Characterization of the Interactions between Calmodulin and Death Receptor 5 in Triple-negative and Estrogen Receptor-positive Breast Cancer Cells: AN INTEGRATED EXPERIMENTAL AND COMPUTATIONAL STUDY

Abstract

Activation of death receptor-5 (DR5) leads to the formation of death inducing signaling complex (DISC) for apoptotic signaling. Targeting DR5 to induce breast cancer apoptosis is a promising strategy to circumvent drug resistance and present a target for breast cancer treatment. Calmodulin (CaM) has been shown to regulate DR5-mediated apoptotic signaling, however, its mechanism remains unknown. In this study, we characterized CaM and DR5 interactions in breast cancer cells with integrated experimental and computational approaches. Results show that CaM directly binds to DR5 in a calcium dependent manner in breast cancer cells. The direct interaction of CaM with DR5 is localized at DR5 death domain. We have predicted and verified the CaM-binding site in DR5 being (354)WEPLMRKLGL(363) that is located at the α2 helix and the loop between α2 helix and α3 helix of DR5 DD. The residues of Trp-354, Arg-359, Glu-355, Leu-363, and Glu-367 in DR5 death domain that are important for DR5 recruitment of FADD and caspase-8 for DISC formation to signal apoptosis also play an important role for CaM-DR5 binding. The changed electrostatic potential distribution in the CaM-binding site in DR5 DD by the point mutations of W354A, E355K, R359A, L363N, or E367K in DR5 DD could directly contribute to the experimentally observed decreased CaM-DR5 binding by the point mutations of the key residues in DR5 DD. Results from this study provide a key step for the further investigation of the role of CaM-DR5 binding in DR5-mediated DISC formation for apoptosis in breast cancer cells.

Keywords: breast cancer; calmodulin (CaM); death domain; death receptor 5; electrostatics; integrated experimental and computational study; protein-protein interaction.

FIGURE 1.

CaM interactions with DR5 in a Ca²⁺-dependent manner in triple-negative MDA-MB-231 and ER-positive ZR-75-1 breast cancer cells. A, co-immunoprecipitation of DR5 and CaM from MDA-MB-231 and ZR-75-1 cell lysates. Lysate lane: whole cell lysate of breast cancer cells; IP TRA-8 lane: IP using TRA-8-conjugated beads; IP Iso-IgG lane: IP using a mouse Iso-IgG-conjugated beads. B, CaM pull-down of DR5 from MDA-MB-231 and ZR-75-1 cell lysates supplemented with 1 mm Ca ²⁺ or 2 mm EGTA. I: input, CaM-S: pull-down using CaM-conjugated Sepharose beads. S: pull-down control using Sepharose beads. Representative results are shown from two independent experiments.

FIGURE 2.

CaM directly interacts with DR5 in DR5 death domain in a Ca²⁺-dependent manner. A, CaM pull-down of purified DR5 cytoplasmic region (DR5 CR) and DR5 death domain (DR5 DD) in a 50 mm Tris, pH 7.6, 120 mm NaCl, 1% Brij buffer with 1 mm Ca ²⁺, or 2 mm EGTA. B, CaM pull-down of purified DR5 CR mutant with the deletion of DR5 death domain (DR5 CR ΔDD) in a 50 mm Tris pH 7.6, 120 mm NaCl, 1 mm Ca²⁺, 1% Brij buffer. I: input, CaM-S: pull-down using CaM-conjugated Sepharose beads. S: pull-down control using Sepharose beads. Representative results are shown from two independent experiments.

FIGURE 3.

Predicted CaM-binding site in DR5 death domain (DR5 DD) and the key residues critical for CaM-DR5 binding. A, predicted CaM-binding site in DR5 DD (³⁵⁴WEPLMRKLGL³⁶³) is underlined. Arrows with solid lines indicate hydrophobic residues, and arrows with dotted lines indicate positively charged residues. Cylinders above the sequence represent α helical regions. B, structural view of DR5 DD with the predicted CaM-binding site (³⁵⁴WEPLMRKLGL³⁶³) shown in yellow color and the predicted key residues of Leu-363, Trp-354, Glu-355, Arg-359, and Glu-367 in DR5 DD shown as licorice.

FIGURE 4.

Validation of the predicted CaM-binding site in DR5 DD and the effect of critical residues in DR5 DD on CaM-DR5 binding. A, CaM pull-down of DR5 cytoplasmic region (DR5 CR) or DR5 CR mutant with the deletion of the predicted CaM-binding site (³⁵⁴WEPLMRKLGL³⁶³) (DR5 CR ΔBSD). B, CaM pull-down of wild type DR5 CR (WT) or DR5 CR mutants: DR5 W354A, DR5 E355K, DR5 R359A, DR5 L363N, and DR5 E367K. I: input, CaM-S: pull-down using CaM-conjugated Sepharose beads. Representative results are shown from two independent experiments.

FIGURE 5.

Electrostatic potential comparison between the CaM-binding site in wild type DR5 DD and DR5 DD mutants. The blue regions show the positive electrostatic potential, whereas the red regions show the negative electrostatic potential. The black dot boxes show the electrostatic potential comparison between the CaM-binding site in wild type DR5 DD and DR5 DD mutants (A) W354A mutant, (B) E355K mutant, (C) R359A mutant, (D) E367K mutant, and (E) L363N mutant.