus PLP-dependent decarboxylase (StPDD), Vibrio parahaemolyticus GAD (VpGAD) and the tryptamine-synthesizing enzyme from the gut bacterium Ruminococcus gnavus (RUMGNA_01526; Williams et al., 2014[Williams, B. B., Van Benschoten, A. H., Cimermancic, P., Donia, M. S., Zimmermann, M., Taketani, M., Ishihara, A., Kashyap, P. C., Fraser, J. S. & Fischbach, M. A. (2014). Cell Host Microbe, 16, 495-503.]). Despite low sequence homology, the R. gnavus enzyme displays high structural similarity to GADL1, suggesting conservation of the fold of PLP-dependent decarboxylases involved in neurotransmitter synthesis. It is interesting to note that the absence of PLP in the active site neither alters the overall tightness of the superposed proteins nor changes the position of the conserved Lys in most structures. In the future, experimental solution-state methods, such as SAXS, may help to distinguish functionally relevant conformational states from possible crystallographic artifacts. These observations conflict with earlier results described for HsDDC in the beginning of its PLP accumulation-dependent conformational landscape, where a more open conformation was evident in the crystalline state with the active-site Lys residue oriented away from the PLP-binding pocket (Giardina et al., 2011[Giardina, G., Montioli, R., Gianni, S., Cellini, B., Paiardini, A., Voltattorni, C. B. & Cutruzzolà, F. (2011). Proc. Natl Acad. Sci. USA, 108, 20514-20519.]).

Table 4
Structural homologues of MmGADL1

The homologues were detected by an SSM analysis using PDBeFold.

Protein Organism PDB entry Reference Chain Q-score R.m.s.d. (Å) Sequence identity (%) UniProtKB entry Aligned residues
MmGADL1 Mus musculus 6enz This study A E9QP13 1–502
HsCSAD Homo sapiens 2jis Unpublished work B 0.586 0.85 62.0 Q9Y600 1–493
HsGAD65 Homo sapiens 2okk Fenalti et al. (2007[Fenalti, G. et al. (2007). Nature Struct. Mol. Biol. 14, 280-286.]) A 0.565 1.06 49.4 Q05329 88–584
HsGAD67 Homo sapiens 2okj Fenalti et al. (2007[Fenalti, G. et al. (2007). Nature Struct. Mol. Biol. 14, 280-286.]) B 0.532 1.15 50.9 Q9925 93–594
StPDD Sphaerobacter thermophilus 4rit Unpublished work B 0.461 1.96 28.9 D1C7D8 1–483
RUMGNA_01526 Ruminococcus gnavus 4obu Williams et al. (2014[Williams, B. B., Van Benschoten, A. H., Cimermancic, P., Donia, M. S., Zimmermann, M., Taketani, M., Ishihara, A., Kashyap, P. C., Fraser, J. S. & Fischbach, M. A. (2014). Cell Host Microbe, 16, 495-503.]) H 0.492 1.79 25.6 A7B1V0 1–490
VpGAD Vibrio parahaemolyticus 2qma Unpublished work B 0.442 2.16 25.8 Q87NC6 464–957
HsHDC Homo sapiens 4e1o Komori et al. (2012[Komori, H., Nitta, Y., Ueno, H. & Higuchi, Y. (2012). J. Biol. Chem. 287, 29175-29183.]) C 0.425 2.13 26.1 P19113 2–477
HsDDC Homo sapiens 3rbl Giardina et al. (2011[Giardina, G., Montioli, R., Gianni, S., Cellini, B., Paiardini, A., Voltattorni, C. B. & Cutruzzolà, F. (2011). Proc. Natl Acad. Sci. USA, 108, 20514-20519.]) A 0.397 2.61 23.3 P20711 1–480
[Figure 4]
Figure 4
Comparison of MmGADL1 with other PLP-dependent decarboxylases. (a) Sequence alignment of MmGADL1 with homologous structures. The conserved PLP-modified lysine is indicated (green triangle). Secondary structure elements and sequence numbering correspond to MmGADL1. (b) Stereo image of a structural superposition of PLP-dependent decarboxylase homologues, viewed towards the active-site cavity. The covalently linked MmGADL1 PLP moiety is depicted as red spheres. The black arrow shows the position of the active-site-covering loop, which is disordered in the MmGADL1 crystal structure. (c) Conservation of PLP conformation in the superposed PLP-dependent decarboxylase structures.

