Identification, activity and disulfide connectivity of C-di-GMP regulating proteins in Mycobacterium tuberculosis

Abstract

C-di-GMP, a bacterial second messenger plays a key role in survival and adaptation of bacteria under different environmental conditions. The level of c-di-GMP is regulated by two opposing activities, namely diguanylate cyclase (DGC) and phosphodiesterase (PDE-A) exhibited by GGDEF and EAL domain, respectively in the same protein. Previously, we reported a bifunctional GGDEF-EAL domain protein, MSDGC-1 from Mycobacterium smegmatis showing both these activities (Kumar and Chatterji, 2008). In this current report, we have identified and characterized the homologous protein from Mycobacterium tuberculosis (Rv 1354c) named as MtbDGC. MtbDGC is also a bifunctional protein, which can synthesize and degrade c-di-GMP in vitro. Further we expressed Mtbdgc in M. smegmatis and it was able to complement the MSDGC-1 knock out strain by restoring the long term survival of M. smegmatis. Another protein Rv 1357c, named as MtbPDE, is an EAL domain protein and degrades c-di-GMP to pGpG in vitro. Rv1354c and 1357c have seven cysteine amino acids in their sequence, distributed along the full length of the protein. Disulfide bonds play an important role in stabilizing protein structure and regulating protein function. By proteolytic digestion and mass spectrometric analysis of MtbDGC, connectivity between cysteine pairs Cys94-Cys584, Cys2-Cys479 and Cys429-Cys614 was determined, whereas the third cysteine (Cys406) from N terminal was found to be free in MtbDGC protein, which was further confirmed by alkylation with iodoacetamide labeling. Bioinformatics modeling investigations also supported the pattern of disulfide connectivity obtained by Mass spectrometric analysis. Cys406 was mutated to serine by site directed mutagenesis and the mutant MtbC406S was not found to be active and was not able to synthesize or degrade c-di-GMP. The disulfide connectivity established here would help further in understanding the structure - function relationship in MtbDGC.

Figure 1. Sequence alignment of MSDGC-1, MtbDGC and MtbEAL was generated by Clustal W and was adjusted manually.

Cysteines at the identical position in both the sequences are indicated by a black background.

Figure 2. Representatives of C-di-GMP specific domain architectures in Mycobacteirum tuberculosis and their subsequent protein purification.

(A) Domain architecture of the protein with GGDEF and EAL domain in Mycobacterium tuberculosis. The length of each sequence is approximately to scale. (B) Purification of C- terminal His₆ tag proteins Lane – 1 Marker, Lane - 2 MtbDGC and Lane – 3 MtbPDE. C-terminal His₆ –tagged Rv1354 is 67.6 KDa and Rv1357c is 33.9 KDa. Purity of the preparations was determined by (10%) SDS- PAGE.

Figure 3. MtbDGC is a bifunctional protein showing both diguanylate cyclase (DGC) and phosphodiesterae acitivity (PDE-A) and MtbPDE is a functional protein showing phosphodiesterae acitivity (PDE-A).

(A) Purified His₆ protein was assayed for the ability to synthesize c-di-GMP. HPLC analysis for the detection of c-di-GMP and pGpG: (a): GTP, (b): Purified c-di-GMP, (c): With protein MtbDGC, separated on reversed phase HPLC. MALDI TOF analysis of the relevant HPLC fractions, in the positive ion detection mode, ions were detected at m/z of 542 (M+H)⁺ for GTP, 691 (M+H)⁺ and 713 (M+Na)⁺ for c-di-GMP, 709 (M+H)⁺ and 731 (M + Na)⁺ for pGpG. (B) Purified His₆ protein was assayed for the ability to degrade c-di-GMP. HPLC analysis for the detection of pGpG. (c): Purified c-di-GMP (C): With Protein (MtbPDE),. MALDI TOF analysis of the relevant HPLC fractions, in the positive ion detection mode, ions were detected at m/z  = 691 (M+H)⁺ and 713 (M+Na)⁺ for c-di-GMP, 709 (M+H)⁺ and 731 (M + Na)⁺ for pGpG.

Figure 4. Complementation of ΔMSDGC-1 with MtbDGC and its long term survival.

(A) ΔMSDGC-1 a strain of M. smegmatis was complemented with MtbDGC and its growth was compared with wildtype as well as ΔMSDGC-1. Cultures were grown in MB7H9 medium with 0.02% v/v glucose and 0.05% Tween-80 as carbon source. Aliquots were withdrawn at different time interval after culture reached stationary phase (48 hours of growth) and plated on MB7H9 agar supplemented with Kanamycin containing 2% w/v glucose as carbon source. The colony forming unit were determined and plotted. (B) Level of MtbDGC increases in stationary phase. ΔMSDGC-1 was transformed with MtbDGC and expression was studied in three phases of growth i.e. exponential, early stationary and late stationary phase. The α subunit (RpoA) of RNA polymerase was used as an control because its level remains unchanged in different phases of growth. Whole cell lysates was separated on 10% SDS-PAGE and analyzed by Western blot technique probed with antibody raised against MtbDGC.

