Solution structure of a low-molecular-weight protein tyrosine phosphatase from Bacillus subtilis

Abstract

The low-molecular-weight (LMW) protein tyrosine phosphatases (PTPs) exist ubiquitously in prokaryotes and eukaryotes and play important roles in cellular processes. We report here the solution structure of YwlE, an LMW PTP identified from the gram-positive bacteria Bacillus subtilis. YwlE consists of a twisted central four-stranded parallel beta-sheet with seven alpha-helices packing on both sides. Similar to LMW PTPs from other organisms, the conformation of the YwlE active site is favorable for phosphotyrosine binding, indicating that it may share a common catalytic mechanism in the hydrolysis of phosphate on tyrosine residue in proteins. Though the overall structure resembles that of the eukaryotic LMW PTPs, significant differences were observed around the active site. Residue Asp115 is likely interacting with residue Arg13 through electrostatic interaction or hydrogen bond interaction to stabilize the conformation of the active cavity, which may be a unique character of bacterial LMW PTPs. Residues in the loop region from Phe40 to Thr48 forming a wall of the active cavity are more flexible than those in other regions. Ala41 and Gly45 are located near the active cavity and form a noncharged surface around it. These unique properties demonstrate that this loop may be involved in interaction with specific substrates. In addition, the results from spin relaxation experiments elucidate further insights into the mobility of the active site. The solution structure in combination with the backbone dynamics provides insights into the mechanism of substrate specificity of bacterial LMW PTPs.

FIG. 1.

Sequence alignment of LMW PTPs with B. subtilis YwlE, Mycobacterium tuberculosis MptpA (PDB code 1U2P), Saccharomyces cerevisiae Ltp1 (PDB code 1D1P), bovine PTP (BPTP; PDB code 1PNT), and human HCPTPA (PDB code 5PNT) by the program CLUSTAL W (22). The conserved residues in the P-loop and others mentioned in the text are highlighted in boxes. The protein sequences were obtained from the PDB database.

FIG. 2.

Solution structure of B. subtilis YwlE. (A). The stereo view diagram of the superimposition of 20 representative structures (blue) with the mean structure (orange) of YwlE. (B). The ribbon diagram of the mean structure shows the secondary-structure elements. The P-loop is in blue.

FIG. 3.

Mapping the amide chemical shift changes on the ribbon diagram of YwlE. The color ranging from white to red corresponds to the composite chemical shift changes of amide ¹H and ¹⁵N from 0 to 0.5 ppm. The missing residues T8, G9, T11, C12, and R13 in the HSQC spectra are shown in black.

FIG. 4.

Conformations of the active site. The active sites of YwlE and BPTP are shown in ball-and-stick style and the other regions of the proteins in the stereo view ribbon diagram. The N, H, C, O, and S atoms are shown in blue, gray, black, red, and yellow, respectively. (A). Active site of YwlE. It contains the P-loop (Cys7 to Ser14) and Asp118. Residues Phe40 and Phe120 are responsible for the selection of a tyrosine-containing substrate, and residue Asp115 stabilizes the local conformation of the active cavity by electrostatic interaction with Arg13, which are also shown. (B). Conformation of the active site of BPTP. Residues Cys12 and Arg18 in the P-loop region and Trp49, Asp92, Tyr131, and Tyr132 are shown.

FIG. 5.

Charge distributions on the surface near the active sites of YwlE, HCPTPA, and BPTP. The positive charged regions are in blue, the negative charged regions are in red, and noncharged regions are in white. The active cavity and surface near it are highlighted in yellow circles.

FIG. 6.

Backbone dynamics of YwlE. (A) R₁, R₂, and {¹H}-¹⁵N heteronuclear NOE and (B) reduced spectral density functions J(0), J(ω_N), and J(0.87ω_H) values versus residue numbers. The regular secondary-structure elements are indicated on the top. The experiments were performed on a 600-MHz NMR spectrometer at 25°C. The sample (0.25 mM) was dissolved in 50 mM Tris-HCl buffer containing 50 mM NaCl and 30 mM DTT at pH 7.5.