A proposed carbon-utilization and virulence protein A, CuvA (Rv1422), from Mycobacterium tuberculosis H37Rv: crystallization, X-ray diffraction analysis and ligand binding

This study presents the crystallization, X-ray diffraction analysis and ligand binding of CuvA (Rv1422) from M. tuberculosis H37Rv and reports that MtCuvA can bind to the cell-wall precursor components uridine diphosphate (UDP)-glucose and UDP-N-acetylglucosamine.

Abstract

Mycobacterium tuberculosis possesses the ability to undergo physiological adaptations in order to persist during the prolonged course of infection despite the active immune response of the host and in order to overcome multiple environmental changes. Previous studies have proposed that M. tuberculosis CuvA (Rv1422; MtCuvA) might play a critical role in the adaptation of the bacterium to environmental changes, such as nutrient utilization and alteration of the growth rate. However, the detailed function of MtCuvA still remains unclear owing to a lack of structural information. To better understand its role in host adaptation, MtCuvA was purified to homogeneity and was crystallized for the first time using the hanging-drop vapor-diffusion method. The crystal of MtCuvA diffracted to a resolution of 2.1 Å and belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 47.27, b = 170.93, c = 178.10 Å. The calculated Matthews coefficient (V _M) was 2.4 ų Da⁻¹, with a solvent content of 48.02%, and thus four molecules appeared to be present in the asymmetric unit. Moreover, it is reported that MtCuvA can bind to the cell-wall precursor components uridine diphosphate (UDP)-glucose and UDP-N-acetylglucosamine.

1. Introduction  

Mycobacterium tuberculosis, an intracellular pathogen that causes tuberculosis (TB), remains the leading cause of morbidity worldwide (World Health Organization, 2020 ▸). In particular, M. tuberculosis is able to maintain a persistent asymptomatic (latent) infection in the human host or to cause TB by activating a latent infection, depending on the immune conditions of the host (Kang et al., 2005 ▸). A latent infection may result from major alterations in gene expression and metabolism that allow persistent minimal replication (Manabe & Bishai, 2000 ▸; McKinney et al., 2000 ▸). The mechanism of switching between the active and latent states of M. tuberculosis is still unclear. Thus, to identify the essential factors that are required in this process, it is necessary to gain insight into the bacterial adaptation mechanism with regard to intra­cellular regulation that is used to respond to environmental changes that occur in the host. Moreover, these essential factors involved in bacterial adaptation may be attractive targets for novel therapeutic approaches to TB.

M. tuberculosis CuvA (Rv1422; MtCuvA) is a hypothetical protein that may play an important role in bacterial adaptation during infection in vivo (Mir et al., 2014 ▸). Previous studies have suggested that MtCuvA might be required for the utilization of nutrients and for peptidoglycan synthesis, and may be involved in maintaining cell morphology and bacterial virulence (Mir et al., 2014 ▸). In addition, it has been reported that M. smegmatis and M. tuberculosis strains in which the cuvA gene was deleted showed growth defects under conditions containing several different carbon sources and have morphological abnormalities such as a bulging and shortened phenotype, suggesting a peptidoglycan defect (Mir et al., 2014 ▸). Additionally, recent studies have reported that Bacillus subtilis YvcK (BsYvcK), an orthologue of MtCuvA, may be essential for growth on gluconeogenic carbon sources and may be involved in the regulation of cell-wall synthesis, including cell morphology and antibiotic sensitivity (Patel et al., 2018 ▸; Foulquier & Galinier, 2017 ▸). The study by Foulquier and Galinier also showed that BsYvcK is a uridine diphosphate (UDP)-sugar-binding protein and that deletion of the gene affects the cell size. However, the molecular mechanism through which the deletion of these genes (cuvA or yvcK) influences cell-wall synthesis and cell morphology is still unknown owing to a lack of structural information.

