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KRas4b is a membrane-bound regulatory protein belonging to the family of small GTPases that function as a molecular switch, facilitating signal transduction from activated membrane receptors to intracellular pathways controlling cell growth and proliferation. Oncogenic mutations locking KRas4b in the active GTP state are responsible for nearly 85% of all Ras-driven cancers. Understanding the membrane-bound state of KRas4b is crucial for designing new therapeutic approaches targeting oncogenic KRas-driven signaling pathways. Extensive research demonstrates the significant involvement of the membrane bilayer in Ras-effector interactions, with anionic lipids playing a critical role in determining protein conformations The preferred topology of KRas4b for interacting with signaling partners has been a long-time question. Computational studies suggest a membrane-proximal conformation, while other biophysical methods like neutron reflectivity propose a membrane-distal conformation. To address these gaps, we employed FRET measurements to investigate the conformation of KRas4b. Using fully post-translationally modified KRas4b, we designed a Nanodisc based FRET assay to study KRas4b-membrane interactions. We suggest an extended conformation of KRas4b relative to the membrane surface. Measurement of FRET donor - acceptor distances reveal that a negatively charged membrane surface weakly favors closer association with the membrane surface. Our findings provide insights into the role of anionic lipids in determining the dynamic conformations of KRas4b and shed light on the predominant conformation of its topology on lipid headgroups. [Display omitted] • Nanodisc based FRET assay quantitates effect of anionic lipids on KRas4b membrane topology. • FRET Efficiency measurement yields average height of KRas4b above bilayer. • KRas4b adopts an extended form with the presence of negatively charge lipids promoting G-domain proximity to the bilayer. [ABSTRACT FROM AUTHOR]

Background: SAAP-148 is an antimicrobial peptide derived from LL-37. It exhibits excellent activity against drug-resistant bacteria and biofilms while resisting degradation in physiological conditions. Despite its optimal pharmacological properties, its mechanism of action at the molecular level has not been explored. Methods: The structural properties of SAAP-148 and its interaction with phospholipid membranes mimicking mammalian and bacterial cells were studied using liquid and solid-state NMR spectroscopy as well as molecular dynamics simulations. Results: SAAP-148 is partially structured in solution and stabilizes its helical conformation when interacting with DPC micelles. The orientation of the helix within the micelles was defined by paramagnetic relaxation enhancements and found similar to that obtained using solid-state NMR, where the tilt and pitch angles were determined based on 15N chemical shift in oriented models of bacterial membranes (POPE/POPG). Molecular dynamic simulations revealed that SAAP-148 approaches the bacterial membrane by forming salt bridges between lysine and arginine residues and lipid phosphate groups while interacting minimally with mammalian models containing POPC and cholesterol. Conclusions: SAAP-148 stabilizes its helical fold onto bacterial-like membranes, placing its helix axis almost perpendicular to the surface normal, thus probably acting by a carpet-like mechanism on the bacterial membrane rather than forming well-defined pores. [ABSTRACT FROM AUTHOR]

Glycoprotein 3 (GP3) serves as a structural protein in equine arteritis virus (EAV), forming a heterotrimeric complex that plays a pivotal role in virus tropism. In this study, we tested the membrane topology of GP3, both when expressed separately and during infection with recombinant tagged EAV GP3-HA. In our antibody accessibility experiment, we made a noteworthy discovery: GP3, when expressed separately, exhibits a dual topology. We introduced an additional N-glycosylation site, which was only partially used, providing further evidence for the dual topology of GP3. Intriguingly, this mutated GP3 was secreted into the medium, a result of the disruption of the ER retention motif RXR. The additional glycosylation site was not used when we examined the recombinant EAV virus with the same mutation. Despite the fact of higher expression levels of mutant GP3-HA, the protein was not secreted, and the recombinant mutant virus did not have growth delay compared to the EAV wild-type virus. This finding suggests that GP3 has a single type one membrane topology in virus infected cells, whereas the expression of GP3 in trans results in the dual topology of this protein. The RXR motif in the C-terminus is a co-factor of ER retention of the protein, but the main retention signal remains elusive. • GP3 in equine arteritis virus shows dual membrane topology if protein is expressed separatelly • A portion of the protein slips into the ER lumen, resulting in hairpin-like topology. • During virus infection, the membrane topology of GP3 is of type I. • Mutation of C-terminal motif RXR is responsible for secretion of portion of GP3 to the supernatant, but only in cells transfected with plasmid carrying GP3 gene. • GP3 with disrupted RXR motif is expressed at higher levels. • Recombinant EAV with a deleted RXR motif in GP3 exhibits only a small growth defect. [ABSTRACT FROM AUTHOR]

