A universal stress protein (USP) in mycobacteria binds cAMP

Abstract

Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG_3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as "sinks" for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell.

Keywords: Crystal Structure; Cyclic AMP (cAMP); Isothermal Titration Calorimetry (ITC); Mycobacteria; Surface Plasmon Resonance (SPR); Universal Stress Protein.

FIGURE 1.

Determination of the bound fraction of cAMP and identification of an abundant cAMP-binding protein in mycobacteria. A, measurement of the bound (gray) and free (white) cAMP concentrations in the culture supernatant of M. smegmatis and M. bovis BCG cultures at log and stationary phases of growth. The total extracellular concentration of cAMP is the sum of the bound and free fractions. Means ± S.E. are plotted for three biological replicates. B, measurement of the bound (gray) and free (white) amounts of cAMP in the cytosolic fraction of M. smegmatis and M. bovis BCG cultures at log and stationary phases of growth. The sum of bound and free fractions indicates the total levels of intracellular cAMP. Means ± S.E. are plotted for three biological replicates. C, pulldown assays to identify cAMP-binding proteins in mycobacteria. Left panel, proteins bound to 6-AH-cAMP-agarose beads interacting with M. tuberculosis CDC1551 cell lysates were eluted with 1 mm 5′-AMP and -cAMP, respectively. The band corresponding to ∼15 kDa in the cAMP elution lane was identified as MT_1672 (orthologs of Rv1636) by mass spectrometry. Right panel, lane showing MSMEG_3811 (identified by mass spectrometry) that eluted with 1 mm cAMP following interaction of M. smegmatis cell lysate with 6-AH-cAMP-agarose beads. Gels were visualized by silver staining. D, multiple sequence alignment of Rv1636 (M. tuberculosis H37Rv) with its orthologs present in other mycobacterial species as follows: BCG_1674 (M. bovis BCG), MT_1672 (M. tuberculosis CDC1551), MRA_1646 (M. tuberculosis H37Ra), ML1390 (M. leprae TN), MSMEG_3811 (M. smegmatis mc²155), and MAV_3137 (M. avium (strain 104)). The conserved Walker A-like ATP-binding motif present in USPs is highlighted in gray.

FIGURE 2.

Analysis of kinetic and thermodynamic parameters of binding of Rv1636 and MSMEG_3811 with cAMP. A, Rv1636 and MSMEG_3811 were expressed and purified as an N-terminal His-tagged protein from E. coli SP850 (cyc⁻) cells. Purified proteins were analyzed by SDS-PAGE and stained with Coomassie dye. Lane M, molecular mass marker. B, SPR sensorgram showing the binding kinetics of Rv1636 (1 μm) and MSMEG_3811 (3 μm) onto 8-AHA-cAMP conjugated to a CM5 sensor chip. Data were analyzed using Langmuir single site-binding model, and data shown are representative of experiments repeated three times. C, either cAMP or cGMP (1 mm) was incubated with Rv1636 (1 μm) following which the mixture was injected over the SPR chip. The RU observed is plotted as a percentage of the RU seen with protein alone. Data shown are the means ± S.D. of experiments repeated twice in duplicate. D, thermodynamic parameters of cAMP-binding were measured using isothermal titration calorimetry, with 50 μm Rv1636 or MSMEG_3811 and 1 mm cAMP as injectant solution. The binding isotherms were plotted using a single site binding model, and binding stoichiometry was determined considering monomeric protein concentrations. Data shown are representative of experiments repeated three times.

FIGURE 3.

Parameters of binding of ATP to Rv1636. A, increasing concentrations (micromolars) of free Mg-ATP were incubated with Rv1636 (1 μm) and applied to the SPR chip. Inset, B_max (binding maximum in RU) versus log[ATP] was plotted to measure the IC₅₀. B, isotherm showing the binding of Mg-ATP (3 mm concentration of injectant solution) to Rv1636 (150 μm) using ITC. The table below represents the changes in enthalpy (ΔH) and entropy (ΔS); the number of binding sites (N) and the dissociation constant (K_d) values were measured. Monomer protein concentrations were used for analysis with a single site binding model. Values represent means ± S.D. from three independent experiments.