To date, only the crystal structure of YvcK (PDB entry 2o2z) from B. halodurans (BhYvcK; Forouhar et al., 2008 ▸), an orthologue of MtCuvA, has been determined. However, MtCuvA shares low sequence identity (approximately 31%) with BhYvcK, implying that MtCuvA may have a different environment in its ligand-binding site that is as yet uncharacterized. Thus, more detailed investigations are required to compare the structure of MtCuvA with the known structure of BhYvcK and to improve our understanding of the molecular function of MtCuvA in bacterial adaptation. As a first step towards the elucidation of its structure, the MtCuvA protein was overexpressed, purified and crystallized for the first time by the hanging-drop vapor-diffusion method. In this study, we present the crystallization, X-ray diffraction analysis and ligand binding of MtCuvA.

2. Materials and methods  

2.1. Protein expression and purification  

The gene for MtCuvA was amplified from M. tuberculosis H37Rv genomic DNA by polymerase chain reaction (PCR) using the forward and reverse primers in Table 1 ▸. The primers contained modifications to add suitable restriction endo­nuclease sites for insertion into the vector; the NdeI site in the forward primer and the XhoI site in the reverse primer are underlined in Table 1 ▸. The PCR-amplified DNA fragment was then digested with NdeI and XhoI and inserted into the bacterial expression vector pET-28a (Novagen, USA) to generate the plasmid pMtCuvA encoding MtCuvA with a hexahistidine tag at the N-terminus. Transformed Escherichia coli BL21(DE3) cells (Novagen, USA) harboring pMtCuvA were grown in Luria–Bertani medium with 50 µg ml⁻¹ kanamycin at 25°C to an optical density at 600 nm of 0.6. Protein expression was induced by the addition of 0.5 mM isopropyl β-d-1-thiogalactopyranoside and incubation at 18°C for a further 18 h. The cells were harvested by centrifugation at 5000g for 20 min at 25°C.

Table 1. Macromolecule-production information.

Source organism	M. tuberculosis H37Rv
DNA source	Genomic DNA
Forward primer†	5′-GTAATACATATGACCGATGGCATCGTCGCG-3′
Reverse primer†	5′-GTAATACTCGAGTCATCGCCACGCGTCGTC-3′
Cloning vector	pET-28a
Expression vector	pET-28a
Expression host	E. coli BL21(DE3)
Complete amino-acid sequence of the construct produced‡	MGSSHHHHHHSSGLVPRGSHMTDGIVALGGGHGLYATLSAARRLTPYVTAVVTVADDGGSSGRLRSELDVVPPGDLRMALAALASDSPHGRLWATILQHRFGGSGALAGHPIGNLMLAGLSEVLADPVAALDELGRILGVKGRVLPMCPVALQIEADVSGLEADPRMFRLIRGQVAIATTPGKVRRVRLLPTDPPATRQAVDAIMAADLVVLGPGSWFTSVIPHVLVPGLAAALRATSARRALVLNLVAEPGETAGFSVERHLRVLAQHAPGFTVHDIIIDAERVPSEREREQLRRTATMLQAEVHFADVARPGTPLHDPGKLAAVLDGVCARDVGASEPPVAATQEIPIDGGRPRGDNPWR

^†Restriction-enzyme sites are underlined.

^‡The extra amino acids introduced into the wild-type MtCuvA protein by cloning are underlined.

