Expression, crystallization and preliminary crystallographic study of GluB from Corynebacterium glutamicum

GluB, a substrate-binding protein from C. glutamicum, was expressed, purified and crystallized, followed by X-ray diffraction data collection and preliminary crystallographic analysis.

Abstract

GluB is a substrate-binding protein (SBP) which participates in the uptake of glutamic acid in Corynebacterium glutamicum, a Gram-positive bacterium. It is part of an ATP-binding cassette (ABC) transporter system. Together with the transmembrane proteins GluC and GluD and the cytoplasmic protein GluA, which couples the hydrolysis of ATP to the translocation of glutamate, they form a highly active glutamate-uptake system. As part of efforts to study the amino-acid metabolism, especially the metabolism of glutamic acid by C. glutamicum, a bacterium that is widely used in the industrial production of glutamic acid, the GluB protein was expressed, purified and crystallized, an X-ray diffraction data set was collected to a resolution of 1.9 Å and preliminary crystallographic analysis was performed. The crystal belonged to space group P3₁21 or P3₂21, with unit-cell parameters a = b = 82.50, c = 72.69 Å.

1. Introduction  

Substrate-binding proteins (SBPs) are part of the ATP-binding cassette transporters (ABC transporters) that couple ATP hydrolysis with signal transduction or active transmembrane transport of a diverse array of substrates (Berntsson et al., 2010 ▶). SBPs usually reside in the periplasmic space of Gram-negative bacteria, where they bind their specific substrates and hand them over to their cognate transmembrane transporters. Gram-positive bacteria, on the other hand, lack the outer membrane and consequently have no periplasmic space where the SBPs could be retained. As a result, SBPs in Gram-positive bacteria are either attached to the cytoplasmic membrane or fused to membrane-anchored domains of ABC importers (Berntsson et al., 2010 ▶).

Previous studies reveal that SBPs share low sequence similarities with each other, yet their structural folds are highly conserved. They usually contain two similarly folded domains connected by a two- or three-stranded β-sheet hinge (Quiocho & Ledvina, 1996 ▶). The substrate-binding sites are located between these two domains. Among SBPs with known structures, the Campylobacter jejuni PEB1a (PDB entry 2v25; Müller et al., 2007 ▶), an aspartate and glutamate receptor, is the most closely related to GluB, with 30% sequence identity.

GluB contains a signal sequence followed by a putative target sequence of signal peptidase II; it is thus likely to be anchored to the membrane as lipoprotein (Kronemeyer et al., 1995 ▶).

The gluB gene shares an operon with gluA, gluC and gluD which encode the ABC transporter. GluA was characterized as the nucleotide-binding protein, whereas GluC and GluD are integral membrane proteins (Kronemeyer et al., 1995 ▶). Based on the organ­ization of the gluABCD operon, it should belong to the class 3 ABC transporter system which contains most SBP-dependent importers with integral membrane and ABC domains as separate polypeptide chains (Davidson et al., 2008 ▶). GluABCD is part of the amino-acid metabolism of C. glutamicum. It is induced when the cells use glutamate as sole carbon source and is down-regulated in media containing salts and glucose or sucrose as carbon source (Kronemeyer et al., 1995 ▶; Trötschel et al., 2003 ▶).

The bacterium Corynebacterium glutamicum is nonpathogenic, fast growing and has few growth requirements. It thus has seen widespread applications in biotechnology, especially in the production of glutamate and other amino acids. In order to better understand the glutamate metabolism of C. glutamicum, we have purified and crystallized C. glutamicum GluB and performed preliminary crystallographic studies of this protein.

2. Materials and methods  

2.1. Cloning, expression and purification of GluB  

The genes encoding full-length and the N-truncation fragment of GluB were amplified by PCR cloning from the chromosome DNA of C. glutamicum res 167 strain using the following primers: 5′-AGCTGACATATGTCTGCAAAGCGTACTTTTACCCGTATCG-3′ and 5′-GACAGTGCGGCCGCGCTTGCGTCGAGGAAGGAGAGGTC­ACC-3′ for the full-length gene and 5′-AGCTGACATATGGACGGATTCCTCGCAGCCATTGAAAATG-3′ and 5′-GACAGTGCGGCCGCGCTTGCGTCGAGGAAGGAGAGGTCACC-3′ for the N-truncation. The N-terminal truncation fragment of GluB, which has the N-terminal 34 residues removed, was cloned into expression vector pET-22b between the NdeI and NotI restriction sites. A six-histidine tag was engineered at the C-terminus. The recombinant GluB contains residues 35–295 of the full-length sequence preceded by M and followed by AAALEHHHHHH. The recombinant plasmid was transformed into Escherichia coli strain BL21 (DE3). The bacteria were grown in LB medium and induced with 0.2 mM isopropyl β-d-1-thiogalactopyranoside for 12 h at 295 K on reaching an OD₆₀₀ of 0.6. The cells were harvested by centrifugation at 4670g for 30 min at 277 K. After resuspension in lysis buffer (50 mM NaH₂PO₄/Na₂HPO₄ pH 7.5, 300 mM NaCl, 10 mM imidazole), cells were lysed on ice using a sonicator (3 s pulse, 9 s pause; repeated 99 times). The lysate was centrifuged at 21 130g for 30 min at 277 K to remove cell debris. The supernatant was loaded onto Ni–NTA agarose beads (Novagen, Darmstadt, Germany) and washed with washing buffer (50 mM NaH₂PO₄/Na₂HPO₄ pH 7.5, 300 mM NaCl, 10 mM imidazole). The GluB protein was eluted with elution buffer (50 mM NaH₂PO₄/Na₂HPO₄ pH 7.5, 300 mM NaCl, 250 mM imidazole). The protein was then further purified by gel-filtration chromatography using a HiLoad 16/60 Superdex 75 column (GE Healthcare) in a buffer consisting of 20 mM Tris–HCl, 150 mM NaCl. The purified GluB protein was concentrated to 20 mg ml⁻¹ by ultrafiltration (Amicon Ultra-15 Centrifugal Filter, Millipore) prior to crystallization. Protein concentration was determined using Bio-Rad Protein Assay Dye Reagent with a BSA standard curve.