Dynamic disorder of the lipid bilayer presents a challenge for establishing structure–function relationships in membrane proteins, especially to those that insert by refolding from soluble states, e.g., bacterial toxins and Bcl-2 apoptotic regulators. Because many high-resolution structural techniques cannot be easily applied to such systems, methods like depth-dependent fluorescence quenching gained prominence. Over three decades ago, Prof. Erwin London and his co-workers revolutionized the studies of membrane protein insertion by introducing a quantitative approach to the analysis of membrane quenching data and inspired many researchers to continue this work. Here, we illustrate how the application of the quantitative analysis yields new insights into previously published results. We have used the method of distribution analysis (DA) to calculate the precise immersion depth of NBD fluorophores selectively attached to various single-cysteine mutants of the apoptotic factor BAX from quenching data obtained with a series of spin-labeled lipids. The original qualitative analysis interpreted the higher quenching determined for shallower probes in positions flanking the helix 9 of BAX as evidence of a transmembrane helix 9 topology. The quantitative DA, however, revealed that a transmembrane topology of helix 9 of BAX is unlikely, as it would require labeling sites that are only 15 residues apart in a helical conformation to be separated by a transverse distance of over 45 Å. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) NS4B protein is a membranotropic multifunctional protein. Despite its versatile functioning, its topology and dynamics are not entirely understood. There is no X-ray or cryo-EM structure available for any flaviviral NS4B full-length protein. In this study, we have investigated the structural dynamics of full-length ZIKV NS4B protein through 3D structure models using molecular dynamics simulations and experimental techniques. Also, we employed a reductionist approach to understand the dynamics of NS4B protein where we studied its N-terminal (residues 1–38), C-terminal (residues 194–251), and cytosolic (residues 131–169) regions in isolation in addition to the full-length protein. Further, using a series of circular dichroism spectroscopic experiments, we validate the cytosolic region as an intrinsically disordered protein region. The microsecond-long all atoms molecular dynamics and replica-exchange simulations complement the experimental observations. Furthermore, we have also studied the NS4B proteins C-terminal regions of four other flaviviruses viz. DENV2, JEV, WNV, and YFV through microsecond simulations to characterize their behaviour in presence and absence of lipid membranes. There are significant differences observed in the conformations of other flavivirus NS4B C-terminal regions in comparison to ZIKV NS4B. Lastly, we have proposed a ZIKV NS4B protein model illustrating its putative topology consisting of various membrane-spanning and non-membranous regions. The illustration of structural dynamics of Zika virus NS4B protein with a reductionist approach demonstrating its N-terminal, Cytosolic, and C-terminal regions in truncated form. [Display omitted] • Microsecond simulations of NS4B N-terminus and cytosolic regions exposed their dynamic nature. • C-terminal region remains intact in presence of lipid bilayer during 1 μs simulations. • Spectroscopic results also reveal the cytosolic region as an intrinsically disordered protein region. • Cytosolic region does not gain structure in presence of macromolecular crowding and liposomes. [ABSTRACT FROM AUTHOR]

The RtxA cytotoxin, a member of the RTX (Repeats in ToXin) family of pore-forming toxins, is the primary virulence factor of the paediatric facultative pathogen Kingella kingae. Although structure-function studies of RTX toxins have defined their characteristic domains and features, the exact membrane topology of RTX toxins remains unknown. Here, we used labelling of cell-bound RtxA with a membrane-impermeable, lysine-reactive reagent and subsequent detection of the labelled lysine residues by mass spectrometry, which revealed that most of the membrane-bound toxin is localised extracellularly. A trypsin protection assay with cell-bound RtxA demonstrated that five of seven transmembrane α-helices, predicted by various algorithms within the N-terminal half of the molecule, are irreversibly embedded in the membrane. Structure-function analysis showed that these α-helices, four of which are arranged as two pairs of back-to-back helices, are essential for the formation of an ion-conducting membrane pore. In contrast, the C-terminal half of RtxA is required for the interaction with the cell surface and for the irreversible insertion of the toxin into the membrane via acyl chains covalently linked to the molecule. These findings advance our understanding of the structure-function relationships of RtxA and enable us to propose a membrane topology model of the toxin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Flavivirus nonstructural protein 2A (NS2A) is a small endoplasmic reticulum (ER)-resident, hydrophobic transmembrane protein that function in viral replication, virion assembly and evasion of the host immune response. Despite previous studies on the role of duck Tembusu virus (DTMUV) NS2A in inhibiting the host immune response, its membrane topology has not been clearly addressed (Zhang et al., 2020; Zhang et al., 2022). Here, we present the first report on the membrane topology model and functional characterization of DTMUV NS2A. Our findings demonstrate that DTMUV NS2A localizes to the endoplasmic reticulum (ER) and associates with viral double-stranded RNA, with a single segment (TMD3, amino acids 72 to 95) spanning the ER membrane. To better delineate the residues in NS2A-TMD3 related to viral properties, specific mutations were introduced to generate DTMUV replicons and infectious cDNA clones. Functional analysis indicates that L77, Q86 and L89 of NS2A are crucial for viral RNA synthesis, while residues M79 and F83 are crucial for the assembly or release of viral particles. Moreover, these mutations attenuated the virulence of DTMUV in vivo. Collectively, our results shed light on the relationship between the transmembrane of DTMUV NS2A and its functions in virus proliferation, providing insights for further understanding the molecular mechanisms of NS2A in the virus life cycle., Competing Interests: Conflicts of interest The authors have no conflicts of interest to declare., (Copyright © 2024. Published by Elsevier Inc.)