The harvested cell pellets were suspended in buffer A [50 mM Tris–HCl pH 8.0, 500 mM NaCl, 5 mM β-mercapto­ethanol (β-ME), 10% glycerol] containing 1 mM phenylmethylsulfonyl fluoride and disrupted by sonication at 4°C. The crude lysate was centrifuged at 25 000g for 20 min at 4°C. The supernatant was loaded onto a Ni²⁺-chelated HiTrap chelating HP column (GE Healthcare, USA) equilibrated in buffer A. The bound protein was eluted with a linear gradient of buffer B (50 mM Tris–HCl pH 8.0, 500 mM NaCl, 5 mM β-ME, 10% glycerol, 500 mM imidazole). Fractions containing MtCuvA were pooled together and dialyzed against buffer consisting of 50 mM Tris–HCl pH 8.0, 150 mM NaCl, 5 mM β-ME, 10% glycerol at 4°C. The buffer for dialysis was exchanged twice in 18 h. The purity of MtCuvA was confirmed by 15% SDS–PAGE and it was concentrated to 2.5 mg ml⁻¹ using Amicon Ultra-15 10K centrifugal filter devices (Millipore, USA) for subsequent crystallization.

2.2. Protein crystallization  

The preliminary crystallization screening of MtCuvA was performed by the sitting-drop vapor-diffusion method (1 µl protein solution and 1 µl reservoir solution equilibrated against 200 µl reservoir solution) using various commercial screening kits (Crystal Screen, Crystal Screen 2, PEGRx 1, PEGRx 2, Natrix, Natrix 2 and MembFac from Hampton Research, USA) in 48-well Intelli-Plates (Hampton Research, USA) at 21°C. The initial crystals were obtained using the following condition: 2% Tacsimate pH 7.0, 5% 2-propanol, 0.1 M imidazole pH 7.0, 8%(w/v) polyethylene glycol (PEG) 3350 (from PEGRx 2; Hampton Research, USA). In the optimized crystal-growth condition each drop was made up of 1 µl protein solution and 1 µl reservoir solution [2% Tacsimate pH 7.0, 4%(w/v) PEG 3350] and was equilibrated against 500 µl reservoir solution by the hanging-drop vapor-diffusion method in 24-well VDX plates (Hampton Research, USA). Crystallization information is summarized in Table 2 ▸.

Table 2. Crystallization conditions for diffracting crystals.

Method	Vapor diffusion in hanging drops
Temperature (K)	294
Protein concentration (mg ml−1)	2.2
Buffer composition of protein solution	50 mM Tris–HCl pH 8.0, 150 mM NaCl, 5 mM β-ME, 10% glycerol
Composition of reservoir solution	2% Tacsimate pH 7.0, 5%(v/v) 2-propanol, 0.1 M imidazole pH 7.0, 4%(w/v) PEG 3350
Volume and ratio of drop	2 µl, 1:1 ratio
Volume of reservoir (µl)	500

2.3. Data collection and processing  

For data collection under cryogenic conditions, the crystals were transferred into a cryoprotectant solution consisting of 25% ethylene glycol in the reservoir solution. The cryoprotected crystals were then directly flash-cooled at −180°C in a stream of nitrogen gas. The diffraction data sets were collected on beamline 7A at the Pohang Light Source (PLS), Pohang, Republic of Korea using an ADSC Quantum 270r CCD detector. A total range of 360° was covered, with an oscillation angle of 1° and 1 s exposure per frame. The distance from the crystal to the detector was 250 mm. The crystal of MtCuvA diffracted to 2.1 Å resolution. All diffraction data sets were indexed to identify the unit cell and space group of the crystal and were scaled after integration of the indexed data using the HKL-2000 software package (Otwinowski & Minor, 1997 ▸). Detailed information on data collection is given in Table 3 ▸.

Table 3. Data-collection statistics for MtCuvA.

Values in parentheses are for the highest resolution shell.

Diffraction source	Beamline 7A at PLS
Wavelength (Å)	1.0000
Temperature (K)	100
Detector	ADSC Quantum 270r CCD
Crystal-to-detector distance (mm)	250
Rotation range per image (°)	1
Total rotation range (°)	360
Exposure time per image (s)	1
Space group	P212121
a, b, c (Å)	47.27, 170.93, 178.10
α, β, γ (°)	90, 90, 90
Resolution range (Å)	50.0–2.1 (2.18–2.10)
Total No. of reflections	81437
No. of unique reflections	7287
Completeness (%)	93.1 (84.3)
Multiplicity	6.3 (3.0)
〈I/σ(I)〉	5.3 (1.9)
CC1/2	0.985 (0.265)
R merge † (%)	15.4 (43.3)

^† R _merge = , where I(hkl) represents the observed intensity, 〈I(hkl)〉 represents the average intensity and i counts through all symmetry-related reflections.

