Paclitaxel binding to human and murine MD-2

Abstract

Paclitaxel (PTX) is an important cancer chemotherapeutic agent that binds to beta-tubulin and prevents mitosis through microtubule overstabilization. Recent evidence also implicates PTX in the induction of apoptosis of cancer cells via the TLR4 innate immune pathway. The TLR4 accessory protein, MD-2, is an essential component for the species-specific proinflammatory activity of PTX on murine cells. However, whether PTX binds to human MD-2 and how MD-2 and TLR4 interact with PTX are not well defined. Recombinant human MD-2 (rhMD-2) was produced in a Pichia pastoris expression system, and the interaction between rhMD-2 and PTX was assessed by an enzyme-linked immunosorbent assay to show that PTX binds rhMD-2. Formation of the latter complex was found to be dose-dependent and inhibited by anti-MD-2 antibody but not by an isotype control antibody. As measured by human tumor necrosis factor alpha production, human THP-1 monocytes expressing TLR4 and MD-2 were poorly responsive to the addition of PTX, but murine macrophages expressing TLR4 and MD-2 responded in a dose-dependent manner. Human embryonic kidney (HEK293) cells transfected with both human TLR4 and human MD-2 or human MD-2 and murine TLR4 were also poorly responsive to PTX (10 microm). However, HEK293 cells transfected with murine MD-2 and human TLR4 or murine MD-2 and murine TLR4 were highly responsive to PTX (10 microm), indicating that the murine MD-2/PTX interaction is required for TLR4 activation. To further define the structural differences for MD-2/TLR4 activation, crystal structures of both murine and human MD-2 were subjected to PTX docking by computational methods. These models indicate that PTX binds in the pocket of both human and mouse MD-2 structures. The species-specific difference between human and murine MD-2 activation of TLR4 by PTX can be explained by alterations of surface charge distribution (i.e. electrostatic potential), binding pocket size, and the locus of PTX binding within the MD-2 pocket, which results in reorganization of the 123-130 amino acid loop. In particular, Phe(126) appears to operate as a bridge for TLR4.MD-2 dimerization in the mouse but not the human protein.

FIGURE 1.

Paclitaxel. a, topological structure of PTX. b, three-dimensional conformation of PTX as observed in β-tubulin (i.e. T-Taxol (6)). This conformer is one of the many possible options for PTX binding to MD-2 as revealed by Glide docking.

FIGURE 2.

MD-2 structures. a, hMD-2 crystal structure, ligand removed (Protein Data Bank code 2Z65). b, mMD-2 crystal structure (Protein Data Bank code 2Z64).

FIGURE 3.

Proinflammatory response to PTX is species-specific. a, RAW264.7 cells (10⁶ cells/ml) were stimulated with serial dilutions of PTX (100-0.19 μm). Murine TNFα production was measured by ELISA. PTX activated murine macrophages cells in a dose-dependent manner. Unstimulated cells were used as a control. Error bars, ±S.D. from the mean. b, differentiated THP-1 monocytes (10⁶ cells/ml) were stimulated with 0.56 pmol of wild-type meningococcal LOS (NMB) or dilutions of PTX (12.5-100 μm). Human TNFα production was measured by ELISA. Human monocytes were poorly responsive to PTX, as measured by TNFα release. Unstimulated cells were used as a control. Error bars, ±S.D. from the mean.

FIGURE 4.

HEK293 cells transfected with mouse but not human TLR4 and MD-2 respond to PTX. HEK293 cells (10⁶ cell/ml) were transfected with plasmids for human TLR4 and human MD-2 or mouse TLR4 and mouse MD-2. Transfectants were stimulated with wild-type meningococcal LOS (NMB) (0.56 nm) or PTX (10 μm). Interleukin-8 production (pg/ml) was measured by ELISA. Both human and murine transfectants responded to NMB; however, only murine transfectants responded to PTX. Untransfected cells were unresponsive. Error bars, ±S.D. from the mean.

FIGURE 5.