The topology of helical membrane proteins is generally defined during insertion of the transmembrane helices, yet it is now clear that it is possible for topology to change under unusual circumstances. It remains unclear, however, if topology reorientation is part of normal biogenesis. For dual topology dimer proteins such as the multidrug transporter EmrE, there may be evolutionary pressure to allow topology flipping so that the populations of both orientations can be equalized. We previously demonstrated that when EmrE is forced to insert in a distorted topology, topology flipping of the first transmembrane helix can occur during translation. Here, we show that topological malleability also extends to the C-terminal helix and that even complete topology inversion of the entire EmrE protein can occur after the full protein is translated and inserted. Thus, topology rearrangements are possible during normal biogenesis. Wholesale topology flipping is remarkable given the physical constraints of the membrane and expands the range of possible membrane protein folding pathways, both productive and detrimental.

Phosphatidylserine (PS) synthase 1 (PSS1) of mammalian cells is a multiple membrane‐spanning protein of the endoplasmic reticulum (ER) and regulated by inhibition with the product PS. Alanine‐scanning mutagenesis of PSS1 has revealed eight amino acid residues as those crucial for its activity and six as those important for its regulation. Furthermore, three missense mutations in the human PSS1 gene, which lead to regulatory dysfunctions of PSS1 and are causative of Lenz–Majewski syndrome, have been identified. In this study, we investigated the membrane topology of PSS1 by means of epitope insertion and immunofluorescence. According to a 10‐transmembrane segment model supported by topology analysis of PSS1, all the 8 amino acid residues crucial for the enzyme activity were localized to the luminal side of the lipid bilayer or the lumen of the ER, whereas all the 9 amino acid residues involved in the enzyme regulation were localized to the cytosol or the cytoplasmic side of the lipid bilayer of the ER. This localization of the functional amino acid residues suggests that PSS1 is regulated by inhibition with PS in the cytoplasmic leaflet of the ER membrane and synthesizes PS at the luminal leaflet. [ABSTRACT FROM AUTHOR]

The Rhodococcus erythropolis gene DYC18_RS18060 (1437 bp) putatively codes for a secondary transporter of the Nucleobase Cation Symporter-1 (NCS-1) protein family (478 amino acids). The DYC18_RS18060 gene was successfully cloned from R. erythropolis genomic DNA with the addition of EcoRI and PstI restriction sites at the 5′ and 3′ ends, respectively, using PCR technology. The amplified gene was introduced into IPTG-inducible plasmid pTTQ18, immediately upstream of the sequence coding for a His6-tag. The construct was transformed into Escherichia coli BL21(DE3). Then, the amplified expression of the DYC18_RS18060-His6 protein was achieved with detection through SDS-PAGE and western blotting. Computational methods predicted that DYC18_RS18060 has a molecular weight of 51.1 kDa and isoelectric point of 6.58. The protein was predicted to be hydrophobic in nature (aliphatic index 113.24, grand average of hydropathicity 0.728). It was also predicted to form twelve transmembrane spanning α-helices, with both N- and C-terminal ends at the cytoplasmic side of the membrane. Database sequence similarity searches and phylogenetic analysis suggested that the substrate of DYC18_RS18060 could be cytosine; however, this was uncertain based on the comparison of residues involved in substrate binding in experimentally characterised NCS-1 proteins. The current study lays the foundations for further structural and functional studies of DYC18_RS18060 and other NCS-1 proteins. Keywords: bioinformatics analysis, gene cloning, membrane topology, NCS-1 family, protein expression, transport protein Copyright(c) The Authors

Wnt gradients elicit distinct cellular responses, such as proliferation, specification, differentiation and survival in a dose-dependent manner. Porcupine (PORCN), a membrane-bound O-acyl transferase (MBOAT) that resides in the endoplasmic reticulum, catalyses the addition of monounsaturated palmitate to Wnt proteins and is required for Wnt gradient formation and signalling. In humans, PORCN mutations are causal for focal dermal hypoplasia (FDH), an X-linked dominant syndrome characterized by defects in mesodermal and endodermal tissues. PORCN is also an emerging target for cancer therapeutics. Despite the importance of this enzyme, its structure remains poorly understood. Recently, the crystal structure of DltB, an MBOAT family member from bacteria, was solved. In this report, we use experimental data along with homology modelling to DltB to determine the membrane topology of PORCN. Our studies reveal that PORCN has 11 membrane domains, comprising nine transmembrane spanning domains and two reentrant domains. The N-terminus is oriented towards the lumen while the C-terminus is oriented towards the cytosol. Like DltB, PORCN has a funnel-like structure that is encapsulated by multiple membrane-spanning helices. This new model for PORCN topology allows us to map residues that are important for biological activity (and implicated in FDH) onto its three-dimensional structure.

Pestiviruses like bovine viral diarrhea virus (BVDV) belong to the family Flaviviridae. A distinctive feature of the Flaviviridae is the importance of nonstructural (NS) proteins for RNA genome replication and virus morphogenesis. For pestiviruses, the NS2 protease-mediated release of NS3 is essential for RNA replication, whereas uncleaved NS2-3 is indispensable for producing viral progeny. Accordingly, in the pestiviral life cycle the switch from RNA replication to virion morphogenesis is temporally regulated by the extent of NS2-3 cleavage, which is catalyzed by the NS2 autoprotease. A detailed knowledge of the structural and functional properties of pestiviral NS2 and NS2-3 is mandatory for a better understanding of these processes. In the present study, we experimentally determined the membrane topology of NS2 of BVDV-1 strain NCP7 by the substituted cysteine accessibility method (SCAM) assay. According to the resulting model, the N terminus of NS2 resides in the endoplasmic reticulum (ER) lumen and is followed by three transmembrane segments (TMs) and a cytoplasmic C-terminal protease domain. We used the resulting model for fine mapping of the minimal autoprotease domain. Only one TM was found to be essential for maintaining residual autoprotease activity. While the topology of pestiviral NS2 is overall comparable to that of hepatitis C virus (HCV) NS2, our data also reveal potentially important differences between the two molecules. The improved knowledge about structural and functional properties of this protein will support future functional and structural studies on pestiviral NS2. [ABSTRACT FROM AUTHOR]

