Neutron reflectometry and NMR spectroscopy of full-length Bcl-2 protein reveal its membrane localization and conformation

Ameeq Ul Mushtaq,Luke A Clifton,Artur P G Dingeldein,Gerhard Gröbner,Mario Campana,Hanna Wacklin-Knecht,Jörgen Ådén,Tobias Sparrman,Cecilia Persson

doi:10.1038/s42003-021-02032-1

Abstract

B-cell lymphoma 2 (Bcl-2) proteins are the main regulators of mitochondrial apoptosis. Anti-apoptotic Bcl-2 proteins possess a hydrophobic tail-anchor enabling them to translocate to their target membrane and to shift into an active conformation where they inhibit pro-apoptotic Bcl-2 proteins to ensure cell survival. To address the unknown molecular basis of their cell-protecting functionality, we used intact human Bcl-2 protein natively residing at the mitochondrial outer membrane and applied neutron reflectometry and NMR spectroscopy. Here we show that the active full-length protein is entirely buried into its target membrane except for the regulatory flexible loop domain (FLD), which stretches into the aqueous exterior. The membrane location of Bcl-2 and its conformational state seems to be important for its cell-protecting activity, often infamously upregulated in cancers. Most likely, this situation enables the Bcl-2 protein to sequester pro-apoptotic Bcl-2 proteins at the membrane level while sensing cytosolic regulative signals via its FLD region.

Highlights

B-cell lymphoma 2 (Bcl-2) proteins are the main regulators of mitochondrial apoptosis
For neutron reflectometry (NR) analyses, full-length human Bcl-2 protein was reconstituted in supported L-α-dimyristoylphosphatidylcholine (DMPC) lipid bilayers at a 70:1 lipid-to-protein molar ratio
The Quartz Crystal Microbalance with Dissipation (QCM-D) technique was used to develop a protocol for membrane deposition and protein incorporation with NR reflectometry data (Fig. 2a) confirming that well-aligned supported DMPC/Bcl-2 and pure DMPC bilayers could be generated

Summary

Introduction

B-cell lymphoma 2 (Bcl-2) proteins are the main regulators of mitochondrial apoptosis. The membrane location of Bcl-2 and its conformational state seems to be important for its cell-protecting activity, often infamously upregulated in cancers Most likely, this situation enables the Bcl-2 protein to sequester pro-apoptotic Bcl-2 proteins at the membrane level while sensing cytosolic regulative signals via its FLD region. Following activation of the intrinsic apoptotic pathway by intracellular stress, mitochondria play major roles in the process through permeabilization of the mitochondrial outer membrane (MOM) system and consequent release of apoptotic proteins such as cytochrome c from their intermembrane spaces[3]. This paves the irreversible way towards cellular selfdestruction[4]. Bcl-2 has a specific groove region which is central in restraining these apoptotic proteins (Bax, Bak, and BH3 only proteins) by binding to their Bcl-2 homology 3 (BH3) motifs

Methods

Results

Conclusion