The substrate specificity and physiological function of GADL1 remain enigmatic. However, the conserved structural details and distinct expression patterns of GADL1 suggest that it plays a specific physiological role. Variants of GADL1 have been linked to lithium response in bipolar disorder patients (Chen et al., 2014[Chen, C.-H. et al. (2014). N. Engl. J. Med. 370, 119-128.]), suggesting a role for GADL1 in lithium pharmacodynamics or kinetics. However, these findings have not been replicated by others, and they have been subject to much controversy (Birnbaum et al., 2014[Birnbaum, R., Shin, J. H. & Weinberger, D. (2014). N. Engl. J. Med. 370, 1855-1856.]; Cruceanu et al., 2015[Cruceanu, C., Alda, M., Dion, P. A., Turecki, G. & Rouleau, G. A. (2015). Am. J. Psychiatry, 172, 94-95.]; Kotambail et al., 2015[Kotambail, A., Mathur, A., Bhat, S. M., Rai, P. S., Sharma, P. S. & Satyamoorthy, K. (2015). Psychiatr. Genet. 25, 39-40.]; Winge et al., 2015[Winge, I., Teigen, K., Fossbakk, A., Mahootchi, E., Kleppe, R., Sköldberg, F., Kämpe, O. & Haavik, J. (2015). Neurochem. Int. 90, 173-184.]; Chen et al., 2016[Chen, C.-K., Lee, C.-S., Chen, H.-Y., Wu, L. S.-H., Chang, J.-C., Liu, C.-Y. & Cheng, A. T.-A. (2016). BJPsych Open, 2, 301-306.]).

Owing to their common mechanistic features, many PLP-dependent enzymes are able to catalyze multiple biochemical reactions, making it difficult to define their primary biological function (Percudani & Peracchi, 2003[Percudani, R. & Peracchi, A. (2003). EMBO Rep. 4, 850-854.]). Of the known GADL1 substrates, Asp appears as the most characteristic substrate for GADL1 (Winge et al., 2015[Winge, I., Teigen, K., Fossbakk, A., Mahootchi, E., Kleppe, R., Sköldberg, F., Kämpe, O. & Haavik, J. (2015). Neurochem. Int. 90, 173-184.]), although the Km is relatively high and the catalytic efficiency is low. Nevertheless, the Km of GADL1 for Asp is in the physiological range, and one could speculate that GADL1 is involved in sensing selected metabolite levels. Relatively low binding affinity is a hallmark of many proteins with signalling roles, which have evolved as sensors of ligand availability; such proteins include, for example, the calcium sensors calmodulin and annexin (Kursula, 2014[Kursula, P. (2014). Amino Acids, 46, 2295-2304.]; Monastyrskaya et al., 2007[Monastyrskaya, K., Babiychuk, E. B., Hostettler, A., Rescher, U. & Draeger, A. (2007). Cell Calcium, 41, 207-219.]). Conformational flexibility, as observed here for GADL1 in solution, might have relevance in such a function.

4. Concluding remarks

The physiological functions and enzymatic properties of GADL1 are subject to further studies. The structure of MmGADL1 and its flexibility in solution, coupled to structural conservation with other PLP-dependent enzymes, point towards functional relevance of these features within the enzyme family. Important future work will concentrate on high-resolution structural details of substrate and inhibitor binding by GADL1, and on comparing these with those of CSAD, GAD and other PLP-dependent decarboxylases. Crucial topics to address will include the fine details of substrate specificity determinants in PLP-dependent decarboxylases, as well as the relevance of the conformational changes observed here to the catalytic cycle of this family of enzymes.

Supporting information


Footnotes

These authors made equal contributions.

Acknowledgements

We gratefully acknowledge access to the synchrotron-radiation facilities and the outstanding beamline support at EMBL/DESY, Hamburg, Germany.

Funding information

Funding for this research was provided by: Helse Vest (grant to Jan Haavik); Norges Forskningsråd (grant to Petri Kursula); Sigrid Juséliuksen Säätiö (grant to Petri Kursula); Stiftelsen Kristian Gerhard Jebsen (grant to Jan Haavik); Seventh Framework Programme (grant No. 602805 to Jan Haavik).

