We developed a nanodisc (ND)-based platform with recombinant acid sphingomyelinase (ASM) embedded in a bilayer of phospholipids that are circumscribed by apolipoprotein E3 (apoE3) for targeted delivery to lysosomes. We designed a fusion protein comprising essential catalytic segments of ASM with a transmembrane helix at one end (TM-ASMcat). Purified TM-ASMcat was reconstituted with phospholipids and the N-terminal (NT) domain of apoE3 to promote the formation of NDs (TM-ASMcat-ND). Physicochemical characterization revealed the presence of both TM-ASMcat (35 kDa) and apoE3NT (24 kDa) in the preparations, and formation of large complexes (400–600 kDa). Transmission electron microscopy of TM-ASMcat-ND revealed discoidal complexes (major/minor axes of 30.9 ± 11.9/8.6 ± 2.0 nm, respectively), while atomic force microscopy showed NDs 14 nm high. The TM-ASMcat-ND displayed a significant ability to hydrolyze sphingomyelin at pH 5. LC-MS/MS of glioblastoma cells treated with TM-ASMcat-ND revealed a significant decrease (22.3
The global challenge of increasing antibiotic resistance development has necessitated the continued development and investigation of novel and existing antimicrobials. Daptomycin is a cyclic lipopeptide isolated from Streptomyces roseosporus, which has been clinically approved to treat a variety of gram-positive infections. Daptomycin is a membrane-active antimicrobial peptide that requires Ca2+ ions and phosphatidylglycerol lipids to exert its antimicrobial activity. To better understand the mechanism of action at the biophysical level, daptomycin interactions with bilayers and bacterial membranes were evaluated across different solution pH values and by varying cholesterol content in the model membranes. The results demonstrate that pH 5–8 does not affect daptomycin binding to model membranes or its structural rearrangement upon binding to the bilayer. Similarly, including cholesterol up to 30 mol
Time-resolved X-ray solution scattering (TR-XSS) provides direct access to protein structural dynamics but has largely been restricted from the microsecond range up to approximately 100 milliseconds. As a result, slower enzymatic systems, including many P-type ATPases, remain difficult to probe. Here, we extend the temporal reach of TR-XSS by sequentially positioning radiation damage-free acquisition windows to enable capturing structural evolution across sub-second to second timescales. Implemented at the CoSAXS beamline at MAX IV Laboratory, this strategy enables continuous tracking of slow protein dynamics while preserving structural sensitivity. Using adenylate kinase (AdK) as a benchmark, we observed a single conformational transition accompanied by signal amplitude decay. In contrast, application to the prokaryotic P-type ATPase LMCA1 revealed clear evolution in scattering profiles, consistent with sequential conformational transitions. Kinetic analysis identified two transitions on the 140 ms and 660 ms timescales, which correspond monitoring rise and decay of a rate-limiting step which can symbolize intermediate dynamics in a slow transport cycle. The results demonstrate that extended-time TR-XSS can resolve multi-step reaction pathways in slow membrane proteins. The approach broadens the accessible timescale of TR-XSS and establishes a general framework for studying slow conformational dynamics in P-type ATPases and related systems.
The viral envelope fuses with the host cell membrane to initiate viral infection. In the case of SARS-CoVs, the fusion process is catalyzed by the spike (S) protein. The S1 subunit interacts with the host cell receptor and assists in docking the viral particle at the cell surface, whereas the fusion process is induced by the insertion of a conserved hydrophobic motif at the N-terminal of the S2 subunit, known as a fusion peptide, into the host cell. Current experimental findings suggest that the fusion peptide of SARS-CoVs displays a strong Ca2+-dependent membrane binding and fusogenic activity, indicating that Ca2+ functions as a critical cofactor during viral entry. Nevertheless, many earlier studies fail to clearly differentiate between fusion driven directly by Ca2+ and that mediated by the fusion peptide, often due to the absence of appropriate controls for different Ca2+ concentrations. In this work, we have assessed the impact of the consensus fusion peptide of SARS-CoVs in polyethylene glycol (PEG)-mediated fusion of small unilamellar vesicles with varying Ca2+ concentration, where appropriate control experiments have been carried out in the absence of the peptide. Our results show that the peptide has no significant effect on Ca2+ in inducing lipid mixing of model membranes.