PTX binds to recombinant human MD-2. a, human rMD-2 was coated on Maxisorp plates. PTX (1 or 10 μm) was added, and unbound rhMD-2 was recognized by anti-MD-2 antibody. NMB (0.3 μg/ml) and BSA were used as controls. Error bars, ±S.D. from the mean. b, meningococcal LOS, PTX, or BSA were coated on Maxisorp plates. The binding of human rMD-2 to wild-type meningococcal LOS (0.17 nmol of NMB; closed diamonds) and PTX (10 μm; closed squares) was inhibited by increasing concentrations of anti-MD-2 antibody (0-2 μg/ml) (x axis). Isotype control antibody (0-2 μg/ml) did not inhibit binding (open symbols). Human rMD-2 did not bind to BSA-coated wells. Error bars, ±S.D. from the mean. c, PTX was coated on Maxisorp plates. Increasing concentrations of PTX (0, 10, and 100 μm) were recognized by human rMD-2 (0-100 μg/ml). Binding of rhMD-2 to PTX was recognized by anti-His₆ antibody. Error bars, ±S.D. from the mean.

FIGURE 6.

Lipid molecules in hMD-2 bind differently to the cavity mouth. a, lipid IVa in hMD-2 (Protein Data Bank code 2E59) clamped by Lys¹²² at the upper lip of the cavity mouth and by Arg⁹⁰ and Glu⁹² at the lower lip. b, eritoran in hMD-2 (Protein Data Bank code 2Z65), illustrating columbic interactions with Lys¹²² and Lys¹²⁵ on the upper lip but no interactions with Arg⁹⁰ and Glu⁹².

FIGURE 7.

Electrostatic potential energy surfaces of MD-2 and MD-2·TLR4 models. Left, mouse; right, human. a, electrostatic surfaces (blue is negative) of the MD-2 proteins in the MD-2·TLR4 x-ray complexes (m2Z64 and h2Z65) (30) were produced with SYBYL software. The surfaces are shown with the binding cavities circled in white and the Cys⁹⁵-Cys¹⁰⁵ loops in yellow; b, complexes of MD-2·TLR4 in which the MD-2 is pictured to the right of each graphic as a solid surface, whereas the TLR4 protein is shown as translucent. The corresponding Cys⁹⁵-Cys¹⁰⁵ loops are circled in yellow. In the human MD-2·TLR4 complex, the latter loop is not in direct van der Waals contact with TLR4 residues.

FIGURE 8.

Lipophilic and hydrophobic surfaces of the MD-2 models. Lipophilic (LP; blue) and hydrophobic (brown) surfaces of MD-2 (2Z64 and 2Z65) were obtained with SYBYL software. The surfaces are shown from the front (top; cavities are circled for clarity) and from the back (bottom).

FIGURE 9.

MD-2·PTX docking poses. a, the top docking poses for PTX in mouse MD-2 (green) and human MD-2 (blue) (Protein Data Bank codes 2Z64 and 2Z65, respectively); b, detailed view of the top PTX docking pose in human MD-2 (Protein Data Bank code 2Z65) illustrating the Phe¹²⁶-benzamido phenyl hydrophobic interaction and the Lys¹²²-C13 phenyl π-cation interaction.

FIGURE 10.

Detailed view of the top PTX docking pose in human MD-2 (Protein Data Bank code 2Z65) illustrating hydrogen bonds for Ser¹²⁰-C1 hydroxyl, Glu⁹²-C7 hydroxyl, and the Arg⁹⁰-C13 hydroxyl.

FIGURE 11.

Comparison between mMD-2 top docking pose (blue) and mMD-2 crystal structure (orange). PTX docking shifts the 123-130 loop (circled in yellow), including Phe¹²⁶ outward (blue) relative to the crystal structure.

FIGURE 12.

Molecular surface of MD-2 models. a, mouse MD-2 protein (Protein Data Bank code 2Z64) with Phe¹²⁶ in red; b, human MD-2 protein (Protein Data Bank code 2Z65) with Phe¹²⁶ in red.