Structure and functional characterization of Vibrio parahaemolyticus thermostable direct hemolysin

Abstract

Thermostable direct hemolysin (TDH) is a major virulence factor of Vibrio parahaemolyticus that causes pandemic foodborne enterocolitis mediated by seafood. TDH exists as a tetramer in solution, and it possesses extreme hemolytic activity. Here, we present the crystal structure of the TDH tetramer at 1.5 A resolution. The TDH tetramer forms a central pore with dimensions of 23 A in diameter and approximately 50 A in depth. Pi-cation interactions between protomers comprising the tetramer were indispensable for hemolytic activity of TDH. The N-terminal region was intrinsically disordered outside of the pore. Molecular dynamic simulations suggested that water molecules permeate freely through the central and side channel pores. Electron micrographs showed that tetrameric TDH attached to liposomes, and some of the tetramer associated with liposome via one protomer. These findings imply a novel membrane attachment mechanism by a soluble tetrameric pore-forming toxin.

FIGURE 1.

Monomeric and tetrameric structures of TDH. A, ribbon representation of the TDH monomer. A disulfide bond is shown by stick representation. B, Cα-tracing of the TDH tetramer. Individual protomers are shown as green, cyan, magenta, and yellow wire models. The molecular surface is shown in transparent representation. Water molecules are indicated by red spheres. The crystallographic 4-fold axis is shown by a black square. Top (C) and side (D) views of TDH tetramer superimposed to three-dimensional reconstruction from 2,516 electron microscopic images of the TDH tetramer. E, intersubunit interactions. Only two protomers are shown in green and cyan ribbon representation. Residues interactions with Arg⁴⁶ are only shown by stick models (left panel). A close-up stereo view (right panel) of intersubunit interactions. Ionic interactions and hydrogen bonds are shown by dashed lines.

FIGURE 2.

Characterization of TDH mutants. Sedimentation velocity analyses of wild-type TDH (A, red) and R46E (B, cyan). Boundary fits to the sedimentation scans are shown for absorbance data at 1.0 mg/ml at a rotor speed of 40,000 (wild-type) or 50,000 rpm (R46E). Electron micrographs of wild-type TDH (C; bar = 60 nm) and R46E (D), with the same magnification as in C. E, size exclusion chromatography analysis of the mutants. F, hemolytic activities of wild-type, R46E, Y140F, and Y140A mutants.

FIGURE 3.

SAXS analysis of TDH. A, red circles indicate the raw data measured at pH 7.0, 20 °C (9). The theoretical profile calculated by 20 models is drawn as a black line (±S.D.). The protein concentration was 9.6 mg/ml. The superimposed volume envelopes of TDH constructed from twenty SAXS models. B, top view. C, side view. Molecular surface from the crystal (blue), the most populated model from SAXS analysis (red sphere, N terminus 11 residues; white sphere; amino acids 12–165), and the second to 20th most highly populated structures calculated for the N-terminal 11 residues (green).

FIGURE 4.

Molecular dynamic simulation of the TDH tetramer. A, profile of the pore diameter. B, the side channel (the hole centered in the gray region) located between β8, β9, β10, the 3₁₀ helix, the C-terminal loop of the cyan protomer and the β3–β4 loop of the green protomer. C, an example of full permeation events during water transport. The surface area of TDH is shown in white. The colored spheres illustrate the position of a water molecule during the process. D, an example of water permeation through the side channel. The colors of the water molecule are changed from blue, green, yellow, and orange to red to indicate the sequential positions of a water molecule passing through the pore.

FIGURE 5.

Attachment of TDH to lipid membrane. TEM analysis of 1 mm liposomes incubated with 0.019 mg/ml TDH (A) or without TDH (B) at 37 °C for 15 min. Black bar = 100 nm. Representative electron microscopic images of TDH and liposome. TDH attached to the liposome with one corner (C), and with two corners (D). Red bar = 10 nm. TDH was traced in red, and liposome was in blue (lower panel).