Latent membrane protein 1 (LMP1) is the primary oncoprotein of Epstein-Barr virus (EBV) and plays versatile roles in the EBV life cycle and pathogenesis. Despite decades of extensive research, the molecular basis for LMP1 folding, assembly, and activation remains unclear. Here, we report cryo-electron microscopy structures of LMP1 in two unexpected assemblies: a symmetric homodimer and a higher-order filamentous oligomer. LMP1 adopts a non-canonical and unpredicted fold that supports the formation of a stable homodimer through tight and antiparallel intermolecular packing. LMP1 dimers further assemble side-by-side into higher-order filamentous oligomers, thereby allowing the accumulation and specific organization of the flexible cytoplasmic tails for efficient recruitment of downstream factors. Super-resolution microscopy and cellular functional assays demonstrate that mutations at both dimeric and oligomeric interfaces disrupt LMP1 higher-order assembly and block multiple LMP1-mediated signaling pathways. Our research provides a framework for understanding the mechanism of LMP1 and for developing potential therapies targeting EBV-associated diseases.

The work presents results of the in vitro and in silico study of formation of amyloid-like structures under harsh denaturing conditions by non-specific OmpF porin of Yersinia pseudotuberculosis (YpOmpF), a membrane protein with β-barrel conformation. It has been shown that in order to obtain amyloid-like porin aggregates, preliminary destabilization of its structure in a buffer solution with acidic pH at elevated temperature followed by long-term incubation at room temperature is necessary. After heating at 95°C in a solution with pH 4.5, significant conformational rearrangements are observed in the porin molecule at the level of tertiary and secondary structure of the protein, which are accompanied by the increase in the content of total β-structure and sharp decrease in the value of characteristic viscosity of the protein solution. Subsequent long-term exposure of the resulting unstable intermediate YpOmpF at room temperature leads to formation of porin aggregates of various shapes and sizes that bind thioflavin T, a specific fluorescent dye for the detection of amyloid-like protein structures. Compared to the initial protein, early intermediates of the amyloidogenic porin pathway, oligomers, have been shown to have increased toxicity to the Neuro-2aCCL-131™ mouse neuroblastoma cells. The results of computer modeling and analysis of the changes in intrinsic fluorescence during protein aggregation suggest that during formation of amyloid-like aggregates, changes in the structure of YpOmpF affect not only the areas with an internally disordered structure corresponding to the external loops of the porin, but also main framework of the molecule, which has a rigid spatial structure inherent to β-barrel.

Aquaporin-0 (AQP0) constitutes 50 % of the lens membrane proteome and plays important roles in lens fiber cell adhesion, water permeability, and lens transparency. Previous work has shown that specific proteins, such as calmodulin (CaM), interact with AQP0 to modulate its water permeability; however, these studies often used AQP0 peptides, rather than full-length protein, to probe these interactions. Furthermore, the specific regions of interaction of several known AQP0 interacting partners, i.e. αA and αB-crystallins, and phakinin (CP49) remain unknown. The purpose of this study was to use crosslinking mass spectrometry (XL-MS) to identify interacting proteins with full-length AQP0 in crude lens cortical membrane fractions and to determine the specific protein regions of interaction. Our results demonstrate, for the first time, that the AQP0 N-terminus can engage in protein interactions. Specific regions of interaction are elucidated for several AQP0 interacting partners including phakinin, α-crystallin, connexin-46, and connexin-50. In addition, two new interacting partners, vimentin and connexin-46, were identified.

SARS-CoV-2 is a highly transmissible virus that causes COVID-19 disease. Mechanisms of viral pathogenesis include excessive inflammation and viral-induced cell death, resulting in tissue damage. We identified the host E3-ubiquitin ligase TRIM7 as an inhibitor of apoptosis and SARS-CoV-2 replication via ubiquitination of the viral membrane (M) protein. Trim7 -/- mice exhibited increased pathology and virus titers associated with epithelial apoptosis and dysregulated immune responses. Mechanistically, TRIM7 ubiquitinates M on K14, which protects cells from cell death. Longitudinal SARS-CoV-2 sequence analysis from infected patients revealed that mutations on M-K14 appeared in circulating variants during the pandemic. The relevance of these mutations was tested in a mouse model. A recombinant M-K14/K15R virus showed reduced viral replication, consistent with the role of K15 in virus assembly, and increased levels of apoptosis associated with the loss of ubiquitination on K14. TRIM7 antiviral activity requires caspase-6 inhibition, linking apoptosis with viral replication and pathology.

Expression and purification of membrane-bound proteins remains a challenge and limits enzymology efforts, contributing to a substantial knowledge gap in the biochemical functions of many proteins found in nature. Accordingly, the study of bacterial UbiA terpene synthases (TSs) has been limited due to the experimental hurdles required to purify active enzymes for characterization in vitro. Previous work employed the use of microsomes or crude membrane fractions to test enzyme activity; however, these methods can be labor intensive, require access to an ultracentrifuge, or may not be suitable for all membrane-bound TSs. We detail here an alternative strategy for the in vivo expression and biochemical characterization of the membrane associated UbiA TSs by employing a precursor overproduction system in Escherichia coli.