The cell surface localization of the Na,K-ATPase (sodium pump) is required for maintaining transmembrane electrochemical gradients. While glycosylation of the β1 subunit facilitates trafficking from the endoplasmic reticulum to the plasma membrane, its role in nanoscale surface organization is not characterized. This study employed GlycoSHIELD computational modeling and DNA-PAINT single-molecule localization microscopy (SMLM) to evaluate how N-glycans influence pump distribution. In-silico simulations indicated that N-glycans sequester the protein core, providing a steric shield that increases with structural complexity. To investigate this experimentally, glycosylation-deficient mutants (3NQ) were generated and confirmed via immunoblotting. Quantitative SMLM analysis of A498 cells demonstrated that wild-type pumps exhibit higher localization density and form larger (144 nm) and more frequent clusters than 3NQ mutants (109 nm). These results indicate that N-glycosylation promotes stable enzyme clustering, supporting a galectin-lattice mechanism of organization rather than steric repulsion.
Syncytin-2, a human endogenous retrovirus-derived fusogen, is essential for placental syncytiotrophoblast formation by mediating trophoblast cell-cell fusion. While Syncytin-2 shares structural similarities with viral envelope proteins, the molecular mechanisms underlying its receptor recognition and membrane fusion remain poorly characterized, largely due to the lack of in vitro reconstitution systems. Here, we established an in vitro reconstitution system to investigate the initial steps of Syncytin-2-mediated membrane fusion. We purified Syncytin-2 and its receptor MFSD2A and confirmed their interaction through co-immunoprecipitation and pull-down assays. Both proteins were successfully reconstituted into liposomes and exhibited specific tethering dependent on the receptor-fusogen interaction, which is the first committed step of fusion. This study provides an in vitro reconstitution of a placental fusogen-receptor pair, establishing a powerful platform for dissecting the molecular mechanism of syncytin-mediated cell-cell fusion and shedding light on understanding placenta development at the molecular level.
The small molecule di-tert-butylhydroquinone (BHQ) is a potent inhibitor of the sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA). It inhibits the enzyme by stabilizing it in its E2 conformation and thereby preventing the transition to the E1 state. To define the structural determinants underlying BHQ inhibition, we examined ten BHQ derivatives with systematic modifications of polar and hydrophobic substituents of different shapes and sizes. Inhibitory potencies were determined in ATPase activity assays, and crystal structures of SERCA1a in complex with eight of the ten derivatives were obtained at resolutions of 2.6 Å or better, aided by co-crystallization of thapsigargin to stabilize the E2 state. The structures revealed that the compounds bind in a pocket formed by transmembrane helices M1-M4, in which the ligand is stabilized by CH-π interactions between the central benzene ring and Leu61 and Pro312, by hydrogen bonds – preferentially involving Asp59 – and by extensive van der Waals contacts mediated by two butyl substituents. Analysis of the SERCA/inhibitor poses showed that while at least one hydrogen bond is required for binding, inhibitory potency correlates strongly with the number and quality of van der Waals contacts mediated by appropriately positioned alkyl groups. Compounds lacking alkyl substituents or containing oversized or mispositioned groups displayed markedly reduced potencies. Although no derivative exceeded BHQ in inhibitory activity, structural analysis suggests that repositioning hydroxy groups on the benzene ring or modifying alkyl substituents may further optimize binding affinity and thus inhibitory potency. Together, these results define the key molecular features of SERCA inhibition by BHQ analogs and suggest strategies for further optimization of this inhibitor class.
Extracellular vesicles (EVs) are central components of fungal biology, yet their isolation commonly relies on ultracentrifugation protocols originally developed for mammalian systems. In Cryptococcus, most EVs carry the capsular polysaccharide glucuronoxylomannan (GXM), raising the possibility that vesicle density, and therefore sedimentation, could be influenced by polysaccharide content. Here, we systematically evaluated EV recovery across sequential ultracentrifugation speeds (20,000 × g to 100,000 × g) in Cryptococcus deuterogattii, Candida auris, and Candida parapsilosis. In C. deuterogattii, GXM was detected across all ultracentrifugation fractions, and each fraction efficiently transferred polysaccharide to acapsular cells, demonstrating that its presence is independent of fraction density and does not correlate with sedimentation behavior. In the three fungal species, transmission electron microscopy and nanoparticle tracking analysis confirmed that bona fide EVs are recoverable across all centrifugation speeds. Qualitative and quantitative proteomic analyses revealed largely overlapping protein compositions among fractions within each species. However, proteomic profiles differed between species. In C. deuterogattii and C. auris, fractions displayed similar proteomic and predicted protein-protein interaction signatures across centrifugation speeds. In contrast, C. parapsilosis exhibited a clear partitioning pattern, with low-speed fractions (20,000 × g and 40,000 × g) clustering together and differing from higher-speed fractions (60,000–100,000 × g). These findings demonstrate that fungal EVs are recoverable across a broad range of ultracentrifugation speeds, but their sedimentation behavior is species-specific. Our study highlights the need for tailored EV isolation strategies and cautions against assuming methodological equivalence across fungal pathogens. Extracellular vesicles (EVs) are central to fungal biology, but their isolation still relies on protocols adapted from mammalian systems, with limited exploration of more appropriate protocols for fungi. Here, we show that EVs from major fungal pathogens are recovered across a broad range of ultracentrifugation speeds, and that their sedimentation behavior is species-specific. These findings challenge the use of fixed ultracentrifugation conditions as a universal standard and highlight the need to tailor EV isolation strategies for different fungal species, with important implications for reproducibility and data interpretation in the field.