References

First citationAfonine, P. V., Grosse-Kunstleve, R. W., Echols, N., Headd, J. J., Moriarty, N. W., Mustyakimov, M., Terwilliger, T. C., Urzhumtsev, A., Zwart, P. H. & Adams, P. D. (2012). Acta Cryst. D68, 352–367.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationBaekkeskov, S., Aanstoot, H.-J., Christgau, S., Reetz, A., Solimena, M., Cascalho, M., Folli, F., Richter-Olesen, H. & Camilli, P.-D. (1990). Nature (London), 347, 151–156.  CrossRef PubMed CAS Google Scholar
First citationBirnbaum, R., Shin, J. H. & Weinberger, D. (2014). N. Engl. J. Med. 370, 1855–1856.  CAS PubMed Google Scholar
First citationBlanchet, C. E., Spilotros, A., Schwemmer, F., Graewert, M. A., Kikhney, A., Jeffries, C. M., Franke, D., Mark, D., Zengerle, R., Cipriani, F., Fiedler, S., Roessle, M. & Svergun, D. I. (2015). J. Appl. Cryst. 48, 431–443.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationChen, C.-H. et al. (2014). N. Engl. J. Med. 370, 119–128.  CrossRef CAS PubMed Google Scholar
First citationChen, C.-K., Lee, C.-S., Chen, H.-Y., Wu, L. S.-H., Chang, J.-C., Liu, C.-Y. & Cheng, A. T.-A. (2016). BJPsych Open, 2, 301–306.  PubMed Google Scholar
First citationChen, V. B., Arendall, W. B., Headd, J. J., Keedy, D. A., Immormino, R. M., Kapral, G. J., Murray, L. W., Richardson, J. S. & Richardson, D. C. (2010). Acta Cryst. D66, 12–21.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationCruceanu, C., Alda, M., Dion, P. A., Turecki, G. & Rouleau, G. A. (2015). Am. J. Psychiatry, 172, 94–95.  CrossRef PubMed Google Scholar
First citationEdgar, R. C. (2004). Nucleic Acids Res. 32, 1792–1797.  Web of Science CrossRef PubMed CAS Google Scholar
First citationEliot, A. & Kirsch, J. (2004). Annu. Rev. Biochem. 73, 383–415.  CrossRef PubMed CAS Google Scholar
First citationEl-Sayed, A. S. & Shindia, A. A. (2011). Targets in Gene Therapy, edited by Y. You, ch. 7. Rijeka: Intech. https://doi.org/10.5772/17449Google Scholar
First citationEmsley, P. & Cowtan, K. (2004). Acta Cryst. D60, 2126–2132.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationFenalti, G. et al. (2007). Nature Struct. Mol. Biol. 14, 280–286.  Web of Science CrossRef CAS Google Scholar
First citationFranke, D., Kikhney, A. G. & Svergun, D. I. (2012). Nucl. Instrum. Methods Phys. Res. A, 689, 52–59.  Web of Science CrossRef CAS Google Scholar
First citationGasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M. R., Appel, R. D. & Bairoch, A. (2005). The Proteomics Protocols Handbook, edited by J. M. Walker, pp. 571–607. Totowa: Humana Press.  Google Scholar
First citationGiardina, G., Montioli, R., Gianni, S., Cellini, B., Paiardini, A., Voltattorni, C. B. & Cutruzzolà, F. (2011). Proc. Natl Acad. Sci. USA, 108, 20514–20519.  CrossRef CAS PubMed Google Scholar
First citationHammarström, M., Woestenenk, E., Hellgren, N., Härd, T. & Berglund, H. (2006). J. Struct. Funct. Genomics, 7, 1–14.  PubMed Google Scholar
First citationKarplus, P. A. & Diederichs, K. (2015). Curr. Opin. Struct. Biol. 34, 60–68.  Web of Science CrossRef CAS PubMed Google Scholar
First citationKomori, H., Nitta, Y., Ueno, H. & Higuchi, Y. (2012). J. Biol. Chem. 287, 29175–29183.  CrossRef CAS PubMed Google Scholar
First citationKonarev, P. V., Petoukhov, M. V., Volkov, V. V. & Svergun, D. I. (2006). J. Appl. Cryst. 39, 277–286.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationKotambail, A., Mathur, A., Bhat, S. M., Rai, P. S., Sharma, P. S. & Satyamoorthy, K. (2015). Psychiatr. Genet. 25, 39–40.  CrossRef PubMed Google Scholar
