Search Results (37)

We explored the pharmacology of the P-glycoprotein (P-gp) efflux pump and its role in multidrug resistance. We used Protein Data Bank (PDB) database mining and the artificial intelligence (AI) model Boltz-2.1.1, developed for simultaneous structure and affinity prediction, to explore the multimeric nature of recent P-gp inhibitors. We construct a MARTINI coarse-grained (CG) force field description of P-gp embedded in a model of the endothelial blood–brain barrier. We found that recent P-gp inhibitors have been captured in either monomeric, dimeric, or trimeric states. Our CG model demonstrates the ability of P-gp substrates to permeate and transition across the BBB bilayer. We report a multimodal binding model of P-gp inhibition in which later generations of inhibitors are found in dimeric and trimeric states. We report analyses of P-gp substrates that point to an extended binding surface that explains how P-gp can bind over 300 substrates non-selectively. Our coarse-grained model of substrate permeation into membranes expressing P-gp shows benchmarking similarities to prior atomistic models and provide new insights on far longer timescales. Full article

BAR domains are a superfamily of widely conserved membrane binding motifs. In fungi, Pil1 family proteins are BAR domain containing proteins involved in organizing the plasma membrane. S. pombe encodes a sporulation-specific Pil1 family protein, Meu14, which has a specialized role in shaping the forespore membrane during sporulation. The functional analog of Meu14 in S. cerevisiae is Ssp1. While Ssp1 has no primary sequence homology to Pil1 or Meu14, AlphaFold predicts that it contains a Pil1-related BAR domain. Consistent with this structural prediction, mutation of residues in the putative lipid binding face of Ssp1 or in a residue implicated in multimerization disrupt sporulation. Characterization of the mutant proteins indicates that the BAR domain is necessary for recruitment of Ssp1 to the highly curved leading edge of the prospore membrane and multimerization of Ssp1 at that location is required for assembly of the leading edge complex. The distribution of Pil1 family proteins across an evolutionary tree of Ascomycetes reveals that Meu14 and Ssp1 arose independently in the lineages leading to S. pombe and S. cerevisiae, respectively. Full article

Background/Objectives: SARS-CoV-2 vaccine candidates comprising the receptor binding domain (RBD) of the spike protein have been shown to confer protection against infection. Previous research evaluating vaccine candidates with SARS-CoV-2 RBD fused to ferritin (RBD-ferritin) and other scaffolds suggested that multimeric assemblies of RBD can enhance antigen presentation to improve the potency and breadth of immune responses. Though RBDs directly fused to a self-assembling scaffold can be delivered as messenger RNA (mRNA) formulated with lipid nanoparticles (LNPs), reports of SARS-CoV-2 vaccine candidates that combine these approaches remain scarce. Methods: Here, we designed RBD fused to AP205 or TIP60 self-assembling nanoparticles following a search of available structures focused on several scaffold properties. RBD-AP205 and RBD-TIP60 were tested for antigenicity following transfection and for immunogenicity and neutralization potency when delivered as mRNA in mice, with RBD-ferritin as a direct comparator. Results: All scaffolded RBD constructs were readily secreted to transfection supernatant and showed antigenicity in ELISA, though clear heterogeneity in assembly was observed. RBD-AP205 and RBD-TIP60 also exhibited robust antibody binding and neutralization titers in mice that were comparable to those elicited by RBD-ferritin or a full-length membrane-bound spike. Conclusions: These data suggest that AP205 and TIP60 can present RBD as effectively as ferritin and induce similar immune responses. By describing additional scaffolds for multimeric display that accommodate mRNA delivery platforms, this work can provide new tools for future vaccine design efforts. Full article

The β-barrel assembly machinery (BAM) is a multimeric protein complex responsible for the folding of outer membrane proteins in gram-negative bacteria. It is essential for cell survival and outer membrane integrity. Therefore, it is of impact in the context of antibiotic resistance and can serve as a target for the development of new antibiotics. The interaction between two of its subunits, BamA and BamD, is essential for its function. Here, a FRET-based assay to quantify the affinity between these two proteins in living bacterial cells is presented. The method was applied to identify two interaction hotspots at the binding interface. BamD^Y184 was identified to significantly contribute to the binding between both proteins through hydrophobic interactions and hydrogen bonding. Additionally, two salt bridges formed between BamD^R94, BamD^R97, and BamA^E127 contributed substantially to the binding of BamA to BamD as well. Two peptides (RFIRLN and VAEYYTER) that mimic the amino acid sequence of BamD around the identified hotspots were shown to inhibit the interaction between BamA and BamD in a dose-dependent manner in the upper micromolar range. These two peptides can potentially act as antibiotic enhancers. This shows that the BamA–BamD interaction site can be addressed for the design of protein–protein interaction inhibitors. Additionally, the method, as presented in this study, can be used for further functional studies on interactions within the BAM complex. Full article

