Structural and Functional Insights into the Stealth Protein CpsY of Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (Mtb) is an important and harmful intracellular pathogen that is responsible for the cause of tuberculosis (TB). Mtb capsular polysaccharides can misdirect the host's immune response pathways, resulting in additional challenges in TB treatment. These capsule polysaccharides are biosynthesized by stealth proteins, including CpsY. The structure and functional mechanism of Mtb CpsY are not completely delineated. Here, we reported the crystal structure of CpsY^201-520 at 1.64 Å. CpsY^201-520 comprises three β-sheets with five α-helices on one side and three on the other. Four conserved regions (CR1-CR4) are located near and at the base of its catalytic cavity, and three spacer segments (S1-S3) surround the catalytic cavity. Site-directed mutagenesis demonstrated the strict conservation of R419 at CR3 and S1-S3 in regulating the phosphotransferase activity of CpsY^201-520. In addition, deletion of S2 or S3 (∆S2 or ∆S3) dramatically increased the activity compared to the wild-type (WT) CpsY^201-520. Results from molecular dynamics (MD) simulations showed that S2 and S3 are highly flexible. Our study provides new insights for the development of new vaccines and targeted immunotherapy against Mtb.

Keywords: Mycobacterium tuberculosis; crystal structure; phosphotransferase activity; stealth protein CpsY.

Figure 1

Crystal structure of CpsY^201−520. (A) Schematic representation of four conserved regions (CR1–CR4, colored in cyan, green, red, and magenta) and three spacer segments (S1–S3, colored in purple, blue, and orange). (B) Preliminary crystals of CpsY^201−520 were obtained by the initial screening (left, in blue frame), followed by extensive optimization, leading to diffracting crystals (right, in red frame). (C) Crystal structure of CpsY^201−520 in ribbon representation, in two orientations, with the four conserved regions (CR1–CR4) and three spacer segments (S1–S3) colored, as presented in Figure 1A. Dashed lines in the structure of CpsY^201−520 represent a loop with invisible electron densities.

Figure 2

Comparative structural analysis of CpsY^201−520 (in cyan) and zebrafish GlcNAc-1-phosphotransferase (zebrafish GNPTAB, PDB code 7SJ2, in gray). Spacer segment 3 (S3) of CpsY^201−520 is a short loop in our structure but forms a protruding domain comprising loops and alpha helices in the zebrafish GNPTAB structure (inset (A)). Spacer segment 1 (S1) is disordered in our structure (shown as a dashed line) but forms a long helix (inset (B)) in the zebrafish GNPTAB.

Figure 3

Comparative analysis of phosphotransferase activity between CpsY^201−520 and CpsY. The phosphotransferase activity of CpsY^201−520 was identical to that of the full-length CpsY, showing that CpsY^201−520 was responsible for all the activity of the full-length CpsY. The activity of wild-type (WT) full-length CpsY was set to 100%.

Figure 4

Substrate UDP-GlcNAc binds deep in CpsY^201−520 based on the homology model of CpsY^201−520:UDP-GlcNAc binary complex. The complex structure is shown in either surface (A) or ribbon (B) representation. The surface is colored by the electric charge: negative charges in red, positive charges in blue, and neutral charges in white. (C) Sequence alignment of conserved residues (in red) from various species. MYCTU, Mycobacterium tuberculosis; NOCFA, Nocardia farcinica; ACTPL, Actinobacillus pleuropneumoniae; NEIME, Neisseria meningitidis; LACPL, Lactiplantibacillus plantarum; NEIMD, Neisseria meningitidis serogroup A. (D) Relative phosphotransferase activities of WT CpsY^201−520 and the indicated mutants. The E305A, N322A, or D324A mutants displayed significantly decreased phosphotransferase activity in comparison to the WT protein, whereas the R419K or R419Q mutants showed a complete loss of activity. The activity of WT CpsY^201−520 was set to 100%.

Figure 5

Effect of conserved regions (CRs) in CpsY^201−520 near the UDP-GlcNAc substrate on the phosphotransferase activity of CpsY^201−520. These regions are highlighted in (A–C): (A) overall structure of WT CpsY^201−520. (B) The 257–260 segment is rendered in blue, and the 311–321 segment is displayed in purple-blue. (C) The 267–280 segment is shown in light pink, and the 201–211 segment is depicted in green. (D–F) Quantified phosphotransferase activities were observed in various variants of CpsY^201−520 with segment deletion. The deletion of 257–260, 267–280, or 311–321 (Δ257–260, Δ267–280, or Δ311–321) completely abolished the activity, whereas the removal of 201–211 (Δ201–211) significantly increased the activity. The reference point for comparison was the 100% activity of WT CpsY^201−520.

Figure 6

Influence of the spacer segments (S1–S3) (A) near the catalytic cavity on the phosphotransferase activity of CpsY^201−520. (B)The right panel is the phosphotransferase activities of various variants of CpsY^201−520 with deletions on the spacer regions. The deletion of S1 (∆S1) almost fully abolished the activity of CpsY^201−520, while the removal of S2 or S3 (∆S2 or ∆S3) dramatically increased the activity. The reference point for comparison was the 100% activity of WT CpsY^201−520.

Figure 7

The high flexibility of S2 and S3 based on the results of the root mean square fluctuation (RMSF) profile of CpsY^201−520. The peaks of segments S2 and S3 were significantly higher, showing that S2 and S3 possess greater volatility compared to that of other segments of CpsY^201−520. RMSF evaluates the flexibility of a residue by measuring the relative fluctuation of atomic location in the backbone structure and determining the mean deviation of amino acid residues from a reference position in a time-bound manner.

Figure 8

Gating regulatory model for CpsY^201−520. Spacer region S2 (in magenta) and S3 (in orange) serve as gates regulating substrate binding. (A) Both S2 and S3 constrict the dimensions of the catalytic pocket, resulting in the inactivation of CpsY^201−520. (B) Both S2 and S3 enlarge the dimensions of the catalytic pocket, consequently activating CpsY^201−520.