Comparison of four different crystal forms of the Mycobacterium tuberculosis ESX-1 secreted protein regulator EspR

Structural comparison of four crystal forms of Mycobacterium tuberculosis EspR protein.

Abstract

The Mycobacterium tuberculosis ESX-1 secreted protein regulator (EspR, Rv3849) is the key protein that delivers bacterial proteins into the host cell during mycobacterial infection. EspR binds directly to the espACD operon and is involved in transcriptional activation. In the current study, M. tuberculosis EspR has been crystallized and its X-ray structure has been determined at 3.3 Å resolution in a P3₂21 crystal form. EspR forms a physiological dimer in the crystal. Each EspR monomer contains an N-terminal helix–turn–helix DNA-binding domain and a C-terminal dimerization domain. The EspR structure in the P3₂21 crystal form was compared with previously determined EspR structures in P3₂, P2₁ and P2₁2₁2₁ crystal forms. Structural comparison analysis indicated that the N-terminal helix–turn–helix domain of EspR acquires a rigid structure in the four crystal forms. However, significant structural differences were observed in the C-terminal domain of EspR in the P2₁ crystal form when compared with the P3₂21 and P3₂ crystal forms. The interaction, stabilization energy and buried surface area analysis of EspR in the four different crystal forms have provided information about the physiological dimer interface of EspR.

1. Introduction  

The protein export systems of Mycobacterium tuberculosis are considered as new drug targets (Feltcher et al., 2010 ▶). The ESX-1 protein secretion system is the principal virulence system of M. tuberculosis, which delivers virulence factors into host macrophages during mycobacterial infection (Stanley et al., 2003 ▶; Hsu et al., 2003 ▶; Pathak et al., 2007 ▶). After macrophage infection, the ESX-1 secretion system is involved in innate immune modulation (Stanley et al., 2003 ▶, 2007 ▶; MacGurn & Cox, 2007 ▶; Volkman et al., 2004 ▶). Precise regulation of the ESX-1 secretion system during infection is essential for pathogenicity of M. tuberculosis.

The precise mechanism of the M. tuberculosis ESX-1 secretion system and its essential role in virulence are currently unknown. The ESAT-6 (early secretory antigenic target, 6 kDa), CFP-10 (culture filtrate protein, 10 kDa) and ESPS (ESX-1 secretion-associated proteins) of the ESX-1 secretion system interfere with host cell metabolism and help mycobacteria to escape phagosomal engulfment (Hsu et al., 2003 ▶; Pathak et al., 2007 ▶; de Jonge et al., 2007 ▶; Singh et al., 2003 ▶; Bitter et al., 2009 ▶; Simeone et al., 2009 ▶).

EspR is a DNA-binding transcriptional regulator and activates the espACD operon (rv3616c–rv3614c) directly upon macrophage infection (Fortune et al., 2005 ▶; Garces et al., 2010 ▶; Raghavan et al., 2008 ▶). EspR is induced upon phagocytosis and activates the expression of downstream M. tuberculosis ESX-1 components. Deletion of EspR affects M. tuberculosis gene expression, including loci critical for function of the ESX-1 secretion system (Raghavan et al., 2008 ▶). The EspR protein contains 132 residues with a molecular mass of ∼14.7 kDa. The N-terminus of EspR harbours a helix–turn–helix DNA-binding domain, while there is a typical dimerization domain at the C-terminus. Deletion of the C-terminal ten amino acids of EspR abolishes the ESX-1 secretion activity, but did not affect the DNA affinity (Raghavan et al., 2008 ▶).

In the current work, we have determined the crystal structure of M. tuberculosis EspR in a P3₂21 crystal form at 3.3 Å resolution (PDB entry 4ndw; two monomers in the asymmetric unit). The current EspR structure was compared with previous EspR structures in (i) a P3₂ crystal form (PDB entry 3r1f; 18 monomers in the asymmetric unit; Rosenberg et al., 2011 ▶), (ii) a P2₁ crystal form (PDB entry 3qf3; six monomers in the asymmetric unit; Blasco et al., 2011 ▶) and (iii) a P2₁2₁2₁ crystal form (PDB entry 3qwg; two monomers in the asymmetric unit; Blasco et al., 2011 ▶). The structural comparison analysis has revealed information on the physiological dimer interface and important structural changes in EspR in different crystal forms.