Enveloped viruses rely on host membranes for trafficking and assembly. A substantial body of literature published over the years supports the involvement of cellular membrane lipids in the enveloped virus assembly processes. In particular, the knowledge regarding the relationship between viral structural proteins and acidic phospholipids has been steadily increasing in recent years. In this review, we will briefly review the cellular functions of plasma membrane-associated acidic phospholipids and the mechanisms that regulate their local distribution within this membrane. We will then explore the interplay between viruses and the plasma membrane acidic phospholipids in the context of the assembly process for two enveloped viruses, the influenza A virus (IAV) and the human immunodeficiency virus type 1 (HIV-1). Among the proteins encoded by these viruses, three viral structural proteins, IAV hemagglutinin (HA), IAV matrix protein-1 (M1), and HIV-1 Gag protein, are known to interact with acidic phospholipids, phosphatidylserine and/or phosphatidylinositol (4,5)-bisphosphate. These interactions regulate the localization of the viral proteins to and/or within the plasma membrane and likely facilitate the clustering of the proteins. On the other hand, these viral proteins, via their ability to multimerize, can also alter the distribution of the lipids and may induce acidic-lipid-enriched membrane domains. We will discuss the potential significance of these interactions in the virus assembly process and the property of the progeny virions. Finally, we will outline key outstanding questions that need to be answered for a better understanding of the relationships between enveloped virus assembly and acidic phospholipids. Full article

ABC toxin complexes are a class of protein toxin translocases comprised of a multimeric assembly of protein subunits. Each subunit displays a unique composition, contributing to the formation of a syringe-like nano-machine with natural cargo carrying, targeting, and translocation capabilities. Many of these toxins are insecticidal, drawing increasing interest in agriculture for use as biological pesticides. The A subunit (TcA) is the largest subunit of the complex and contains domains associated with membrane permeation and targeting. The B and C subunits, TcB and TcC, respectively, package into a cocoon-like structure that contains a toxic peptide and are coupled to TcA to form a continuous channel upon final assembly. In this review, we outline the current understanding and gaps in the knowledge pertaining to ABC toxins, highlighting seven published structures of TcAs and how these structures have led to a better understanding of the mechanism of host tropism and toxin translocation. We also highlight similarities and differences between homologues that contribute to variations in host specificity and conformational change. Lastly, we review the biotechnological potential of ABC toxins as both pesticides and cargo-carrying shuttles that enable the transport of peptides into cells. Full article

Vacuolar-type ATPase (v-ATPase) is a multimeric protein complex that regulates H⁺ transport across membranes and intra-cellular organelle acidification. Catabolic processes, such as endocytic degradation and autophagy, strictly rely on v-ATPase-dependent luminal acidification in lysosomes. The v-ATPase complex is expressed at high levels in the brain and its impairment triggers neuronal dysfunction and neurodegeneration. Due to their post-mitotic nature and highly specialized function and morphology, neurons display a unique vulnerability to lysosomal dyshomeostasis. Alterations in genes encoding subunits composing v-ATPase or v-ATPase-related proteins impair brain development and synaptic function in animal models and underlie genetic diseases in humans, such as encephalopathies, epilepsy, as well as neurodevelopmental, and degenerative disorders. This review presents the genetic and functional evidence linking v-ATPase subunits and accessory proteins to various brain disorders, from early-onset developmental epileptic encephalopathy to neurodegenerative diseases. We highlight the latest emerging therapeutic strategies aimed at mitigating lysosomal defects associated with v-ATPase dysfunction. Full article

