Overexpression, purification, crystallization and preliminary X-ray analysis of uracil N-glycosylase from Mycobacterium tuberculosis in complex with a proteinaceous inhibitor

Uracil N-glycosylase from M. tuberculosis has been crystallized in complex with a proteinaceous inhibitor (Ugi) and X-ray diffraction data have been collected.

Abstract

Uracil N-glycosylase is an enzyme which initiates the pathway of uracil-excision repair of DNA. The enzyme from Mycobacterium tuberculosis was co-expressed with a proteinaceous inhibitor from Bacillus subtilis phage and was crystallized in monoclinic space group C2, with unit-cell parameters a = 201.14, b = 64.27, c = 203.68 Å, β = 109.7°. X-ray data from the crystal have been collected for structure analysis.

1. Introduction

The exocyclic amino group of cytosine is highly susceptible to deamination, a process that occurs in response to normal physio­logical reactions or environmental pollutants. Such a chemical change in DNA results in the conversion of a G–C base pair to a promutagenic G–U wobble pair. If the uracils from such wobble pairs are not repaired prior to replication, an inevitable consequence is that G–C to A–­T mutations will accumulate in the progeny DNA. DNA polymerases may also erroneously incorporate dUMP against adenosine (as an A–U base pair) and hamper the recognition of the regulatory sequences by the proteins. To maintain genomic integrity, cells possess uracil-DNA glycosylases (UDGs), which initiate the uracil-excision repair pathway (Lindahl, 1974 ▶). Among these, the uracil N-glycosylases (Ungs), belonging to family 1 of the UDGs, are a highly efficient, ubiquitous and conserved class of proteins. Escherichia coli Ung (EcUng), the founder member of the family 1 UDGs, is encoded by the ung gene (Lindahl et al., 1977 ▶; Varshney et al., 1988 ▶). Mycobacterium tuberculosis Ung (MtUng; GenBank accession No. Rv2976c) is a 227-amino-acid protein with a molecular weight of 24.5 kDa. X-ray crystal structures of the Ung protein from E. coli and from a number of other sources are available (Savva et al., 1995 ▶; Mol, Arvai, Slupphaug et al., 1995 ▶; Ravishankar et al., 1998 ▶; Saikrishnan et al., 2002 ▶; Moe et al., 2004 ▶; Leiros et al., 2005 ▶)

Ung proteins are inhibited by free uracil and some of its derivatives (Krokan & Wittwer, 1981 ▶; Blaisdell & Warner, 1983 ▶; Focher et al., 1993 ▶; Jiang et al., 2005 ▶). Another category of Ung inhibitors is represented by the Bacillus subtilis phage PBS-1/PBS-2-encoded early gene product Ugi (Cone et al., 1980 ▶; Warner et al., 1980 ▶; Wang & Mosbaugh, 1988 ▶). Ugi (GenBank accession No. J04434) is a heat-stable acidic 84-amino-acid protein with a molecular weight of 9.5 kDa, which interacts with Ung with a 1:1 molar stoichiometry. Biochemical characterization of complexes of Ugi with the Ung proteins from M. tuberculosis and M. smegmatis shows that unlike the EcUng–Ugi complex, which is stable to treatment with 8 M urea, the mycobacterial Ung–Ugi complexes dissociate in 5–6 M urea (Acharya et al., 2003 ▶). However, Ugi is still a potent inhibitor of Ung activity in mycobacteria.

Pathogenic mycobacteria which multiply inside the host macrophages are exposed to reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI), agents which are known to inflict major damage to the genome (Wink et al., 1991 ▶). Further, because of the high G+C richness of their genomes, mycobacteria (e.g. M. tuberculosis) are naturally at high risk of cytosine deamination, making UDGs a crucial group of DNA-repair enzymes. We showed that ung ⁻ strains of M. smegmatis were indistinguishable from the wild-type strain from their growth under standard culture conditions. Interestingly, in contrast to there being no overt consequences (for at least 1 y) of the ung ⁻/ung ⁻ genotype in mice (Nilsen et al., 2000 ▶), ung ⁻ strains of M. smegmatis showed increased mutator phenotype and poor endurance in mouse macrophages, especially under conditions of increased RNI production (Venkatesh et al., 2003 ▶). Another study, in which a library of randomly integrated transposons containing M. tuberculosis were tested for survival in mouse macrophages, the bacteria that survived failed to reveal the presence of transposons within the ung locus, indicating that Ung is highly crucial for the survival of mycobacteria in animal models (Sassetti & Rubin, 2003 ▶).

As a part of our structural genomics program on mycobacterial proteins (Datta et al., 2000 ▶; Roy et al., 2004 ▶; Saikrishnan et al., 2003 ▶, 2005 ▶; Das et al., 2006 ▶; Krishna et al., 2006 ▶; Selvaraj et al., 2006 ▶), we report here the overexpression, purification and crystallization of the MtUng–Ugi complex with the aim of understanding the specific features of M. tuberculosis Ung and the mechanism of Ugi–inhibitor interaction. Distinctive properties of the mycobacterial Ung may allow the design of selective inhibitors against it.

