Structural and functional insight into the Mycobacterium tuberculosis protein PrpR reveals a novel type of transcription factor

Abstract

The pathogenicity of Mycobacterium tuberculosis depends upon its ability to catabolize host cholesterol. Upregulation of the methylcitrate cycle (MCC) is required to assimilate and detoxify propionyl-CoA, a cholesterol degradation product. The transcription of key genes prpC and prpD in MCC is activated by MtPrpR, a member of a family of prokaryotic transcription factors whose structures and modes of action have not been clearly defined. We show that MtPrpR has a novel overall structure and directly binds to CoA or short-chain acyl-CoA derivatives to form a homotetramer that covers the binding cavity and locks CoA tightly inside the protein. The regulation of this process involves a [4Fe4S] cluster located close to the CoA-binding cavity on a neighboring chain. Mutations in the [4Fe4S] cluster binding residues rendered MtPrpR incapable of regulating MCC gene transcription. The structure of MtPrpR without the [4Fe4S] cluster-binding region shows a conformational change that prohibits CoA binding. The stability of this cluster means it is unlikely a redox sensor but may function by sensing ambient iron levels. These results provide mechanistic insights into this family of critical transcription factors who share similar structures and regulate gene transcription using a combination of acyl-CoAs and [4Fe4S] cluster.

Figure 1.

Genome organization of ramB/icl1 and prp regulons and structure of MtPrpR. (A) Genome organization of ramB-icl1 and prpR-prpDC regulons. Dashed lines indicate the presence of genes in between. Distances of the intergenic regions are indicated. The tandem repeats are represented as boxes. (B) Domain organization of MtPrpR: gray-scaled boxes and the numbers above indicate the predicted domain borders; color-filled boxes and the numbers below indicate the domains observed in the structure. The D_III domain is colored in light yellow; the GAF-like domain is colored in blue; the C-terminal region is colored in green. The hatched boxes indicate the domains that were either truncated (HTH) from or not visible (D_II) in the protein construct. (C) Crystal structure of the MtPrpR_81–486 tetramer. The four chains are shown in different colors. Arrows and the central ellipse indicate the 2-fold rotational relationships between each pair of polypeptide chains. (D) The top half of the MtPrpR tetramer. Chain A is colored by domain organization in the same way as in B; Chain B is colored in pink. (E) Close up of the [4Fe4S] cluster-binding site. The 2mFo-DFc electron density of the cluster is contoured at 1 σ.

Figure 2.

MtPrpR CoA binding cavity. (A) The CoA binding cavity in MtPrpR_81–486; the protein chain is colored in gray. The binding cavity is formed by the D_III (left) and the GAF-like (right) domains. Key interacting residues are labeled. CoA is shown in stick and ball; the 2mFo-DFc map of CoA is contoured at 1 σ. (B) Electron density of CoA in A is displayed by a 90-degree rotation. (C and D) Mass spectra of CoA and its derivatives extracted from purified MtPrpR_81–486 expressed in LB (C) or M9-dextrose-iron (D) media. M/z values and identities are labeled (ACO for acetyl-CoA). (E) CoA interacting residues. The π–π interactions are indicated by bold broken lines and colored in green; hydrogen bonds (also including electrostatic interactions) are indicated by dashed lines (blue for interactions between protein and CoA; magenta for interactions to stabilize the side-chain rotamer conformation); orange arc line indicates van der Waals interactions. (F) CoA-binding cavity of Chain B (pink for protein surface, cyan for CoA) covered by the neighboring Chain A (blue).

Figure 3.

Effect of [4Fe4S] cluster upon MtPrpR. (A) Transcription levels of prpD (N = 3) and prpR (N = 3) in MtPrpR variants under acetate or propionate conditions. Values presented as mean ± SD (Data were analyzed with two-way ANOVA followed by Sidak's test, **** P < 0.0001). (B) Conformational change of MtPrpR in the presence (blue and orange for the two neighboring chains of MtPrpR_81–486) or absence (yellow for MtPrpR_155–440) of the [4Fe4S] cluster binding domain. The arrow indicates the open and closed conformations of the loop; Regions within the solid and dashed line boxes are zoomed in with detailed interactions shown in (C) and (D).

Figure 4.

Proposed model of MtPrpR conformational change and transcriptional activation. (A) Location of helix α1 in Chains A and B in MtPrpR_81–486 tetramer. The N-termini of the visible portion of the protein are shown. (B) Model of propionyl-CoA bound in the CoA-binding cavity of Chain A (light blue). Atoms within 4 Å to C1 and C2 of the propionyl group are colored in purple. Rotamers of Phe155 are shown as black wires. A minimum of 15° movement of α1 (dark blue for the new position) is required to overcome the clashes with propionyl-CoA. (C) Schematic of MtPrpR-mediated transcriptional regulation. Binding of propionyl-CoA is proposed to induce a conformational change of MtPrpR via helix α1, which may alter the distances between the adjacent HTH domains and bend the recognition DNA, leading to gene activation.

Figure 5.

The polymorphism of residue 155 (number as in MtPrpR) in the MtPrpR homologs controls the ligand selectivity. (A) Sequence alignment of the helix α1 between MtPrpR and MtRamB, highlighting Phe155 in MtPrpR and its counterpart His143 in MtRamB. (B) Transcription levels of prpD (N = 3) and prpR (N = 3) in MtPrpR_F155 variants under acetate or propionate conditions. Values presented as mean ± SD (Data were analyzed with two-way ANOVA followed by Sidak's test, **** P < 0.0001). (C) Mass spectrum of the ligands. M/z and ligand identities are labeled (SCA for succinyl-CoA; SCA+Na⁺ for the sodium adduct form of SCA). (D) The 2mFo-DFc electron density of succinyl-CoA bound by MtPrpR_81–486_F155H mutant, electron density contoured at 1 σ. (E) The succinyl-CoA binding environment. The dashed lines indicate the interactions between the protein and the succinyl moiety.

Figure 6.

Transcriptional regulation by MtPrpR and MtRamB on prp operon and icl1. (A) Transcription levels of ramB in Mtb H37Rv or ΔprpR strains using acetate or propionate carbon sources with or without CRISPRi induction by anhydrous tetracycline (ATc). (B–D) Transcription levels of icl1,prpR and prpD, respectively, under the same treatment as in A. The black and red bars are WT H37Rv and H37RvΔprpR, respectively, transformed with an ATc inducible vector. N = 2 for each strain, treatment and carbon source combination. Data were analyzed with two-way ANOVA followed by Tukey's test. *** P < 0.0002, ns: not significant.

Figure 7.

Schematic of MtPrpR/MtRamB regulation mediated by short-chain fatty acyl-CoAs. Left panel: MtPrpR-mediated transcriptional activation via propionyl-CoA binding. The upregulation of the prp operon and icl1 leads to a robust MCC to efficiently assimilate and detoxify propionyl-CoA. Right panel: MtRamB-mediated transcriptional repression via succinyl-CoA binding. Succinyl-CoA can be produced at different levels depending on the carbon sources and the metabolic pathways including the glyoxylate shunt and the TCA cycle. Binding to succinyl-CoA by MtRamB leads to the transcriptional repression of icl1 but not the prp operon.