**1. Introduction**

The regulation step in mitochondrial apoptosis is the permeabilization of the Mitochondrial Outer Membrane (MOM) [1]. It is under the control of the Bcl-2 family of proteins, whose members carry between 1 and 4 Bcl-2 protein homology (BH) regions [2]. The BH3 region, present in all Bcl-2 family proteins, is thought to act as a "death ligand" mediating heterodimerization between family members [3]. Members of the Bcl-2 family have been classified into four different subgroups [2,4,5]. Pro-apoptotic multidomain family members, Bax and Bak, are the executors of the permeabilization process. They oligomerize and form pores in the MOM in response to apoptotic signals. They can be activated by pro-apoptotic BH3-only family members (such as truncated Bid—known as tBid, Bim and Puma [6–8]), through a process thought to involve a direct physical interaction [9–11]. The Bcl-2 family also includes anti-apoptotic multidomain members (e.g., Bcl-2, Bcl-XL, Mcl-1), which work by either directly inhibiting Bax and Bak, or sequestering BH3-only proteins. The last subgroup in the family is made of the so-called sensitizer proteins (e.g., Bad), which regulates MOM permeabilization by inhibiting anti-apoptotic Bcl-2 family members.

The fate of cells is dictated by the intricate balance of interactions between the different groups of Bcl-2 family proteins, a balance often found to be dysregulated in drug-resistant

**Citation:** Rose, M.; Kurylowicz, M.; Mahmood, M.; Winkel, S.; Moran- Mirabal, J.M.; Fradin, C. Direct Measurement of the Affinity between tBid and Bax in a Mitochondria-Like Membrane. *Int. J. Mol. Sci.* **2021**, *22*, 8240. https:// doi.org/10.3390/ijms22158240

Academic Editor: Masoud Jelokhani-Niaraki

Received: 8 June 2021 Accepted: 22 July 2021 Published: 31 July 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

cancer cells and therefore a sought-after target for pharmacological intervention against cancer [12–15]. At the centre of the Bcl-2 family interaction network are several competing interactions: the activator-effector interaction (e.g., between tBid and Bax), the repressoreffector interaction (e.g., between Bcl-XL and Bax) and the activator-repressor interaction (e.g., between between tBid and Bcl-XL), each of them able to tip the balance either towards death or survival. Several models have been put forward that try and capture the main features of the activation of Bax, each giving more or less importance to these different interactions, and with the most recent models incorporating the influence of the lipid membrane, increasingly recognized as a main player in the process [16–19]. One way to discriminate between these different models would be to measure the affinity between different pairs of Bcl-2 family proteins in conditions as close as possible as those encountered in the MOM.

Quantification of the affinity between different pairs of Bcl-2 family proteins has been achieved using ensemble fluorescence resonance energy transfer [10,20,21]. However, it has since become clear that a number of Bcl-2 family proteins (e.g., Bax, tBid, Bcl-XL) exist in a dynamic equilibrium between solution and membrane [22–24]. Ensemble measurements, which do not distinguish between the soluble and membrane forms of the proteins, can thus only return an apparent affinity, which depends on the amount and type of lipids present in the system. Interactions between different pairs of Bcl-2 family proteins embedded in the membrane of giant unilamellar vesicles have also been detected using linear scan cross-correlation spectroscopy [25–27]. Yet these experiments cannot distinguish between the different conformations or stoichiometries these proteins are known to adopt in lipid membranes [28,29]. In contrast, single particle experiments allow measuring affinities between pairs of proteins with specific conformations and stoichiometries, and have been successfully used by us and others to characterize the oligomerization of tBid [29] and Bax [30] in planar membranes. It is therefore the approach we have chosen here to characterize Bcl-2 family protein interactions. One challenge associated with such measurements is that a low surface density of proteins, typically below the 2D-*KD*, must be used for single particle detection, and therefore interacting protein pairs are rare events—a needle in a haystack. A second issue is that coincidental co-localization events may be observed, leading to an underestimate of the 2D-*KD* [31]. We solved both issues by combining the ideas of single particle detection and image correlation spectroscopy (building on previous ideas by others [32]), and calculating a single particle cross-correlation coefficient for each detected pair of particles to identify true binding interactions [33].

Here we focus on the activator-effector interaction, using tBid and Bax as a model system. Bid is a BH3-only direct activator of Bax [10,34,35]. It is unique amongs<sup>t</sup> the BH3- only proteins because in solution it adopts a alpha-helical structure packed into a globular fold which is structurally homologous to that of multidomain family members Bcl-2 and Bax [36,37]. Other BH3-only proteins, on the other hand, are known or predicted to be unstructured or to have a structure distinct from that of other Bcl-2 family proteins [38–41]. Bid gets cleaved by caspase-8 in response to ligand binding to death receptors. The larger of the two resulting Bid fragments, called truncated Bid (tBid), then inserts into the MOM [42]. Upon contact with the membrane, tBid changes conformation and increases its affinity for Bax, which in turn inserts into the MOM [10,43]. Bax then forms oligomers that increase the membrane permeability [6,7]. To fully characterize the interaction between the two proteins at the membrane, we used a reconstituted system capturing all facets of this interaction—full-length fluorescently-labelled purified proteins and a Supported Lipid Bilayer (SLB) with mitochondria-like lipid composition. We used single particle detection on confocal images to sort them into different categories according to their stoichiometry and their mobility, as we have done previously for tBid alone [29], and to detect associations between particles. This allowed us to measure the 2D-*KD* for each protein subpopulation, which shows the tBid-Bax interaction becomes stronger as the proteins insert deeper into the membrane.