First citationKozin, M. B. & Svergun, D. I. (2001). J. Appl. Cryst. 34, 33–41.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationKrissinel, E. & Henrick, K. (2004). Acta Cryst. D60, 2256–2268.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationKursula, P. (2014). Amino Acids, 46, 2295–2304.  CrossRef CAS PubMed Google Scholar
First citationLiu, P., Ge, X., Ding, H., Jiang, H., Christensen, B. M. & Li, J. (2012). J. Biol. Chem. 287, 40898–40906.  CrossRef CAS PubMed Google Scholar
First citationMcCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni, L. C. & Read, R. J. (2007). J. Appl. Cryst. 40, 658–674.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationMin, J., Senut, M., Rajanikant, K., Greenberg, E., Bandagi, R., Zemke, D., Mousa, A., Kassab, M., Farooq, M. U., Gupta, R. & Majid, A. (2008). J. Neurosci. Res. 86, 2984–2991.  CrossRef PubMed CAS Google Scholar
First citationMonastyrskaya, K., Babiychuk, E. B., Hostettler, A., Rescher, U. & Draeger, A. (2007). Cell Calcium, 41, 207–219.  CrossRef PubMed CAS Google Scholar
First citationPaiardini, A., Contestabile, R., Buckle, A. M. & Cellini, B. (2014). Biomed. Res. Int. 2014, 856076.  CrossRef PubMed Google Scholar
First citationPanjkovich, A. & Svergun, D. I. (2016). Phys. Chem. Chem. Phys. 18, 5707–5719.  Web of Science CrossRef CAS PubMed Google Scholar
First citationPark, H., Han, K., Shin, J., Park, J., Song, K. & Kim, D. (2014). J. Korean. Neurosurg. Soc. 55, 125–130.  CrossRef PubMed Google Scholar
First citationPercudani, R. & Peracchi, A. (2003). EMBO Rep. 4, 850–854.  Web of Science CrossRef PubMed CAS Google Scholar
First citationPetoukhov, M. V., Franke, D., Shkumatov, A. V., Tria, G., Kikhney, A. G., Gajda, M., Gorba, C., Mertens, H. D. T., Konarev, P. V. & Svergun, D. I. (2012). J. Appl. Cryst. 45, 342–350.  Web of Science CrossRef CAS IUCr Journals Google Scholar
First citationPettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C. & Ferrin, T. E. (2004). J. Comput. Chem. 25, 1605–1612.  Web of Science CrossRef PubMed CAS Google Scholar
First citationRobert, X. & Gouet, P. (2014). Nucleic Acids Res. 42, W320–W324.  Web of Science CrossRef CAS PubMed Google Scholar
First citationSköldberg, F., Rorsman, F., Perheentupa, J., Landin-Olsson, M., Husebye, E., Gustafsson, J. & Kämpe, O. (2004). J. Clin. Endocrinol. Metab. 89, 1636–1640.  PubMed Google Scholar
First citationSvergun, D. I. (1992). J. Appl. Cryst. 25, 495–503.  CrossRef CAS Web of Science IUCr Journals Google Scholar
First citationSvergun, D., Barberato, C. & Koch, M. H. J. (1995). J. Appl. Cryst. 28, 768–773.  CrossRef CAS Web of Science IUCr Journals Google Scholar
First citationSvergun, D. I., Petoukhov, M. V. & Koch, M. H. J. (2001). Biophys. J. 80, 2946–2953.  Web of Science CrossRef PubMed CAS Google Scholar
First citationUnni, S., Huang, Y., Hanson, R., Tobias, M., Krishnan, S., Li, W. W., Nielsen, J. E. & Baker, N. A. (2011). J. Comput. Chem. 32, 1488–1491.  CrossRef CAS PubMed Google Scholar
First citationWilliams, B. B., Van Benschoten, A. H., Cimermancic, P., Donia, M. S., Zimmermann, M., Taketani, M., Ishihara, A., Kashyap, P. C., Fraser, J. S. & Fischbach, M. A. (2014). Cell Host Microbe, 16, 495–503.  Web of Science CrossRef CAS PubMed Google Scholar
First citationWinge, I., Teigen, K., Fossbakk, A., Mahootchi, E., Kleppe, R., Sköldberg, F., Kämpe, O. & Haavik, J. (2015). Neurochem. Int. 90, 173–184.  CrossRef CAS PubMed Google Scholar

This is an open-access article distributed under the terms of the Creative Commons Attribution (CC-BY) Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.

Journal logoSTRUCTURAL BIOLOGY
COMMUNICATIONS
ISSN: 2053-230X
Follow Acta Cryst. F
Sign up for e-alerts
Follow Acta Cryst. on Twitter
Follow us on facebook
Sign up for RSS feeds