2.2. Crystallization  

Crystallization screening was performed using the hanging-drop vapour-diffusion method with Hampton Research Crystal Screen kits. 1 µl GluB solution (20 mg ml⁻¹) was mixed with 1 µl reservoir solution and the drop was equilibrated against 450 µl reservoir solution at 293 K. The initial condition for crystal growth was further optimized by altering the pH and the concentration of the precipitant. A heavy-atom screen was performed using the Hampton Research Heavy Atom Screen kit. The heavy-atom stock solutions were diluted to 50 or 100 mM and added directly to the hanging drops to a final concentration of 5 or 10 mM, followed by 3 d of soaking.

2.3. Data collection and processing  

The X-ray diffraction data set of the mercury derivative was collected on beamline 3W1A at the Beijing Synchrotron Radiation Facility (BSRF; Beijing, People’s Republic of China) at a wavelength of 1.0090 Å. The crystal was soaked in reservoir solution supplemented with saturated Li₂SO₄ for 15 s before flash-cooling in liquid nitrogen on a nylon loop. The cryoprotectant was prepared by adding a saturating amount of Li₂SO₄ powder to the reservoir solution and removing the extra powder by centrifugation. A total of 360 images were collected with an oscillation of 1° per image. The diffraction data were indexed and integrated with the program MOSFLM (Leslie & Powell, 2007 ▶) and scaled with SCALA (Evans, 2006 ▶) from the CCP4 program suite (Winn et al., 2011 ▶).

3. Results  

The crystals of GluB appear in several conditions of the Hampton Research Index kit (Nos. 3–6) that use 2.0 M ammonium sulfate as precipitant. After optimization, the best crystal was obtained by mixing 1 µl GluB solution (20 mg ml⁻¹) with 1 µl reservoir solution consisting of 0.1 M Tris–HCl pH 7.5, 2.9 M ammonium sulfate. Within 2–3 d, the crystal grew to a size of 0.20 × 0.20 × 0.05 mm (Fig. 1 ▶).

Figure 1.

Crystals of GluB with typical dimensions of 0.20 × 0.20 × 0.05 mm.

Of the known structures of glutamate-binding proteins such as E. coli DEBP (PDB entry 2vha; Hu et al., 2008 ▶) and Campylobacter jejuni PEB1a (PDB entry 2v25; Müller et al., 2007 ▶), the proteins always acquire their cognate ligands from the environment and are purified as complexes even in the absence of added ligands (Müller et al., 2007 ▶; Hu et al., 2008 ▶). The same is true for the SBPs of other amino acids. It is therefore very likely that the GluB samples used in our studies are in the form of complexes with glutamate.

The structures of several glutamate-binding proteins have been solved, but all of them have sequence identities with GluB of 30% or lower. Attempts to solve the structure of GluB by the molecular-replacement method failed, probably owing to the low sequence identity between GluB and the search model. We then carried out a heavy-atom screen. The best derivatized crystals of GluB were obtained using 10 mM mercury acetate solution and were found to be suitable for data collection. Data-collection statistics are listed in Table 1 ▶.

Table 1. Data-collection statistics of the GluB crystal.

Values in parentheses are for the outermost resolution shell.

Space group	P3121 or P3221
Unit-cell parameters (Å)	a = b = 82.50, c = 72.69
Wavelength (Å)	1.0090
Resolution (Å)	19.92–1.90 (2.00–1.90)
No. of reflections	22911
R merge † (%)	8.0 (51.4)
Multiplicity	21.7 (21.7)
Mean I/σ(I)	28.0 (6.3)
Completeness (%)	99.9 (100)

^† R _merge = , where 〈I(hkl)〉 is the mean of the observations I_i(hkl) of reflection hkl.

The mercury acetate derivatized crystal diffracted to a resolution of 1.9 Å (Fig. 2 ▶) and the space group is P3₁21 or P3₂21 with unit-cell parameters of a = b = 82.50, c = 72.69 Å. The asymmetric unit contains one GluB molecule with a calculated Matthews coefficient of 2.38 ų Da⁻¹, corresponding to a solvent content of 48.36% (Matthews, 1968 ▶). The anomalous difference Patterson map calculated with FFT from the CCP4 program suite shows prominent Harker peaks at 15σ (Fig. 3 ▶), indicating that the data set from the mercury-derivatized crystal contains strong anomalous scattering signals and could be used for phase calculations using SAD (single-wavelength anomalous dispersion) or SIRAS (single isomorphous replacement with anomalous signal) techniques. The research reported here provides us with a good foundation for structure determination and further functional studies of GluB.

Figure 2.

Diffraction pattern of C. glutamicum GluB crystal.

Figure 3.

Anomalous difference Patterson map of GluB crystal on Harker section w = 2/3. The two peaks are at 15σ levels.