2.4. Ligand-binding assay using protein thermal shift  

A thermal shift assay was performed using an Applied Biosystems ABI Fast 7500 system. The reaction samples were distributed in a 0.1 ml PCR multi-strip (Applied Biosystems, USA) sealed with cap strips. The MtCuvA protein (at a final concentration of 0.15 mg ml⁻¹) was equilibrated with or without 5 mM of each ligand in a reaction mixture (20 µl in each well) containing 5 µl Protein Thermal Shift buffer (Thermo Fisher Scientific, USA) and 2 µl Protein Thermal Shift dye (diluted 10×) (Thermo Fisher Scientific, USA). Each melt curve was programmed as follows: 25°C for 2 min followed by a 1°C increase per minute from 25 to 85°C and finally 85°C for 2 min. Arbitrary fluorescence was plotted as a function of temperature. The denaturation temperature (T _m) is defined as the temperature with the highest fluorescence, coinciding with the maximum amount of dye binding exposed by thermal denaturation. No significant background fluorescence was observed in the absence of protein. The T _m was obtained from the melt-curve peak using Protein Thermal Shift Software version 1.4 (Applied Biosystems, USA).

3. Results and discussion  

The CuvA protein (Rv1422) from M. tuberculosis H37Rv was successfully cloned into the pET-28a expression vector in order to obtain soluble production in the E. coli BL21(DE3) strain. The recombinant MtCuvA protein was purified using a Ni²⁺-chelated HiTrap HP column and was monitored by 15% SDS–PAGE. The purified MtCuvA protein showed a single band indicating a calculated molecular weight of 38 kDa, corresponding to 342 amino acids, and was estimated to have a purity of 95% or greater (Fig. 1 ▸). MtCuvA crystals that were suitable for X-ray diffraction were obtained within five days by the hanging-drop vapor-diffusion method using an optimized reservoir solution consisting of 2% Tacsimate pH 7.0, 5% 2-propanol, 0.1 M imidazole pH 7.0, 4%(w/v) PEG 3350. The dimensions of a thin plate-shaped MtCuvA crystal were approximately 0.3 × 0.6 × 0.1 mm (Fig. 2 ▸). The crystals of MtCuvA grew in a ‘wing’ form in which crystals were stuck to each other at their edges. To avoid overlap when mounting the crystal, we tried to tear off the edges of the stuck crystals using an Ultra Micro-Needle (Hampton Research, USA).

Figure 1.

15% SDS–PAGE analysis of MtCuvA. Lane 1, molecular-weight markers (labeled in kDa); lane 2, purified MtCuvA protein. The expected molecular weight of MtCuvA is approximately 38.0 kDa.

Figure 2.

A crystal of CuvA (Rv1422) from M. tuberculosis H37Rv. The dimensions of the MtCuvA crystal are approximately 0.3 × 0.6 × 0.1 mm.

