Experimental and computational approaches for membrane protein insertion and topology determination.

Membrane proteins play pivotal roles in a wide array of cellular processes and constitute approximately a quarter of the protein-coding genes across all organisms. Despite their ubiquity and biological significance, our understanding of these proteins remains notably less comprehensive compared to their soluble counterparts. This disparity in knowledge can be attributed, in part, to the inherent challenges associated with employing specialized techniques for the investigation of membrane protein insertion and topology. This review will center on a discussion of molecular biology methodologies and computational prediction tools designed to elucidate the insertion and topology of helical membrane proteins.

Computational design of BclxL inhibitors that target transmembrane domain interactions.

Several methods have been developed to explore interactions among water-soluble proteins or regions of proteins. However, techniques to target transmembrane domains (TMDs) have not been examined thoroughly despite their importance. Here, we developed a computational approach to design sequences that specifically modulate protein-protein interactions in the membrane. To illustrate this method, we demonstrated that BclxL can interact with other members of the B cell lymphoma 2 (Bcl2) family through the TMD and that these interactions are required for BclxL control of cell death. Next, we designed sequences that specifically recognize and sequester the TMD of BclxL. Hence, we were able to prevent BclxL intramembrane interactions and cancel its antiapoptotic effect. These results advance our understanding of protein-protein interactions in membranes and provide a means to modulate them. Moreover, the success of our approach may trigger the development of a generation of inhibitors targeting interactions between TMDs.

Intra-Helical Salt Bridge Contribution to Membrane Protein Insertion.

Salt bridges between negatively (D, E) and positively charged (K, R, H) amino acids play an important role in protein stabilization. This has a more prevalent effect in membrane proteins where polar amino acids are exposed to a hydrophobic environment. In transmembrane (TM) helices the presence of charged residues can hinder the insertion of the helices into the membrane. It is possible that the formation of salt bridges could decrease the cost of membrane integration. However, the presence of intra-helical salt bridges in TM domains and their effect on insertion has not been properly studied yet. In this work, we show that potentially salt-bridge forming pairs are statistically over-represented in TM-helices. We then selected some candidates to experimentally determine the contribution of these electrostatic interactions to the translocon-assisted membrane insertion process. Using both in vitro and whole cell systems, we confirm the presence of intra-helical salt bridges in TM segments during biogenesis and determined that they contribute ∼0.5 kcal/mol to the apparent free energy of membrane insertion (ΔGapp). Our observations suggest that salt bridge interactions can be stabilized during translocon-mediated insertion and thus could be relevant to consider for the future development of membrane protein prediction software.

Methodological approaches for the analysis of transmembrane domain interactions: A systematic review.

The study of protein-protein interactions (PPI) has proven fundamental for the understanding of the most relevant cell processes. Any protein domain can participate in PPI, including transmembrane (TM) segments that can establish interactions with other TM domains (TMDs). However, the hydrophobic nature of TMDs and the environment they occupy complicates the study of intramembrane PPI, which demands the use of specific approaches and techniques. In this review, we will explore some of the strategies available to study intramembrane PPI in vitro, in vivo, and, in silico, focusing on those techniques that could be carried out in a standard molecular biology laboratory regarding its previous experience with membrane proteins.

The importance of transmembrane domain interactions in the viral control of apoptosis.

Viral control of apoptosis occurs through the expression of viral encoded anti-apoptotic B-cell lymphoma 2 (BCL2) analogs. These proteins are thought to restrain apoptosis by interacting with cellular BCL2 family members. We identified that protein-protein interactions between cellular and viral BCL2 transmembrane domains are crucial for the viral protein's function.

Viral Bcl2s' transmembrane domain interact with host Bcl2 proteins to control cellular apoptosis.

Viral control of programmed cell death relies in part on the expression of viral analogs of the B-cell lymphoma 2 (Bcl2) protein known as viral Bcl2s (vBcl2s). vBcl2s control apoptosis by interacting with host pro- and anti-apoptotic members of the Bcl2 family. Here, we show that the carboxyl-terminal hydrophobic region of herpesviral and poxviral vBcl2s can operate as transmembrane domains (TMDs) and participate in their homo-oligomerization. Additionally, we show that the viral TMDs mediate interactions with cellular pro- and anti-apoptotic Bcl2 TMDs within the membrane. Furthermore, these intra-membrane interactions among viral and cellular proteins are necessary to control cell death upon an apoptotic stimulus. Therefore, their inhibition represents a new potential therapy against viral infections, which are characterized by short- and long-term deregulation of programmed cell death.

Mcl-1 and Bok transmembrane domains: Unexpected players in the modulation of apoptosis.

The Bcl-2 protein family comprises both pro- and antiapoptotic members that control the permeabilization of the mitochondrial outer membrane, a crucial step in the modulation of apoptosis. Recent research has demonstrated that the carboxyl-terminal transmembrane domain (TMD) of some Bcl-2 protein family members can modulate apoptosis; however, the transmembrane interactome of the antiapoptotic protein Mcl-1 remains largely unexplored. Here, we demonstrate that the Mcl-1 TMD forms homooligomers in the mitochondrial membrane, competes with full-length Mcl-1 protein with regards to its antiapoptotic function, and induces cell death in a Bok-dependent manner. While the Bok TMD oligomers locate preferentially to the endoplasmic reticulum (ER), heterooligomerization between the TMDs of Mcl-1 and Bok predominantly takes place at the mitochondrial membrane. Strikingly, the coexpression of Mcl-1 and Bok TMDs produces an increase in ER mitochondrial-associated membranes, suggesting an active role of Mcl-1 in the induced mitochondrial targeting of Bok. Finally, the introduction of Mcl-1 TMD somatic mutations detected in cancer patients alters the TMD interaction pattern to provide the Mcl-1 protein with enhanced antiapoptotic activity, thereby highlighting the clinical relevance of Mcl-1 TMD interactions.

SARS-CoV-2 envelope protein topology in eukaryotic membranes.

Coronavirus E protein is a small membrane protein found in the virus envelope. Different coronavirus E proteins share striking biochemical and functional similarities, but sequence conservation is limited. In this report, we studied the E protein topology from the new SARS-CoV-2 virus both in microsomal membranes and in mammalian cells. Experimental data reveal that E protein is a single-spanning membrane protein with the N-terminus being translocated across the membrane, while the C-terminus is exposed to the cytoplasmic side (Ntlum/Ctcyt). The defined membrane protein topology of SARS-CoV-2 E protein may provide a useful framework to understand its interaction with other viral and host components and contribute to establish the basis to tackle the pathogenesis of SARS-CoV-2.

A Bimolecular Multicellular Complementation System for the Detection of Syncytium Formation: A New Methodology for the Identification of Nipah Virus Entry Inhibitors.

Fusion of viral and cellular membranes is a key step during the viral life cycle. Enveloped viruses trigger this process by means of specialized viral proteins expressed on their surface, the so-called viral fusion proteins. There are multiple assays to analyze the viral entry including those that focus on the cell-cell fusion induced by some viral proteins. These methods often rely on the identification of multinucleated cells (syncytium) as a result of cell membrane fusions. In this manuscript, we describe a novel methodology for the study of cell-cell fusion. Our approach, named Bimolecular Multicellular Complementation (BiMuC), provides an adjustable platform to qualitatively and quantitatively investigate the formation of a syncytium. Furthermore, we demonstrated that our procedure meets the requirements of a drug discovery approach and performed a proof of concept small molecule high-throughput screening to identify compounds that could block the entry of the emerging Nipah virus.