2. Materials and methods

2.1. Expression and purification of M. tuberculosis Ung–Ugi complex

To purify the MtUng–Ugi complex, a pRSETB-based bicistronic construct (Acharya et al., 2003 ▶) was modified as follows to shorten the N-terminal tag attached to MtUng. The pRSETB MtUng–Ugi construct was digested with NheI and NcoI and the resulting staggered ends were filled in using T4-DNA polymerase and self-ligated (Sambrook et al., 1989 ▶). This procedure resulted in the removal of 26 amino acids from pre-Ung sequence, leaving behind Met-His-His-His-His-His-His-Gly-Met-Ala-Ser as presequence to MtUng. The above construct was transformed into E. coli BL21(DE3) cells and a loopful of transformants were inoculated into 1.8 l Luria–Bertani (LB) medium containing 100 µg ml⁻¹ ampicillin and grown at 310 K until they reached the late exponential phase. Cells were harvested by centrifugation and resuspended in 15 ml 10 mM Tris–HCl pH 7.5 containing 10%(v/v) glycerol and 500 mM NaCl and disrupted by sonication. The lysate was spun in an SS-34 rotor at 15 000 rev min⁻¹ using a Sorvall centrifuge for 30 min at 277 K. The supernatant was filtered through a 0.45 µm filter (Sartorious), loaded onto a pre-equilibrated Ni–NTA column (5 ml capacity) and eluted with a 0–500 mM imidazole gradient in the above buffer. Fractions containing near-homogenous MtUng–Ugi complex were pooled, dialyzed against 10 mM Tris–HCl pH 7.5 and estimated by Bradford’s method using bovine serum albumin as standard (Sedmak & Grossberg, 1977 ▶). The final preparation was concentrated to 10 mg ml⁻¹ and used for crystallization.

2.2. Crystallization

Initial crystallization screening using Hampton Crystal Screens I and II failed to give any positive results. Therefore, other commercially available screening kits, i.e. Index, SaltRx and PEG/Ion Screen from Hampton Research, and Wizard I and II from Emerald Biostructures, were used. Additionally, screening was also performed with commonly used precipitating agents (McPherson, 2004 ▶). Hanging-drop vapour-diffusion as well as microbatch methods were used in these experiments. Hanging drops were prepared using 4 µl protein solution and 1 µl reservoir solution and were equilibrated against 500 µl reservoir solution using 24-well Laxbro plates. In the case of the microbatch method, 3 µl protein solution and 3 µl precipitant solution were mixed in a Microtestplate (Sigma). Hampton paraffin oil and silicon oil were used in a 1:1 ratio. The protein solution contained 10 mg ml⁻¹ MtUng–Ugi complex in 10 mM Tris–HCl pH 7.5. Crystallization plates were stored at 294 K. Eventually, after ten months, a single crystal appeared in a drop with reservoir containing 10%(w/v) PEG 8000 and 0.2 M NaCl in 0.1 M phosphate buffer pH 6.2. Experiments designed to improve the quality using trials around the conditions mentioned above did not yield better crystals.

2.3. X-ray data collection and processing

Diffraction data were collected at low temperature (100 K) from a single crystal using a MAR Research image-plate system (diameter 345 mm) with Osmic mirrors and a Rigaku RU-200 rotating-anode X-­ray generator. The crystal was soaked in 20%(v/v) ethylene glycol and reservoir solution for 5 min prior to data collection. The crystal-to-detector distance was kept at 220 mm. Intensity data were processed and scaled using DENZO and SCALEPACK from the HKL program package (Otwinowski & Minor, 1997 ▶). The crystal belongs to space group C2, with unit-cell parameters a = 201.14, b = 64.27, c = 203.68 Å, β = 109.7°. The crystal diffracted to a resolution of 3.1 Å (Fig. 1 ▶). Intensities were converted to structure-factor amplitudes using TRUNCATE (Collaborative Computational Project, Number 4, 1994 ▶). Data-collection statistics are given in Table 1 ▶.

Figure 1.

A typical X-ray diffraction image of the MtUng–Ugi complex using an oscillation of 1°.

Table 1. Crystal data and data-collection statistics.

Values in parentheses are for the highest resolution shell.

Temparature (K)	100
Space group	C2
Unit-cell parameters
a (Å)	201.14
b (Å)	64.27
c (Å)	203.68
β (°)	109.7
Packing density (VM; Å3 Da−1)	2.6
Solvent content (%)	52.8
No. of molecules in ASU	7
Resolution range (Å)	30.0–3.1 (3.21–3.1)
Observed reflections	426407
No. of unique reflections	43871 (4095)
Completeness (%)	97.6 (91.8)
Multiplicity	9.7
〈I/σ(I)〉	8.2 (2.4)
Rsym† (%)	14.9 (42.1)

^† R _sym = , where I_l is the lth observation of reflection h and 〈I _h〉 is the weighted average intensity for all observations l of reflection h.

3. Results and discussion

The MtUng–Ugi complex was crystallized and X-ray data were collected. On the basis of the solvent content (Matthews, 1968 ▶), the crystal contains seven molecules in the asymmetric unit. MtUng has a sequence identity in the range 41–44% with EcUng, human Ung (HsUng) and Ung from herpes simplex virus (HsvUng) (Fig. 2 ▶). The crystal structures of these three proteins in complex with Ugi are available (Mol, Arvai, Sanderson et al., 1995 ▶; Ravishankar et al., 1998 ▶; Putnam et al., 1999 ▶; Savva & Pearl, 1995 ▶). They were used for molecular-replacement calculations using the program Phaser (Storoni et al., 2004 ▶) from CCP4 (Collaborative Computational Project, Number 4, 1994 ▶). The calculations led to an acceptable solution with a Z score of 22.9 and a log-likelihood gain of 1743.9 for seven crystallographically independent molecules. Further structural analysis is in progress. The presence of multiple independent copies of the molecules in the crystal is expected to partially compensate for the limited resolution of the data. It also provides a handle for exploring the structural plasticity of the molecule and its implications for eventual inhibitor design.

Figure 2.

Multiple sequence alignment of MtUng and Ungs of known three-dimensional structure using ClustalW (Thompson et al., 1994 ▶).