2. Materials and methods  

2.1. Protein expression and purification  

The gene encoding EspR (Met1–Ala132) was amplified from M. tuberculosis H37Rv strain by polymerase chain reaction using the forward primer 5′-GATCCATATGATGAGCACGACGTTCGCT-3′ and the reverse primer 5′-CATGCTCGAGCTAAGCGTCGATCCCTCC-3′. The amplified EspR gene was digested and ligated into pET28a(+) expression vector (Novagen) using NdeI and XhoI restriction sites. The resulting EspR plasmid was transformed in Escherichia coli BL21(DE3) cells and the protein was expressed in the soluble fraction. The EspR was purified using Ni–NTA and size-exclusion chromatography as described in Gangwar et al. (2011 ▶). The recombinant EspR protein contains 152 residues [132 residues from native EspR and 20 residues from the pET28a(+) vector containing a 6×His tag and a thrombin cleavage site].

2.2. Preparation of selenomethionine derivative of EspR  

The selenomethionine derivative of EspR was prepared using SelenoMethionine Base plus Nutrient Mix medium obtained from Molecular Dimensions. 30 mg of seleno-dl-methionine (Sigma–Aldrich) was added to 1 l of the selenomethionine medium. The expression and purification of selenomethionine-substituted EspR protein were similar to those of native EspR described in Gangwar et al. (2011 ▶).

2.3. Crystallization and X-ray structure determination  

The purified EspR was concentrated to 10–12 mg ml⁻¹ in buffer consisting of 20 mM Tris–HCl pH 7.5 for crystallization experiments. Initial crystallization screenings were performed on native and selenomethionine-substituted EspR as described in Gangwar et al. (2011 ▶). The selenomethionine-substituted EspR crystals were obtained in several conditions using the sitting-drop vapour-diffusion technique. The best selenomethionine-substituted EspR crystals appeared in buffer consisting of 24%(w/v) PEG 3350, 200 mM sodium malonate, 100 mM bis-tris propane pH 6.5 after 10–15 d.

For intensity data collection, the native EspR crystals were transferred into mother liquor consisting of 35%(w/v) PEG 3350, 200 mM sodium malonate, 100 mM bis-tris propane pH 6.5. These crystals were cooled directly in liquid nitrogen as 35%(w/v) PEG 3350 acts as a good cryoprotectant for data collection at cryogenic temperature. The native EspR crystal diffracted to 3.3 Å resolution and a full intensity data set was collected at 100 K on the BM14 synchrotron beamline at the ESRF, Grenoble, France. The selenomethionine-substituted EspR crystal diffracted to 3.5 Å resolution and a complete single anomalous dispersion data set (SAD) was collected at the selenium edge. The native and selenomethionine SAD data sets were processed using DENZO and SCALEPACK from the HKL-2000 suite (Otwinowski & Minor, 1997 ▶). The details of intensity data collection and processing are given in Table 1 ▶.

Table 1. Intensity data-collection and refinement statistics of four EspR crystal forms.

Values in parentheses are for the highest resolution shell.

Data set	P3221, SeMet	P3221, native	P32	P21	P212121
PDB code		4ndw	3r1f (Rosenberg et al., 2011 ▶)	3qf3 (Blasco et al., 2011 ▶)	3qwg (Blasco et al., 2011 ▶)
Space group	P3221	P3221	P32	P21	P212121
Wavelength ()	0.97833	0.97625	1.11588	1.000	1.0097
X-ray source	BM14, ESRF	BM14, ESRF	ALS, BI 8.3.1	SLS, PSI Switzerland	SLS, X06DA, Switzerland
Resolution ()	503.55 (3.613.55)	483.30 (3.483.30)	48.852.50 (2.542.50)	36.922.41 (2.562.41)	44.861.99 (2.111.99)
Unit-cell parameters (, )	a = b = 84.9, c = 129.9	a = b = 83.9, c = 130.75	a = b = 148.4, c = 129.6	a = 52.2, b = 81.7, c = 124.8, = 95.8	a = 46.5, b = 55.4, c = 76.6
Unique reflections	12707	8535	100727	40265	24707
Completeness	100 (100)	100 (100)	91.05 (86.9)	98.9 (96.6)	94.4 (82.6)
R merge † (%)	11.0 (38.9)	15.9 (55.3)	14.0 (73.0)	7.7 (65.1)	10.4 (38.7)
Average I/(I)	21.5 (5.8)	11.1 (2.6)	3.9 (1.5)	13.3 (2.6)	9.8 (3.1)
Multiplicity	11.7 (11.8)	11.8 (12.1)	2.1 (2.0)	3.2 (3.1)	3.4 (3.0)
Monomers in asymmetric unit	2	2	18	6	2
Solvent (%)	68	69	59	59	32
V M (3Da1)	3.88	3.91	3.02	3.0	1.81
Refinement
Resolution ()		48.693.30	48.852.50	36.922.41	44.861.99
R work/R free ‡ (%)		27/33	19.7/23.2	18/24	18.5/23.6
Protein atoms		2020	17207	6135	1441
Water atoms			19	232	132
R.m.s.d. bonds ()		0.009	0.010	0.008	0.007
R.m.s.d. angle ()		1.33	1.2	0.9	0.954
Ramachandran plot
Favoured (%)		85.5	87.8	94.2	94.3
Allowed (%)		13.8	11.7	5.8	5.7
Generous (%)		0.4	0.5	0.0	0.0
Forbidden (%)		0.0	0.0	0.0	0.0

