Heterodimerization of BAK and MCL-1 Activated by Detergent Micelles

Qian Liu,Kalle Gehring

doi:10.1074/jbc.m110.144857

Abstract

BAK is a key protein mediating mitochondrial outer membrane permeabilization; however, its behavior in the membrane is poorly understood. Here, we characterize the conformational changes in BAK and MCL-1 using detergents to mimic the membrane environment and study their interaction by in vitro pulldown experiments, size exclusion chromatography, titration calorimetry, and NMR spectroscopy. The nonionic detergent IGEPAL has little impact on the structure of MCL-1 but induces a conformational change in BAK, whereby its BH3 region is able to engage the hydrophobic groove of MCL-1. Although the zwitterionic detergent CHAPS induces only minor conformational changes in both proteins, it is still able to initiate heterodimerization. The complex of MCL-1 and BAK can be disrupted by a BID-BH3 peptide, which acts through binding to MCL-1, but a mutant peptide, BAK-BH3-L78A, with low affinity for MCL-1 failed to dissociate the complex. The mutation L78A in BAK prevented binding to MCL-1, thus demonstrating the essential role of the BH3 region of BAK in its regulation by MCL-1. Our results validate the current models for the activation of BAK and highlight the potential value of small molecule inhibitors that target MCL-1 directly.

Highlights

PUMA, NOXA, HRK/DPS, NIP3, bNIP3, and MULE) share only a common BH3 region and initiate apoptosis indirectly
When apoptosis is induced by factors such as staurosporine, etoposide, cisplatin, anti-Fas antibody, or detergents [3, 7,8,9], BAK undergoes a conformational change, termed BAK activation, that is characterized by the insertion of additional ␣-helical elements into the membrane and the assembly of BAK into higher order oligomers [10, 11]
In the crystal structure of BAK [6], the BH3 region is partially buried in the protein interior and unavailable for binding without a major conformational change

Summary

Introduction

PUMA, NOXA, HRK/DPS, NIP3, bNIP3, and MULE) share only a common BH3 region and initiate apoptosis indirectly. Neither condition resulted in the formation of a MCL-11⁄7BAK complex, as indicated by the failure of cMCL-1 to be retained on Ni2ϩ-NTA resin by FLAG-BAK-HMK-⌬TM-His6 in a pulldown experiment, the absence of elution shifts in analytical size exclusion chromatography, and the absence of chemical shift changes in the NMR spectra of 15N-labeled cMCL-1 or 15N-labeled cBAK upon the addition of unlabeled partner (data not shown).

Results

Conclusion