Reflectin is a cationic, block copolymeric protein that mediates the dynamic fine-tuning of color and brightness of light reflected from nanostructured Bragg reflectors in iridocyte skin cells of squids. In vivo, the neuronally activated phosphorylation of reflectin triggers its assembly, driving osmotic dehydration of the membrane-bounded Bragg lamellae containing the protein to simultaneously shrink the lamellar thickness and spacing while increasing their refractive index contrast, thus tuning the wavelength and increasing the brightness of reflectance. In vitro, we show that the reduction in repulsive net charge of the purified, recombinant reflectin—either (for the first time) by generalized anionic screening with salt or by pH titration—drives a finely tuned, precisely calibrated increase in the size of the resulting multimeric assemblies. The calculated effects of phosphorylation in vivo are consistent with these effects observed in vitro. The precise proportionality between the assembly size and charge neutralization is enabled by the demonstrated rapid dynamic arrest of multimer growth by a continual, equilibrium tuning of the balance between the protein’s Coulombic repulsion and short-range interactive forces. The resulting stability of reflectin assemblies with time ensures a reciprocally precise control of the particle number concentration, encoding a precise calibration between the extent of neuronal signaling, osmotic pressure, and the resulting optical changes. The charge regulation of reflectin assembly precisely fine-tunes a colligative property-based nanostructured biological machine. A physical mechanism is proposed. Full article

Vasorin (VASN), a transmembrane protein heavily expressed in endothelial cells, has garnered recent interest due to its key role in vascular development and pathology. The oligomeric state of VASN is a crucial piece of knowledge given that receptor clustering is a frequent regulatory mechanism in downstream signaling activation and amplification. However, documentation of VASN oligomerization is currently absent. In this brief report, we describe the measurement of VASN oligomerization in its native membranous environment, leveraging a class of fluorescence fluctuation spectroscopy. Our investigation revealed that the majority of VASN resides in a monomeric state, while a minority of VASN forms homodimers in the cellular membrane. This result raises the intriguing possibility that ligand-independent clustering of VASN may play a role in transforming growth factor signaling. Full article

Myelin basic protein (MBP) is the second most abundant protein in the central nervous system and is responsible for structural maintenance of the myelin sheath covering axons. Previously, we showed that MBP has a more proactive role in the oligodendrocyte homeostasis, interacting with membrane-associated proteins, including integral membrane protein 2B (ITM2B or Bri2) that is associated with familial dementias. Here, we report that the molecular dynamics of the in silico-generated MBP-Bri2 complex revealed that MBP covers a significant portion of the Bri2 ectodomain, assumingly trapping the furin cleavage site, while the surface of the BRICHOS domain, which is responsible for the multimerization and activation of the Bri2 high-molecular-weight oligomer chaperone function, remains unmasked. These observations were supported by the co-expression of MBP with Bri2, its mature form, and disease-associated mutants, which showed that in mammalian cells, MBP indeed modulates the post-translational processing of Bri2 by restriction of the furin-catalyzed release of its C-terminal peptide. Moreover, we showed that the co-expression of MBP and Bri2 also leads to an altered cellular localization of Bri2, restricting its membrane trafficking independently of the MBP-mediated suppression of the Bri2 C-terminal peptide release. Further investigations should elucidate if these observations have physiological meaning in terms of Bri2 as a MBP chaperone activated by the MBP-dependent postponement of Bri2 membrane trafficking. Full article

Semaphorins belong to a group of membrane and secretory proteins that act as ligands for several receptor families and are involved in modulating cell signaling pathways. They bind multimeric receptor complexes on the cell membrane to exert their effects and initiate unique intracellular signal transduction cascades. These proteins can influence several processes that are very important for cell function, such as cell division and differentiation. Semaphorins are involved in cell migration, apoptosis, cell adhesion, aggregation, and numerous immune processes due to their immunoregulatory effects. Semaphorins are expressed in keratinocytes, which is why they have become a target for studies on the pathogenesis of skin diseases. Most studies to date on the role of semaphorins in the pathogenesis of skin diseases have been carried out in cellular or animal models, and there are few clinical studies evaluating the role of semaphorins in the pathogenesis and therapy of skin diseases. In this narrative review, we summarized the current state of knowledge on the role of semaphorins in the pathogenesis of skin diseases and their potential importance as targets for therapy. We also tried to present the key findings and weaknesses of previous research in this field. The novelty of this article lies in the comprehensive presentation of the role of semaphorins in the pathogenesis of skin diseases, including the results of studies on cell cultures and animal models, elucidating the mechanisms and signaling pathways through which semaphorins affect the development of skin diseases, as well as on the presentation of the results of existing clinical trials evaluating the role of semaphorins in the pathogenesis of skin diseases, and as potential therapeutic targets. Full article