^† R _merge = , where I_i(hkl) is the ith intensity measurement of reflection hkl and I(hkl) is the average intensity of that reflection.

^‡ R _work/R _free = .

The selenium sites were identified using SHELXD and SHELXE from the HKL2MAP suite (Pape & Schneider, 2004 ▶). Initial phases were obtained by AutoSol from the PHENIX suite (Adams et al., 2010 ▶). The selenomethionine-substituted EspR model was built by AutoBuild from the PHENIX suite. The obtained EspR structure was transferred into the native data set collected at 3.3 Å resolution using Phaser from the CCP4 suite (Winn et al., 2011 ▶). The EspR structure was refined using the REFMAC5 program (Murshudov et al., 2011 ▶) of the CCP4 suite and further model building was done using Coot (Emsley & Cowtan, 2004 ▶). Figures were generated using PyMOL v.1.3 (DeLano, 2002 ▶).

3. Results and discussion  

3.1. Structure determination  

We recombinantly expressed EspR protein in E. coli and purified it using Ni–NTA affinity and size-exclusion chromatography as described in Gangwar et al. (2011 ▶). The native EspR eluted as a dimer (molecular mass of ∼30 kDa) from a Superdex 75 (16/60) column (Fig. 1 ▶). The native EspR was crystallized in space group P3₂21 and an intensity data set was collected to 3.3 Å resolution. We crystallized selenomethionine-substituted EspR in the same condition as used for native EspR. The EspR structure was determined using a single anomalous dispersion data set collected at 3.5 Å resolution using the selenium edge (Supplementary Fig. S11). The obtained structure was placed in the EspR native data set collected to 3.3 Å resolution using Phaser from the CCP4 suite.

Figure 1.

The size-exclusion chromatogram and SDS–PAGE of purified recombinant EspR protein. The recombinant EspR elutes as a dimer from a Superdex 75 (16/60) column. The calculated molecular mass of EspR is denoted in the figure.

The P3₂21 form of EspR contains two monomers in the asymmetric unit packed as a functional homodimer. The P3₂ form of EspR contains 18 monomers in the asymmetric unit that are packed as nine closely interacting dimers (Rosenberg et al., 2011 ▶). The P2₁ form of EspR contains six monomers in the asymmetric unit that are packed into three functional EspR dimers (Blasco et al., 2011 ▶). The P2₁2₁2₁ form of the C-terminal deletion mutant of EspR contains two monomers in the asymmetric unit (Blasco et al., 2011 ▶). The EspR dimer interface in the P2₁2₁2₁ form was quite different to the EspR dimer interface observed in the P2₁, P3₂21 and P3₂ forms (Blasco et al., 2011 ▶). It indicates that the P2₁2₁2₁ form of the C-terminal deletion mutant of EspR lacks the key residues involved in the formation of the physiological dimer interface of EspR (Blasco et al., 2011 ▶).

3.2. Overall structure  

The P3₂21 form of EspR contains two monomers in the asymmetric unit with a Matthews coefficient (V _M) of 3.91 ų Da⁻¹ and a solvent content of 69% (Matthews, 1968 ▶). In the P3₂21 form of EspR, two monomers of EspR are arranged as a physiological dimer (Fig. 2 ▶). Analysis of the EspR dimer in the P3₂21 form using the PISA server (Krissinel & Henrick, 2007 ▶) indicated that both EspR monomers contain 4217 Ų buried surface area and have 21.1 kcal mol⁻¹ free energy of dissociation. The interacting residues between two EspR monomers are shown in Fig. 3 ▶. These data indicate that the EspR dimer in the P3₂21 form is thermodynamically stable.