The crystal of MtCuvA diffracted to 2.1 Å resolution at a synchrotron source (Fig. 3 ▸). The crystal belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 47.27, b = 170.93, c = 178.10 Å. The Matthews coefficient V _M (Kantardjieff & Rupp, 2003 ▸) and solvent content were calculated to be 2.4 ų Da⁻¹ and 48.02%, respectively, indicating the presence of four molecules in the asymmetric unit of this crystal form. We attempted to solve the crystal structure of MtCuvA by molecular replacement (MR) with MOLREP (Vagin & Teplyakov, 2010 ▸) from the CCP4 suite (Winn et al., 2011 ▸) using the crystal structure of BhYvcK (PDB entry 2o2z; Forouhar et al., 2008 ▸) as a search model. The structure solution by MR showed four monomers in the asymmetric unit, as expected. After rigid-body refinement and several rounds of restrained refinement using Phenix (Liebschner et al., 2019 ▸), the R factor and R _free values were approximately 45.9% and 47.2%, respectively. However, further refinement did not decrease the R values, although the electron-density map showed good consistency (FOM = 0.77) with the model structure. To solve this problem, the diffraction data were analysed with phenix.xtriage (Adams et al., 2010 ▸). The crystal of MtCuvA was revealed to be a pseudo-merohedral twin, with twin law −h, l, k, and further refinement of the model structure is currently in progress.

Figure 3.

X-ray diffraction pattern of an MtCuvA crystal obtained using an ADSC Quantum 270r CCD detector. A resolution circle is shown at 2.1 Å and the red box shows an enlargement of an area containing high-resolution spots.

The biochemical properties of the MtCuvA protein remain unclear, but it harbors a Rossmann fold, a structural motif that is commonly known to be a binding site for nucleotides (Rossmann & Argos, 1981 ▸). To investigate protein–ligand inter­actions, the ability of MtCuvA to bind to ligands such as dinucleotide cofactors (NAD⁺ and NADP⁺) and mononucleotides (ADP and ATP) was analyzed. In addition, since previous studies have suggested that MtCuvA may be involved in nutrient metabolism that affects the cell-wall structure (Mir et al., 2014 ▸), we carried out a thermal shift assay to define the binding of MtCuvA to the cell-wall precursor components UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucose (UDP-Glc). Ligand binding to MtCuvA was estimated by the effect of the ligand on its melting temperature (T _m). No binding of NAD(P)⁺, ADP or ATP to MtCuvA was detected, but UDP-GlcNAc and UDP-Glc increased the T _m, indicating that the binding of UDP-sugars contributed to stabilization of the structure of MtCuvA (Fig. 4 ▸). Moreover, we observed that the ΔT _m value for UDP-GlcNAc was increased by approximately 2.7-fold compared with that for UDP-Glc (Table 4 ▸). These results imply that UDP-GlcNAc, which is a key precursor of peptidoglycan, may be the physiological ligand of MtCuvA. Based on the ligand-binding properties of MtCuvA, we are making progress in further crystallization trials to determine the structure of the binary complex of MtCuvA and an UDP-sugar.

Figure 4.

Ligand-binding properties of MtCuvA determined by thermal shift assay. (a) ADP, (b) ATP, (c) NAD⁺, (d) NADP⁺, (e) UDP-Glc, (f) UDP-GlcNAc. A derivative plot of the change in fluorescence versus temperature is shown. The median derivative T _m values are shown as black vertical dotted lines. MtCuvA at a final concentration of 0.15 mg ml⁻¹ was mixed with a ligand (each at 5 mM) and Protein Thermal Shift dye. Four replicate reactions with and without ligand were performed on a real-time PCR system using the melt curve, continuous data collection and a ramp rate of 0.05°C s⁻¹ from 25 to 85°C.

Table 4. The melting temperatures (T _m) of MtCuvA interacting with various ligands (each at 5 mM).

	T m (°C)	ΔT m (°C)
MtCuvA	56.95 ± 0.18
+ ADP	57.91 ± 0.28	0.96 ± 0.33
+ ATP	58.03 ± 0.27	1.08 ± 0.32
+ NAD+	58.00 ± 0.26	1.05 ± 0.31
+ NADP+	57.85 ± 0.10	0.90 ± 0.21
+ UDP-Glc	60.29 ± 0.32	3.34 ± 0.37
+ UDP-GlcNAc	65.83 ± 0.12	8.88 ± 0.22

Funding Statement

This work was funded by National Research Foundation of Korea grant NRF-2018R1D1A1B07047424.