Dopamine neurotransmission plays critical roles in regulating complex cognitive and behavioral processes including reward, motivation, reinforcement learning, and movement. Dopamine receptors are classified into five subtypes, widely distributed across the brain, including regions responsible for motor functions and specific areas related to cognitive and emotional functions. Dopamine also acts on astrocytes, which express dopamine receptors as well. The discovery of direct receptor–receptor interactions, leading to the formation of multimeric receptor complexes at the cell membrane and providing the cell decoding apparatus with flexible dynamics in terms of recognition and signal transduction, has expanded the knowledge of the G-protein-coupled receptor-mediated signaling processes. The purpose of this review article is to provide an overview of currently identified receptor complexes containing dopamine receptors and of their modulatory action on dopamine-mediated signaling between neurons and between neurons and astrocytes. Pharmacological possibilities offered by targeting receptor complexes in terms of addressing neuropsychiatric disorders associated with altered dopamine signaling will also be briefly discussed. Full article

A detailed comprehension of protein function requires information on the spatial structure of the protein, which is often gathered from X-ray crystallography. However, conformational dynamics often also plays an important functional role in proteins and can be directly investigated by complementary quasielastic neutron scattering. A classic example for dynamics–function correlations is Photosystem II, which is a multimeric pigment–protein complex responsible for catalyzing the light-induced photosynthetic water splitting into protons and oxygen. Several functional subprocesses of photosynthetic electron transfer and water splitting are strongly dependent on temperature and hydration, two factors also known to affect protein dynamics. Photosystem II is often investigated in the form of membrane fragments, where the protein complex remains embedded into its native lipid environment. However, experiments on protein function are often carried out in solution state, while direct investigations of molecular dynamics by quasielastic neutron scattering are mainly performed using specifically hydrated membrane fragments only. The present study provides the first quasielastic neutron scattering investigation of the molecular dynamics of Photosystem II membrane fragments (PSIImf) in solution over a wide temperature range from 50 to 300 K. At physiological temperatures above the melting point of water, we observed that the dynamics of PSIImf are significantly activated, leading to larger atomic mean square displacement values compared to those of specifically hydrated membrane stacks. The QENS data can be described by two dynamical components: a fast one, most probably corresponding to methyl group rotation; and a slower one, representing localized conformational dynamics. The latter component could be fitted by a jump-diffusion model at 300 K. The dynamics observed characterize the level of flexibility necessary for the proper PS II functionality under physiological conditions. In contrast, we observe a severe restriction of molecular dynamics upon freezing of the solvent below ~276 K. We associate this unexpected suppression of dynamics with a substantial aggregation of PSIImf caused by ice formation. Full article

β barrels are ubiquitous proteins in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. These transmembrane proteins (TMPs) execute a wide variety of tasks. For example, they can serve as transporters, receptors, membrane-bound enzymes, as well as adhesion, structural, and signaling elements. In addition, multimeric β barrels are common structural scaffolds among many pore-forming toxins. Significant progress has been made in understanding the functional, structural, biochemical, and biophysical features of these robust and versatile proteins. One frequently encountered fundamental trait of all β barrels is their voltage-dependent gating. This process consists of reversible or permanent conformational transitions between a large-conductance, highly permeable open state and a low-conductance, solute-restrictive closed state. Several intrinsic molecular mechanisms and environmental factors modulate this universal property of β barrels. This review article outlines the typical signatures of voltage-dependent gating. Moreover, we discuss recent developments leading to a better qualitative understanding of the closure dynamics of these TMPs. Full article

Positive-strand RNA virus RNA genome replication occurs in membrane-associated RNA replication complexes (RCs). Nodavirus RCs are outer mitochondrial membrane invaginations whose necked openings to the cytosol are “crowned” by a 12-fold symmetrical proteinaceous ring that functions as the main engine of RNA replication. Similar protein crowns recently visualized at the openings of alphavirus and coronavirus RCs highlight their broad conservation and functional importance. Using cryo-EM tomography, we earlier showed that the major nodavirus crown constituent is viral protein A, whose polymerase, RNA capping, membrane interaction and multimerization domains drive RC formation and function. Other viral proteins are strong candidates for unassigned EM density in the crown. RNA-binding RNAi inhibitor protein B2 co-immunoprecipitates with protein A and could form crown subdomains that protect nascent viral RNA and dsRNA templates. Capsid protein may interact with the crown since nodavirus virion assembly has spatial and other links to RNA replication. Using cryoelectron tomography and complementary approaches, we show that, even when formed in mammalian cells, nodavirus RC crowns generated without B2 and capsid proteins are functional and structurally indistinguishable from mature crowns in infected Drosophila cells expressing all viral proteins. Thus, the only nodaviral factors essential to form functional RCs and crowns are RNA replication protein A and an RNA template. We also resolve apparent conflicts in prior results on B2 localization in infected cells, revealing at least two distinguishable pools of B2. The results have significant implications for crown structure, assembly, function and control as an antiviral target. Full article