Figure 2.

Overall structure of EspR. The EspR adopts an S-shaped structure. The EspR dimer is formed through the C-terminal domain.

Figure 3.

The physiological dimer interface of EspR. Residues involved in hydrogen-bonding interactions at the EspR dimer interface are shown.

Analysis of the P3₂21 form of EspR using the ESBRI server (Costantini et al., 2008 ▶) indicated that three salt bridges and 26 hydrophobic interactions are involved in the dimer interface (Table 2 ▶). In the P3₂21 form of EspR, 0.2 kcal mol⁻¹ hydrogen-bond energy, −16.5 kcal mol⁻¹ electrostatic energy and a total of −72 kcal mol⁻¹ stabilizing energy were observed at the dimer interface (Table 2 ▶). The COILCHECK program (Alva et al., 2008 ▶) identified the three side-chain hydrogen bonds at the EspR dimer interface: (i) between Glu87 (O^∊1) of chain A and Arg100 (NH1) of chain B, (ii) between Glu87 (O^δ1) of chain A and Arg100 (NH2) of chain B, and (iii) between His107 (N^δ1) of chain A and Thr93 (O^γ1) of chain B (Fig. 3 ▶).

Table 2. Stabilization energy and number of interactions between two monomers in the four EspR crystal forms.

	P3221	P32	P21	P212121
Hydrogen-bond energy (kJmol1)	5.9	10.7	11.2	13.9
Electrostatic energy (kJmol1)	69.1	186.3	144.2	39.1
Van der Waals energy (kJmol1)	242.5	296.7	321.1	101.1
Total stabilizing energy (kJmol1)	305.7	472.3	454.1	126.3
Energy per residue (kJmol1)	1.2	1.8	1.7	0.7
Short contacts	2	0	1	1
Hydrophobic interactions	26	24	25	0
Van der Waals pairs	4078	4337	4827	1589
Salt bridges	4	8	9	2
Hydrogen bonds	3	2	7	0
Electrostatic interactions	60	59	61	18

3.3. Conformational changes  

The EspR crystal structure has been determined in four different crystal forms: (i) in a P3₂21 form at 3.3 Å resolution (PDB entry 4ndw, current structure), (ii) in a P3₂ form at 2.5 Å resolution (PDB entry 3r1f; Rosenberg et al., 2011 ▶), (iii) in a P2₁ form at 2.4 Å resolution (PDB entry 3qf3; Blasco et al., 2011 ▶) and (iv) an EspR mutant in a P2₁2₁2₁ form at 1.9 Å resolution (PDB entry 3qwg; Blasco et al., 2011 ▶). Superposition of the EspR dimer in the P3₂21 form on the P3₂ form gave an r.m.s.d. of 0.54 Å for 239 C^α atoms (Fig. 4 ▶). Superposition of the EspR dimer of the P3₂21 form on the P2₁ form gave an r.m.s.d. of 0.75 Å for 189 C^α atoms; however, significant structural differences were observed in the C-terminal domain of EspR.

Figure 4.

Superposition of EspR structures obtained in four different crystal forms. The EspR structures in the P3₂, P2₁ and P2₁2₁2₁ crystal forms were superposed on that in the P3₂21 crystal form.

Hinge angles between the α7 and α8 helices of EspR of 102° in the P3₂21 form, 101° in the P3₂ form and 124° in the P2₁ form were observed. Hinge angles between the α6 and α7 helices of EspR of 37° in the P3₂21 form, 30° in the P3₂ form and 47° in the P2₁ form were observed (Fig. 4 ▶). These data indicate that the conformation of the C-terminal domain of EspR is identical in the P3₂21 and P3₂ forms but changed significantly in the P2₁ form.