FIGURE 4.

Crystal structure of MSMEG_3811 bound to cAMP. A, overall structure of the MSMEG_3811/cAMP homodimer. cAMP is shown in stick presentation and colored according to atom type. Secondary structure elements of monomer 1 are labeled. B, nucleotide binding pocket of the MSMEG_3811-cAMP complex with relevant residues shown in stick presentation. Hydrogen bonds are shown as dashed lines. cAMP is shown as sticks colored according to atom type and overlaid with 2F_o − F_c density contoured at 1 σ. C, cAMP ligand of the MSMEG_3811 complex overlaid with F_o − F_c density contoured at 3 σ (green) and −3 σ (red). cAMP is shown as sticks colored according to atom type. D, scheme of the interactions between cAMP and MSMEG_3811. All atoms are colored according to atom type. Hydrogen bonds are shown as dashed lines labeled with the distance between relevant atoms. MSMEG_3811 residues involved in hydrophobic contacts are shown as half-circles. The figure was generated using LigPlot+ (68). E, topology diagrams of Rv1636 apo (left) and MSMEG_3811/cAMP (right), generated with TopDraw and Pro-Origami (67, 69). Cylinders represent α-helices, and arrows correspond to β-strands. Green-shaded secondary structure elements belong to the corresponding second monomer (Fig. 4A). The gray-shaded α-helix (α2) in Rv1636 apo is not visible in the structure, but it will likely form a helix according to secondary structure prediction and sequence alignment with MSMEG_3811 (Fig. 1D). F, overlay of MSMEG_3811/cAMP (blue) with Rv1636 apo (PDB code 1TQ8; green). 84 Cα atoms overlaid with a root mean square deviation of 0.95 Å. cAMP is shown in stick presentation and colored according to atom type. Hydrogen bonds are shown as dashed lines. Residues of the conserved Walker A-like motif are underlined. G, overlay of MSMEG_3811/cAMP (blue) with the ATP-binding universal stress protein TTHA0895 (yellow) used for molecular replacement. 79 Cα atoms overlaid with a root mean square deviation of 1.022 Å. Ligands are shown as sticks and colored according to atom type. Relevant residues for ATP binding of TTHA0895 are shown in stick presentation and colored according to atom type. The ATP-binding region of TTHA0895 with Gly-109, Gly-111, Ser-120, Gln-121, and Ser-122, as well as two magnesium ions (green), coordinate the β- and γ-phosphate groups of ATP. Residues of the conserved Walker A-like motif are underlined.

FIGURE 5.

Potential binding of other cyclic nucleotides to MSMEG_3811. A, overlay of MSMEG_3811/cAMP with the hypothetical ligand cGMP, with relevant residues shown in stick presentation and colored according to atom type. Hypothetical hydrogen bond interactions are shown as dashed lines. The guanine moiety of cGMP would experience some slight clashes with Ala-38 indicated by an outward arrow. The unfavorable interaction between the carbonyl groups of the guanine moiety and of Ala-40 is highlighted by an orange circle. B, placing the cyclic nucleotide c-di-AMP (PDB code 2BA) into the MSMEG_3811/cAMP complex (blue) would lead to a strong clash of one phosphate group with Val-116. Ligand cAMP and hypothetical ligand c-di-AMP are shown as sticks and colored according to atom type. Relevant residues of MSMEG_3811 are labeled and shown in stick presentation.

FIGURE 6.

Phylogenetic tree depicting the relationships between USPs in mycobacteria. USP domain-containing proteins were identified in the genomes of M. tuberculosis (Rv), M. bovis BCG (BCG), M. smegmatis (MSMEG), and M. leprae (ML). Alignment and tree building was performed by MEGA 6. Highlighted as an arc are orthologs of Rv1636, described in this study. Tandem USP domains in proteins are named as USP1 and USP2, respectively.