ABSTRACTZika virus (ZIKV) can cause devastating congenital Zika syndromes in pregnant women and Guillain-Barre syndrome in adults. Understanding the molecular mechanism of ZIKV replication is essential for antiviral and vaccine development. Here we report the structural and functional characterization of ZIKV NS2A protein. Biochemical structural probing suggests that ZIKV NS2A has a single segment that traverses the ER membrane and six segments that peripherally associate with the ER membrane. Functional analysis has defined distinct NS2A residues essential for viral RNA synthesis or virion assembly. Only the virion assembly-defective mutants, but not the RNA synthesis-defective mutants, could be rescued through trans complementation with a wide-type NS2A protein. These results suggest that the NS2A molecules in virion assembly complex could be recruited in trans, whereas the NS2A molecules in viral replication complex must be recruited in cis. Together with previous results, we propose a flavivirus assembly model where NS2A plays a central role in modulating viral structural and nonstructural proteins as well as genomic RNA during virion assembly.

Mitochondrial cristae are dynamic invaginations of the inner membrane and play a key role in its metabolic capacity to produce ATP. Structural alterations caused by either genetic abnormalities or detrimental environmental factors impede mitochondrial metabolic fluxes and lead to a decrease in their ability to meet metabolic energy requirements. While some of the key proteins associated with mitochondrial cristae are known, very little is known about how the inner membrane dynamics are involved in energy metabolism. In this study, we present a computational strategy to understand how cristae are formed using a phase-based separation approach of both the inner membrane space and matrix space, which are explicitly modeled using the Cahn–Hilliard equation. We show that cristae are formed as a consequence of minimizing an energy function associated with phase interactions which are subject to geometric boundary constraints. We then extended the model to explore how the presence of calcium phosphate granules, entities that form in calcium overload conditions, exert a devastating inner membrane remodeling response that reduces the capacity for mitochondria to produce ATP. This modeling approach can be extended to include arbitrary geometrical constraints, the spatial heterogeneity of enzymes, and electrostatic effects to mechanize the impact of ultrastructural changes on energy metabolism. [ABSTRACT FROM AUTHOR]

Background: The human proteins TMTC1, TMTC2, TMTC3 and TMTC4 have been experimentally shown to be components of a new O-mannosylation pathway. Their own mannosyl-transferase activity has been suspected but their actual enzymatic potential has not been demonstrated yet. So far, sequence analysis of TMTCs has been compromised by evolutionary sequence divergence within their membrane-embedded N-terminal region, sequence inaccuracies in the protein databases and the difficulty to interpret the large functional variety of known homologous proteins (mostly sugar transferases and some with known 3D structure). Results: Evolutionary conserved molecular function among TMTCs is only possible with conserved membrane topology within their membrane-embedded N-terminal regions leading to the placement of homologous long intermittent loops at the same membrane side. Using this criterion, we demonstrate that all TMTCs have 11 transmembrane regions. The sequence segment homologous to Pfam model DUF1736 is actually just a loop between TM7 and TM8 that is located in the ER lumen and that contains a small hydrophobic, but not membrane-embedded helix. Not only do the membrane-embedded N-terminal regions of TMTCs share a common fold and 3D structural similarity with subgroups of GT-C sugar transferases. The conservation of residues critical for catalysis, for binding of a divalent metal ion and of the phosphate group of a lipid-linked sugar moiety throughout enzymatically and structurally well-studied GT-Cs and sequences of TMTCs indicates that TMTCs are actually sugar-transferring enzymes. We present credible 3D structural models of all four TMTCs (derived from their closest known homologues 5ezm/5f15) and find observed conserved sequence motifs rationalized as binding sites for a metal ion and for a dolichyl-phosphate-mannose moiety. Conclusions: With the results from both careful sequence analysis and structural modelling, we can conclusively say that the TMTCs are enzymatically active sugar transferases belonging to the GT-C/PMT superfamily. The DUF1736 segment, the loop between TM7 and TM8, is critical for catalysis and lipid-linked sugar moiety binding. Together with the available indirect experimental data, we conclude that the TMTCs are not only part of an O-mannosylation pathway in the endoplasmic reticulum of upper eukaryotes but, actually, they are the sought mannosyl-transferases. [ABSTRACT FROM AUTHOR]

A fundamental first step in the evolution of eukaryotes was infolding of the chemiosmotic membrane of the endosymbiont. This allowed the proto-eukaryote to amplify ATP generation while constraining the volume dedicated to energy production. In mitochondria, folding of the inner membrane has evolved into a highly regulated process that creates specialized compartments (cristae) tuned to optimize function. Internalizing the inner membrane also presents complications in terms of generating the folds and maintaining mitochondrial integrity in response to stresses. This review describes mechanisms that have evolved to regulate inner membrane topology and either preserve or (when appropriate) rupture the outer membrane.