3.4. Dimer interface analysis  

As shown in Table 3 ▶, the EspR dimer interface in the P3₂21 form contains 4220 Ų of buried surface area and has 21 kcal mol⁻¹ dissociation free energy. In the P3₂ form of EspR, 4580 Ų of buried surface area and 18.8 kcal mol⁻¹ free energy of dissociation were observed at the dimer interface. In the P2₁ form of EspR, 4540 Ų of buried surface area and 23.1 kcal mol⁻¹ free energy of dissociation were observed at the dimer interface. In the P2₁2₁2₁ form of truncated EspR, only 585 Ų of buried surface area was observed at the dimer interface. These results indicate that EspR monomers do not form a physiological dimer in the P2₁2₁2₁ crystal form and packed as a result of crystallographic packing (Blasco et al., 2011 ▶). The stabilizing energy and various interactions at the EspR dimer interface are shown in Table 2 ▶. The residues interacting at the EspR dimer interface differ from one crystal form to the other, but do not affect the EspR dimer formation (Table 4 ▶).

Table 3. Accessible surface area in the EspR dimer in the four crystal forms.

Crystal form	Total area per subunit (2)	Contact area (2)	Contact area (%)	Dissociation energy (kcalmol1)
P3221	13528.3	3975.1	29.4	21.1
P32	13297.3	3905.1	29.4	18.2
P21	13483.6	3961.3	29.4	22.7
P212121	10131.2	2987.9	29.5	1.3

Table 4. Interactions occurring in the EspR dimer interfaces in the four crystal forms.

Crystal form	Residue (atom)	Residue (atom)	Distance (2)
P3221	Glu87 (O1)	Arg100 (NH1)	2.7
	Glu87 (O1)	Arg100 (NH2)	3.1
	His107 (N2)	Thr93 (O1)	3.7
	His107 (N1)	Asp96 (O1)	3.5
P32	Glu87 (O1)	Arg100 (NH1)	3.4
	Glu87 (O1)	Arg100 (NH2)	2.9
	Glu87 (O2)	Arg100 (NH1)	3.2
	Arg100 (NH2)	Glu87 (O1)	3.4
	Arg105 (N)	Asp122 (O1)	2.9
	H107 (N1)	Gln89 (N1)	3.4
	Asp122 (O2)	Arg105 (O2)	3.2
P21	Arg101 (NH1)	Glu88 (O1)	2.9
	Arg101 (NH1)	Glu88 (O2)	3.3
	Arg101 (NH2)	Glu88 (O2)	3.2
	Gln116 (O1)	Arg102 (NH2)	3.1
	Asp123 (O1)	Arg106 (N)	2.8
	Asp123 (O1)	Arg100 (NH2)	2.7
	Asp132 (O2)	Arg106 (NH2)	2.8
P212121	Thr5 (O1)	Asn53 (N2)	3.1
	Ser51 (O)	Asn11 (N2)	3.1
	Asn53 (N2)	Thr5 (O1)	3.3

3.5. Crystallographic contacts  

Each crystal form of EspR contains distinct crystallographic contacts, which are given in Supplementary Table S1. In the EspR P2₁ crystal form, two intermolecular crystal contacts, Arg54–Asn11 and Arg54–Asp15, were observed in the N-terminal domain of EspR. In the EspR P3₂21 crystal form, the Arg120, Glu123, Leu124, Arg126 and Ala127 residues of the EspR monomer interact with their symmetry mates. In the P3₂ form, no residues of the EspR monomers are involved in crystallographic contacts; however, the structure aligns very well with the P3₂21 crystal form. These results indicate that crystal contacts do not induce conformational change in the C-terminal domain of EspR in the P2₁ crystal form, which is quite different from the observed structures in the P3₂ and P3₂21 crystal forms.

In the current study, we have determined the structure of EspR in the P3₂21 crystal form and compared it with the structures of EspR in the P3₂, P2₁ and P2₁2₁2₁ crystal forms. These results indicate that the N-terminal domain of EspR acquires a rigid structure and significant structural changes are observed in the C-terminal domain of EspR. The crystallographic contact analysis in various EspR crystal forms indicates that they are not involved in inducing structural change in the C-terminal domain of EspR. In the P3₂ and P2₁ crystal forms, high atomic fluctuations and structural changes are observed in the C-terminal domain of various EspR subunits in the asymmetric unit (Blasco et al., 2011 ▶). The α6–α8 helices of the C-terminal domain of EspR form tight intermolecular interactions and are involved in homodimer formation, which is essential for activation of the espACD operon and EsxA secretion. However, the orientations of these helices are different in the various EspR crystal forms, which may form divergent oligomeric arrangements of EspR upon DNA binding.

Supplementary Material

PDB reference: EspR, 4ndw