Figure 5. Schematic diagram of experimental setup to study disulfide connectivity pattern.

Figure 6. LC-ESI-MS of intact MtbDGC protein.

(A) LC-ESI-MS of Histidine tagged intact MtbDGC protein and corresponding inset zoom of deconvoluted mass m/z 68475.99 of the protein. (B) LC-ESI-MS of DTT reduced MtbDGC intact protein and corresponding inset zoom of deconvoluted mass (m/z 68482.99). The shift in seven Dalton was observed in comparison of unreduced protein.

Figure 7. LC-ESI-MS of MtbDGC in reduced and oxidized condition, after digestion with chymotrypsin and trypsin.

(A). LC-ESI-MS of DTT reduced and iodoacetamide alkylated MtbDGC digested with trypsin and chymotrypsin. The singly charged ions at m/z 3796.2, 828, 833.7, 639.6, 3448.0 and 2775.0 show the mass of cysteine peptides at Cys², Cys⁹⁴, Cys⁴⁰⁶, Cys⁴²⁹, Cys⁴⁷⁹ and Cys⁶¹⁴ respectively (B) LC-ESI-MS of unreduced and iodoacetamide alkylated MtbDGC digested with trypsin and chymotrypsin. The singly charged ions at m/z 833.7, 1053.2 and 1399.5 [M +2H]²⁺ show the free Cys⁴⁰⁵ residue at third position and disulfide bond at Cys⁹⁴-Cys⁵⁸⁴ and Cys⁴²⁹-Cys⁶¹⁴ position respectively. Corresponding insect zoom shows the disulfide bonded Cys²-Cys⁴⁷⁸, the dominant ion is the doubly charged ion of [M +2H]²⁺ at m/z 3566.5.

Figure 8. LC-ESI-MS/MS of peptide containing free Cysteine406.

(A). LC-ESI-MS/MS of m/z 776.6, containing free Cys⁴⁰⁶ from DTT treated, trypsin and chymotrypsin digested MtbDGC. Inset shows the sequence derived from this MS/MS spectrum. (B) LC-ESI-MS/MS of m/z 833.7, containing free Cys⁴⁰⁶ from unreduced trypsin and chymotrypsin digested MtbDGC. Inset shows the sequence derived from this MS/MS spectrum. (C) LC-ESI-MS/MS of m/z 1396, containing free Cys⁴⁰⁶ from DTT treated, chymotrypsin digested MtbDGC. Inset shows the sequence derived from this MS/MS spectrum.

Figure 9. LC-ESI-MS/MS of disulfide connected dipeptide.

(A) LC-ESI-MS/MS m/z 3566.5 (doubly charge), containing disulfide bonded Cys²-Cys⁴⁷⁹ peptide from trypsin and chymotrypsin digested MtbDGC. Inset shows the sequence derived from this MS/MS spectrum. (B) LC-ESI-MS/MS of m/z 1053.2, containing disulfide bonded Cys⁹⁴-Cys⁵⁸⁴ peptide from trypsin and chymotrypsin digested intact unreduced MtbDGC. Inset shows the sequence derived from this MS/MS spectrum. (C) LC-ESI-MS/MS of m/z 2799.1, containing disulfide bonded Cys⁴²⁹-Cys⁶¹⁴ peptide from trypsin and chymotrypsin digested MtbDGC. Inset shows the sequence derived from this MS/MS spectrum.

Figure 10. Modeled MtbDGC.

(A) Distribution of seven cysteines on full length MtbDGC. (B) Modeled MtbDGC with the disulfide bonds. (a) Modeled protein with exposed Cys406 residue. GGDEF motif (residue number 261–265) and is shown in black color. (b) Disulfide bond between Cys residue Cys2-Cys479 (c) Disulfide bond between Cys residue 894-584. (d) free and exposed Cys406 (e) disulfide between Cys residue 429-614.

Figure 11. Part of EAL domain with the residues of loop 6.

Hydrogen bond between residues is shown in the black broken line.

Figure 12. EAL motif shown in yellow color and the conserved residues in close proximity to the motif is highlighted.

Figure 13. Functional activity of MtbC406S mutated protein.

(A) LC-ESI-MS of DTT reduced MtbC406S digested with trypsin and chymotrypsin. The m/z 760.9 shows the substitution of Cys⁴⁰⁶ to Serine⁴⁰⁶. (B) (a) MtbC406S protein: MALDI TOF analysis, in the positive ion detection mode. No peaks are obtained at 691 (M+H)⁺ and 713 (M+Na)⁺ for c-di-GMP, 709 (M+H)⁺ and 731 (M + Na)⁺ for pGpG (b) MtbDGC protein for control: MALDI TOF analysis, in the positive ion detection mode, ions were detected at 691 (M+H)⁺ and 713 (M+Na)⁺ for c-di-GMP (c) 709 (M+H)⁺ and 731 (M + Na)⁺ for pGpG.

Figure 14. Circular Dichorism spectra of MtbDGC and MtbC406S.