Dengue virus is an important human pathogen threating people, especially in tropical and sub-tropical regions. The viral genome has one open reading frame and encodes one polyprotein which can be processed into structural and nonstructural (NS) proteins. Four of the seven nonstructural proteins, NS2A, NS2B, NS4A and NS4B, are membrane proteins. Unlike NS3 or NS5, these proteins do not harbor any enzymatic activities, but they play important roles in viral replication through interactions with viral or host proteins to regulate important pathways and enzymatic activities. The location of these proteins on the cell membrane and the functional roles in viral replication make them important targets for antiviral development. Indeed, NS4B inhibitors exhibit antiviral activities in different assays. Structural studies of these proteins are hindered due to challenges in crystallization and the dynamic nature of these proteins. In this review, the function and membrane topologies of dengue nonstructural membrane proteins are presented. The roles of solution NMR spectroscopy in elucidating the structure and dynamics of these proteins are introduced. The success in the development of NS4B inhibitors proves that this class of proteins is an attractive target for antiviral development.

A fundamental first step in the evolution of eukaryotes was infolding of the chemiosmotic membrane of the endosymbiont. This allowed the proto-eukaryote to amplify ATP generation while constraining the volume dedicated to energy production. In mitochondria, folding of the inner membrane has evolved into a highly regulated process that creates specialized compartments (cristae) tuned to optimize function. Internalizing the inner membrane also presents complications in terms of generating the folds and maintaining mitochondrial integrity in response to stresses. This review describes mechanisms that have evolved to regulate inner membrane topology and either preserve or (when appropriate) rupture the outer membrane. [ABSTRACT FROM AUTHOR]

The NADPH oxidase Nox4 is a multi-pass membrane protein responsible for the generation of reactive oxygen species that are implicated in cellular signaling but may also cause pathological situations when dysregulated. Although topological organization of integral membrane protein dictates its function, only limited experimental data describing Nox4's topology are available. To provide deeper insight on Nox4 structural organization, we developed a novel method to determinate membrane protein topology in their cellular environment, named Topological Determination by Ubiquitin Fusion Assay (ToDUFA). It is based on the proteolytic capacity of the deubiquitinase enzymes to process ubiquitin fusion proteins. This straightforward method, validated on two well-known protein's topologies (IL1RI and Nox2), allowed us to discriminate rapidly the topological orientation of protein's domains facing either the nucleocytosolic or the exterior/luminal compartments. Using this method, we were able for the first time to determine experimentally the topology of Nox4 which consists of 6 transmembrane domains with its N- and C-terminus moieties facing the cytosol. While the first, third and fifth loops of Nox4 protein are extracellular; the second and fourth loops are located in the cytosolic side. This approach can be easily extended to characterize the topology of all others members of the NADPH oxidase family or any multi-pass membrane proteins. Considering the importance of protein topology knowledge in cell biology research and pharmacological development, we believe that this novel method will represent a widely useful technique to easily uncover complex membrane protein's topology. • Technical validation of ToDUFA as new method to uncover membrane protein's topology. • Experimental determination of Nox4 membrane's topology. • Nox4 has 6 transmembrane domains (5 loops, N- and C-terminus facing the cytosol). [ABSTRACT FROM AUTHOR]

Experimental determination of membrane protein topology can be achieved using various techniques. Here, we present the pho-lac dual reporter system, a simple, convenient, and reliable tool to analyze the topology of membrane protein in vivo. The system is based on the use of two topological markers with complementary properties: The Escherichia coli β-galactosidase, LacZ, which is active in the cytoplasm, and the E. coli alkaline phosphatase, PhoA, which is active in the bacterial periplasm. Specifically, in this pho-lac gene system, the reporter molecule is a chimera composed of the mature PhoA that is in-frame with the β-galactosidase α-peptide (LacZα). Hence, when targeted to the periplasm, the PhoA-LacZα dual reporter displays high alkaline phosphatase activity but no β-galactosidase activity. Conversely, when located in the cytoplasm, PhoA-LacZα has no phosphatase activity but exhibits high β-galactosidase activity in E. coli cells expressing the α-fragment of LacZ, LacZα (via the α-complementation phenomenon). The dual nature of the PhoA-LacZα reporter allows a simple way to normalize both enzymatic activities to obtain readily interpretable information about the subcellular location of the fusion site between the membrane protein under study and the reporter. In addition, the PhoA-LacZα reporter permits the utilization of dual indicator agar plates to easily discriminate between colonies bearing cytoplasmic fusions, periplasmic fusions, or out-of-frame fusions. In total, the phoA-lacZα fusion reporter approach is a direct and rather inexpensive method to characterize the topology of membrane proteins in vivo., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The 17β-hydroxysteroid dehydrogenases (17β-HSDs) comprise enzymes initially identified by their ability to interconvert active and inactive forms of sex steroids, a vital process for the tissue-specific control of estrogen and androgen balance. However, most 17β-HSDs have now been shown to accept substrates other than sex steroids, including bile acids, retinoids and fatty acids, thereby playing unanticipated roles in cell physiology. This functional divergence is often reflected by their different subcellular localization, with 17β-HSDs found in the cytosol, peroxisome, mitochondria, endoplasmic reticulum and in lipid droplets. Moreover, a subset of 17β-HSDs are integral membrane proteins, with their specific topology dictating the cellular compartment in which they exert their enzymatic activity. Here, we summarize the present knowledge on the subcellular localization and membrane topology of the 17β-HSD enzymes and discuss the correlation with their biological functions. • 17β-HSDs exert diverse functions, spanning from sex steroid and retinoid metabolism to fatty acid synthesis/oxidation. • 17β-HSDs are present in the cytosol, endoplasmic reticulum, lipid droplets, peroxisomes or mitochondria. • The topology of the 17β-HSDs at the endoplasmic reticulum and lipid droplets has now been resolved. • Subcellular localization and membrane topology of the 17β-HSDs is tightly correlated with their biological function. [ABSTRACT FROM AUTHOR]