Membrane proteins are essential components of biological membranes with key roles in cellular processes such as nutrient transport, cell communication, signaling, or energy conversion. Due to their crucial functions, membrane proteins and their complexes are often targets for therapeutic interventions. Expression and purification of membrane proteins are often a bottleneck to yield sufficient material for structural studies and further downstream characterization. Taking advantage of the Expi293 expression system for the production of eukaryotic proteins, we present a very efficient and fast protocol for the co-expression of a membrane complex. Here, we use transient transfection to co-express the membrane transporter PHT1 with its adaptor protein TASL. To allow the simultaneous screening of different proteins, constructs, or interaction partners, we make use of the Twin-Strep magnetic system. The protocol can be applied for small-scale screening of any membrane protein alone or co-expressed with interacting partners followed by large-scale production and purification of a potential membrane protein complex.

The MC1R protein is a receptor found in melanocytes that plays a role in melanin synthesis. Mutations in this protein can impact hair color, skin tone, tanning ability, and increase the risk of skin cancer. The MC1R protein is activated by the alpha-melanocyte-stimulating hormone (α-MSH). Previous studies have shown that mutations affect the interaction between MC1R and α-MSH; however, the mechanism behind this process is poorly understood. Our study aims to shed light on this mechanism using molecular dynamics (MD) simulations to analyze the Asp84Glu and Asp294His variants. We simulated both the wild-type (WT) protein and the mutants with and without ligand. Our results reveal that mutations induce unique conformations during state transitions, hindering the switch between active and inactive states and decreasing cellular levels of cAMP. Interestingly, Asp294His showed increased ligand affinity but decreased protein activity, highlighting that tighter binding does not always lead to increased activation. Our study provides insights into the molecular mechanisms underlying the impact of MC1R mutations on protein activity.

NMR spectroscopy has played a pivotal role in fragment-based drug discovery by coupling detection of weak ligand-target binding with structural mapping of the binding site. Fragment-based screening by NMR has been successfully applied to many soluble protein targets, but only to a limited number of membrane proteins, despite the fact that many drug targets are membrane proteins. This is partly because of difficulties preparing membrane proteins for NMR-especially human membrane proteins-and because of the inherent complexity associated with solution NMR spectroscopy on membrane protein samples, which require the inclusion of membrane-mimetic agents such as micelles, nanodiscs, or bicelles. Here, we developed a generalizable protocol for fragment-based screening of membrane proteins using NMR. We employed two human membrane protein targets, both in fully protonated detergent micelles: the single-pass C-terminal domain of the amyloid precursor protein, C99, and the tetraspan peripheral myelin protein 22 (PMP22). For both we determined the optimal NMR acquisition parameters, protein concentration, protein-to-micelle ratio, and upper limit to the concentration of D6-DMSO in screening samples. Furthermore, we conducted preliminary screens of a plate-format molecular fragment mixture library using our optimized conditions and were able to identify hit compounds that selectively bound to the respective target proteins. It is hoped that the approaches presented here will be useful in complementing existing methods for discovering lead compounds that target membrane proteins.

In silico validation of de novo designed proteins with deep learning (DL)-based structure prediction algorithms has become mainstream. However, formal evidence of the relationship between a high-quality predicted model and the chance of experimental success is lacking. We used experimentally characterized de novo water-soluble and transmembrane β-barrel designs to show that AlphaFold2 and ESMFold excel at different tasks. ESMFold can efficiently identify designs generated based on high-quality (designable) backbones. However, only AlphaFold2 can predict which sequences have the best chance of experimentally folding among similar designs. We show that ESMFold can generate high-quality structures from just a few predicted contacts and introduce a new approach based on incremental perturbation of the prediction (\"in silico melting\"), which can reveal differences in the presence of favorable contacts between designs. This study provides a new insight on DL-based structure prediction models explainability and on how they could be leveraged for the design of increasingly complex proteins; in particular membrane proteins which have historically lacked basic in silico validation tools.

Liquid crystal (LC) biosensors have received significant attention for their potential applications for point-of-care devices due to their sensitivity, low cost, and easy read-out. They have been employed to detect a wide range of important biological molecules. However, detecting the function of membrane proteins has been extremely challenging due to the difficulty of integrating membrane proteins, lipid membranes, and LCs into one system. In this study, we addressed this challenge by monitoring the proton-pumping function of bacteriorhodopsin (bR) using a pH-sensitive LC thin film biosensor. To achieve this, we deposited purple membranes (PMs) containing a 2D crystal form of bRs onto an LC-aqueous interface. Under light, the PM patches changed the local pH at the LC-aqueous interface, causing a color change in the LC thin film that is observable through a polarizing microscope with crossed polarizers. These findings open up new opportunities to study the biofunctions of membrane proteins and their induced local environmental changes in a solution using LC biosensors.