Crystal structure of low-molecular-weight protein tyrosine phosphatase from Mycobacterium tuberculosis at 1.9-A resolution

Abstract

The low-molecular-weight protein tyrosine phosphatase (LMWPTPase) belongs to a distinctive class of phosphotyrosine phosphatases widely distributed among prokaryotes and eukaryotes. We report here the crystal structure of LMWPTPase of microbial origin, the first of its kind from Mycobacterium tuberculosis. The structure was determined to be two crystal forms at 1.9- and 2.5-A resolutions. These structural forms are compared with those of the LMWPTPases of eukaryotes. Though the overall structure resembles that of the eukaryotic LMWPTPases, there are significant changes around the active site and the protein tyrosine phosphatase (PTP) loop. The variable loop forming the wall of the crevice leading to the active site is conformationally unchanged from that of mammalian LMWPTPase; however, differences are observed in the residues involved, suggesting that they have a role in influencing different substrate specificities. The single amino acid substitution (Leu12Thr [underlined below]) in the consensus sequence of the PTP loop, CTGNICRS, has a major role in the stabilization of the PTP loop, unlike what occurs in mammalian LMWPTPases. A chloride ion and a glycerol molecule were modeled in the active site where the chloride ion interacts in a manner similar to that of phosphate with the main chain nitrogens of the PTP loop. This structural study, in addition to identifying specific mycobacterial features, may also form the basis for exploring the mechanism of the substrate specificities of bacterial LMWPTPases.

FIG. 1.

Crystal structure of M. tuberculosis low-molecular-weight MPtpA. (A) Ribbon diagram of the structure of MPtpA (A). The disposition of the three invariant catalytic residues is shown with a 2Fo-Fc electron density map contoured at 1.1 σ. N-ter, N terminus; C-ter, C terminus. (B) Structural superposition of MPtpA (A) (green) with HCPTPA (purple). Large structural deviations could be noted among the β4-to-α5 (insertion), α4-to-β4 (deletion), α3-to-β3 regions, and the extended α5 helix. N-ter and C-ter labels apply to the MPtpA structure. (C) Stereo diagram showing the modeled chloride ion in the phosphate-binding site of the active-site region. Thr12, being involved in additional stabilization of the PTP loop through its interaction with His93, is indicated. (D) Superposition of the active-site region of MPtpA (B) with HCPTPA and BPTP shown in stereo view. The residues implicated in substrate specificity are His49 (Glu in HCPTPA and Asn in BPTP), Ser52 (Asn in HCPTPA and Arg in BPTP), and Glu56 (Tyr in HCPTPA and Pro in BPTP) and are indicated with glycerol in the active-site region. The side-chain atoms of MPtpA and glycerol are shown in atomic-color mode (O, N, and S atoms are in red, blue, and green, respectively), while HCPTPA and BPTP side chains are shown in red and green, respectively. The depictions in panels A to D were generated using the SETOR program (13). (E) View of the active site showing the surface charge distributions of MPtpA (A) (left) and HCPTPA (right), which are displayed with crucial residues that determine the indicated substrate specificities. For MPtpA, the crevice leading to the active site is indicated by tryptophan and aromatic residues positioned on either side of the active site. This depiction was generated using the GRASP program (30).

FIG. 2.

Structure-based sequence alignment of the low-molecular-weight PTPases MPtpA, BPTP, HCPTPA, and LTP1, along with those from E. coli, and S. enterica serovar Typhi. The active-site signature motifs of the PTP loop and the variable-loop residues in MPtpA are shown in boxes. The residues presumed to determine substrate specificities and that differ from those of mammalian PTPases are shaded. The residues involved in MPtpA PTP loop stabilization are underlined. This figure was created using the program ALSCRIPT (4).