Fatty acyl-CoA reductase 1 (Far1) is a ubiquitously expressed peroxisomal membrane protein that generates the fatty alcohols required for the biosynthesis of ether lipids. Lipid droplet localization of exogenously expressed and endogenous human Far1 was observed by fluorescence microscopy under conditions of increased triglyceride synthesis in tissue culture cells. This unexpected finding was supported further by correlative light electron microscopy and subcellular fractionation. Selective permeabilization, protease sensitivity and N-glycosylation tagging suggested that Far1 is able to assume two different membrane topologies, differing in the orientation of the short hydrophilic C-terminus towards the lumen or the cytosol, respectively. Two closely spaced hydrophobic domains are contained within the C-terminal region. When analyzed separately, the second domain was sufficient for the localization of a fluorescent reporter to lipid droplets. Targeting of Far1 to lipid droplets was not impaired in either Pex19 or ASNA1 (also known as TRC40) CRISPR/Cas9 knockout cells. In conclusion, our data suggest that Far1 is a novel member of the rather exclusive group of dual topology membrane proteins. At the same time, Far1 shows lipid metabolism-dependent differential subcellular localizations to peroxisomes and lipid droplets. [ABSTRACT FROM AUTHOR]

Syndecans are single‐span membrane proteins playing important roles in cell–cell and cell–matrix interactions. The transmembrane domain of syndecans is critical for signal transduction across the cell membrane. Here, the structure of the transmembrane domain of syndecan‐2 in detergent micelles was investigated using solution NMR spectroscopy. Backbone resonance assignment was obtained, and NMR studies show that the transmembrane domain forms a helix in detergent micelles, which is also supported by the hydrogen and deuterium exchange experiment. A study of the dynamics revealed the rigid structure of the transmembrane domain formed in solution, and paramagnetic relaxation enhancement defined the topology of the transmembrane domain in detergent micelles. This structural analysis may facilitate a better understanding of the role of the syndecan‐2 transmembrane domain in signal transduction. [ABSTRACT FROM AUTHOR]

Substantial progress has been made in our understanding of the nongenomic actions, ligand binding, intracellular signaling pathways, and functions of membrane progesterone receptors (mPRs) in reproductive and nonreproductive tissues since their discovery 20 years ago. The five mPRs are members of the progestin adipoQ receptor (PAQR) family which also includes adiponectin receptors (AdipoRs). However, unlike AdipoRs, the 3-D structures of mPRs are unknown, and their structural characteristics remain poorly understood. The mechanisms regulating mPR functions and their trafficking to the cell surface have received little attention and have not been systematically reviewed. This paper summarizes some structural aspects of mPRs, including the ligand binding pocket of mPRα recently derived from homology modeling with AdipoRs, and the proposed topology of mPRs from the preponderance of positively charged amino acid residues in their intracellular domains. The mechanisms of trafficking membrane receptors to the cell surface are discussed, including the amino acid motifs involved with their export to the cell surface, the roles of adaptor proteins, and post-translational glycosylation and palmitoylation modifications that promote cell surface expression and retention. Evidence for similar mechanisms regulating the expression and functions of mPRs on the cell surface is discussed, including the identification of potential export motifs on mPRα required for its trafficking to the cell membrane. Collectively, these results have identified several potential mechanisms regulating the expression and functions of mPRs on the cell membrane for further investigation. [ABSTRACT FROM AUTHOR]

The immune system serves as a crucial line of defense from infection and cancer, while also contributing to tissue homeostasis. Communication between immune cells is mediated by small soluble factors called cytokines, and also by direct cellular interactions. Cell-cell interactions are particularly important for T cell activation. T cells direct the adaptive immune response and therefore need to distinguish between self and foreign antigens. Even though decades have passed since the discovery of T cells, exactly why and how they are able to recognize and discriminate between antigens is still not fully understood. Early imaging of T cells was very successful in capturing the early stages of conjugate formation of T cells with antigen-presenting cells upon recognition of peptide-loaded major histocompatibility complexes by the T cell receptor (TCR). These studies lead to the discovery of a “supramolecular activation cluster” now known as the immunological synapse, followed by the identification of microclusters of TCRs formed upon receptor triggering, that eventually coalesce at the center of the synapse. New developments in light microscopy have since allowed attention to turn to the very earliest stages of T cell activation, and to resting cells, at high resolution. This includes single-molecule localization microscopy, which has been applied to the question of whether TCRs are pre-clustered on resting T cells, and lattice light-sheet microscopy that has enabled imaging of whole cells interacting with antigen-presenting cells. The utilization of lattice light-sheet microscopy has yielded important insights into structures called microvilli, which are small membrane protrusions on T cells that seem likely to have a large impact on T cell recognition and activation. Here we consider how imaging has shaped our thinking about T cell activation. We summarize recent findings obtained by applying more advanced microscopy techniques and discuss some of the limitations of these methods.

Gap junction channels facilitate the intercellular exchange of ions and small molecules, a process that is critical for the function of many different kinds of cells and tissues. Recent crystal structures of channels formed by one connexin isoform (connexin26) have been determined, and they have been subjected to molecular modeling. These studies have provided high-resolution models to gain insights into the mechanisms of channel conductance, molecular permeability, and gating. The models share similarities, but there are some differences in the conclusions reached by these studies. Many unanswered questions remain to allow an atomic-level understanding of intercellular communication mediated by connexin26. Because some domains of the connexin polypeptides are highly conserved (like the transmembrane regions), it is likely that some features of the connexin26 structure will apply to other members of the family of gap junction proteins. However, determination of high-resolution structures and modeling of other connexin channels will be required to account for the diverse biophysical properties and regulation conferred by the differences in their sequences.

The glycoprotein GP3 of the Arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) consists of a cleaved signal peptide, a highly glycosylated domain, a short hydrophobic region and an unglycosylated C-terminal domain. GP3 is supposed to form a complex with GP2 and GP4 in virus particles, but secretion of GP3 from cells has also been reported. We analyzed the membrane topology of GP3 from various PRRSV strains. A fraction of the protein is secreted from transfected cells; GP3 from PRRSV-1 strains to a greater extent than GP3 from PRRSV-2 strains. This secretion behavior is reversed after exchange of the variable C-terminal domain. A fluorescence protease protection assay shows that the C-terminus of GP3, fused to GFP, is resistant against proteolytic digestion in permeabilized cells. Furthermore, glycosylation sites inserted into the C-terminal part of GP3 are used. Both experiments indicate that the C-terminus of GP3 is translocated into the lumen of the endoplasmic reticulum. Deletion of the conserved hydrophobic region greatly enhances secretion of GP3 and fusion of this domain to GFP promotes membrane anchorage. Bioinformatics suggests that the hydrophobic region might form an amphipathic helix. Accordingly, exchanging only a few amino acids in its hydrophilic face prevents and in its hydrophobic face enhances secretion of GP3. Exchanging the latter amino acids in the context of the viral genome did not affect release of virions, but released particles were not infectious. In sum, GP3 exhibits an unusual hairpin-like membrane topology that might explain why a fraction of the protein is secreted. IMPORTANCE The porcine reproductive and respiratory syndrome virus (PRRSV) is the most important pathogen in the pork industry. It causes persistent infections that lead to reduced weight gain of piglets; highly pathogenic strains even kill 90% of an infected pig population. PRRSV cannot be eliminated from pig farms by vaccination due to the large amino acid variability between the existing strains, especially in the glycoproteins. Here we analyzed basic structural features of glycoprotein 3 (GP3) from various PRRSV strains. We show that the protein exhibits an unusual hairpin-like membrane topology; membrane anchoring might occur via an amphipathic helix. This rather weak membrane anchor explains why a fraction of the protein is secreted from cells. Interestingly, PRRSV-1 strains secrete more GP3 than PRRSV-2. We speculate that secreted GP3 might play a role during PRRSV infection of pigs; it might serve as a decoy to distract antibodies away from virus particles. [ABSTRACT FROM AUTHOR]

Dengue virus (DENV) non-structural (NS) 4A is a membrane protein essential for viral replication. The N-terminal region of NS4A contains several helices interacting with the cell membrane and the C-terminal region consists of three potential transmembrane regions. The secondary structure of the intact NS4A is not known as the previous structural studies were carried out on its fragments. In this study, we purified the full-length NS4A of DENV serotype 4 into dodecylphosphocholine (DPC) micelles. Solution NMR studies reveal that NS4A contains six helices in DPC micelles. The N-terminal three helices are amphipathic and interact with the membrane. The C-terminal three helices are embedded in micelles. Our results suggest that NS4A contains three transmembrane helices. Our studies provide for the first time structural information of the intact NS4A of DENV and will be useful for further understanding its role in viral replication. [ABSTRACT FROM AUTHOR]

Organophosphate hydrolase (OPH) is a membrane-associated lipoprotein. It translocates across the inner membrane via the twin-arginine transport pathway and remains anchored to the periplasmic face of the inner membrane through a diacylglycerol moiety linked to the invariant cysteine residue found at the junction of a SpaseII cleavage site. Due to the existence of a transmembrane helix at the C-terminus of the mature OPH, an inner-membrane topology was predicted suggesting the C-terminus of OPH is cytoplasmic. The predicted topology was validated by generating OPH variants either fused in-frame with ß-lactamase or with unique cysteine residues. Sphingopyxis wildii cells expressing OPH variants with Bla fused at the N-terminal, C-terminal or central regions all grew in the presence of ampicillin. Supporting the ß-lactamase reporter assay, the OPH variants having unique cysteine residues at different strategic locations were accessible to the otherwise membrane-impermeant PEG-Mal (methoxypolyethylene glycol maleimide) revealing that, with the exception of the lipoprotein anchor, the entire OPH is in the periplasmic space. [ABSTRACT FROM AUTHOR]

Euonymus alatus diacylglycerol acetyltransferase ( Ea DAcT) catalyzes the transfer of an acetyl group from acetyl-CoA to the sn-3 position of diacylglycerol to form 3-acetyl-1,2-diacyl- sn-glycerol (acetyl- TAG). Ea DAcT belongs to a small, plant-specific subfamily of the membrane bound O-acyltransferases ( MBOAT) that acylate different lipid substrates. Sucrose gradient density centrifugation revealed that Ea DAcT colocalizes to the same fractions as an endoplasmic reticulum ( ER)-specific marker. By mapping the membrane topology of Ea DAcT, we obtained an experimentally determined topology model for a plant MBOAT. The Ea DAcT model contains four transmembrane domains ( TMDs), with both the N- and C-termini orientated toward the lumen of the ER. In addition, there is a large cytoplasmic loop between the first and second TMDs, with the MBOAT signature region of the protein embedded in the third TMD close to the interface between the membrane and the cytoplasm. During topology mapping, we discovered two cysteine residues (C187 and C293) located on opposite sides of the membrane that are important for enzyme activity. In order to identify additional amino acid residues important for acetyltransferase activity, we isolated and characterized acetyltransferases from other acetyl- TAG-producing plants. Among them, the acetyltransferase from Euonymus fortunei possessed the highest activity in vivo and in vitro. Mutagenesis of conserved amino acids revealed that S253, H257, D258 and V263 are essential for Ea DAcT activity. Alteration of residues unique to the acetyltransferases did not alter the unique acyl donor specificity of Ea DAcT, suggesting that multiple amino acids are important for substrate recognition. [ABSTRACT FROM AUTHOR]

Glutathione peroxidases (GPXs) are enzymes that are present in almost all organisms with the primary function of limiting peroxide accumulation. In mammals, two of the eight members (GPX7 and GPX8) reside in the endoplasmic reticulum (ER). A peculiar feature of GPX8 is the concomitant presence of a conserved N-terminal transmembrane domain (TMD) and a C-terminal KDEL-like motif for ER localization. Aims: Investigating whether and how GPX8 impacts Ca2+ homeostasis and signaling. Results: We show that GPX8 is enriched in mitochondria-associated membranes and regulates Ca2+ storage and fluxes. Its levels correlate with [Ca2+]ER, and cytosolic and mitochondrial Ca2+ fluxes. GPX7, which lacks a TMD, does not share these properties. Deleting or replacing the GPX8 TMD with an unrelated N-terminal membrane integration sequence abolishes all effects on Ca2+ fluxes, whereas appending the GPX8 TMD to GPX7 transfers the Ca2+-regulating properties. Innovation and Conclusion: The notion that the TMD of GPX8, in addition to its enzymatic activity, is essential for regulating Ca2+ dynamics reveals a novel level of integration between redox-related proteins and Ca2+ signaling/homeostasis. Antioxid. Redox Signal. 27, 583-595. [ABSTRACT FROM AUTHOR]

CD38 catalyzes the synthesis of the Ca2+ messenger, cyclic ADP-ribose (cADPR). It is generally considered to be a type II protein with the catalytic domain facing outside. How it can catalyze the synthesis of intracellular cADPR that targets the endoplasmic Ca2+ stores has not been resolved. We have proposed that CD38 can also exist in an opposite type III orientation with its catalytic domain facing the cytosol. Here, we developed a method using specific nanobodies to immunotarget two different epitopes simultaneously on the catalytic domain of the type III CD38 and firmly established that it is naturally occurring in human multiple myeloma cells. Because type III CD38 is topologically amenable to cytosolic regulation, we used yeast-two-hybrid screening to identify cytosolic Ca2+ and integrin-binding protein 1 (CIB1), as its interacting partner. The results from immunoprecipitation, ELISA, and bimolecular fluorescence complementation confirmed that CIB1 binds specifically to the catalytic domain of CD38, in vivo and in vitro. Mutational studies established that the N terminus of CIB1 is the interacting domain. Using shRNA to knock down and Cas9/guide RNA to knock out CIB1, a direct correlation between the cellular cADPR and CIB1 levels was demonstrated. The results indicate that the type III CD38 is functionally active in producing cellular cADPR and that the activity is specifically modulated through interaction with cytosolic CIB1. [ABSTRACT FROM AUTHOR]

The topologies of α-helical membrane proteins are generally thought to be determined during their cotranslational insertion into the membrane. It is typically assumed that membrane topologies remain static after this process has ended. Recent findings, however, question this static view by suggesting that some parts of, or even the whole protein, can reorient in the membrane on a biologically relevant time scale. Here, we focus on antiparallel homo- or heterodimeric small multidrug resistance proteins and examine whether the individual monomers can undergo reversible topological inversion (flip flop) in the membrane until they are trapped in a fixed orientation by dimerization. By perturbing dimerization using various means, we show that the membrane orientation of a monomer is unaffected by the presence or absence of its dimerization partner. Thus, membrane-inserted monomers attain their final orientations independently of dimerization, suggesting that wholesale topological inversion is an unlikely event in vivo. [ABSTRACT FROM AUTHOR]