Recent experiments have shown that incorporating polyproline segments into arginine (R)-rich cell-penetrating peptides (CPPs) enhances membrane penetration. Here, we employ molecular dynamics (MD) simulations combined with the weighted ensemble approach to investigate how a polyproline segment influences the free-energy barrier for membrane translocation in the designed peptide P9R9. Our results indicate that the extended, conformationally constrained nature of the P9 segment facilitates early membrane engagement and promotes the formation of a hydrated translocation pathway. This behavior is associated with a reduced desolvation penalty during insertion of the arginine-rich (R9) segment. Consistent with this interpretation, the solvent-accessible surface area (SASA) of R9 exhibits a non-monotonic trend, suggesting partial rehydration within the membrane interior. Together, these findings support a sequential translocation mechanism in which P9 interacts with the membrane prior to R9, thereby facilitating subsequent insertion and lowering the free-energy barrier relative to peptides lacking polyproline segments. This work provides a molecular-level perspective on how polyproline segments modulate membrane translocation and offers useful insights for designing more effective CPPs.
Phosphatidylserine (PS) externalization is a hallmark of apoptosis but can also be triggered within seconds by pulsed electric fields (PEFs). Whether PS reaches the outer leaflet by lateral migration through electropores or indirectly through Ca2+-dependent signaling and scramblase activation remains debated. We used total internal reflection fluorescence (TIRF) microscopy with Annexin V-Alexa Fluor 568 and/or bovine lactadherin-FITC to resolve PS externalization in HEK293 cells placed on an indium tin oxide (ITO) transparent electrode. PEF exposures produced discrete PS-positive puncta (“freckles”) within 10 s after a 20-µs pulse (1.2–2.5 kV/cm) or with a 1-min delay after a 400-ns pulse (9.3–13.7 kV/cm). Their number gradually increased, reaching up to 80–120 freckles per 100 µm2 of cell membrane area within 5 min. Freckles were round, brightest in the center, and well described by a Gaussian intensity profile, with a consistent full width at half maximum (FWHM) of 0.35 µm despite up to 50-fold differences in peak intensity. Freckle formation was inhibited in Ca2+-free medium but could be partially restored by applying 20-µs PEFs at higher strengths. Many freckles moved laterally and axially in an apparently stochastic manner, whereas others remained spatially confined. The uniform size and shape of freckles and their mobility patterns suggested that they are PS-positive vesicles rather than PS membrane patches. Further analyses identified freckles as extracellular vesicles of 200-nm estimated diameter whose formation is triggered by applying PEFs at intensities above the electroporation threshold.
The group of inhibitors known as Potassium Competitive Acid Blockers (PCABs) has become one of the main topics of current research into reducing gastric H,K-ATPase activity. The design of novel PCABs relies on structure-based drug design strategies that integrate structural data with molecular dynamics simulations. A key aspect in conventional molecular dynamics simulations is the assignment of residue charge states, since the local physicochemical environment influences the pKa of amino acids. This computational work investigates the impact of the protonation state of key residues on the interactions between PCABs and the gastric H,K-ATPase. The study focusses on Glu343, Glu795, and Glu820, from the cation binding cavity, and vonoprazan and tegoprazan; two chemically unrelated PCABs. The results show how protonation states generate changes in interactions, especially with the charged group of inhibitors. Simulations in which Glu343 or Glu795 were protonated showed that the charged secondary amine of vonoprazan could rotate freely, favoring hydrogen bonding with some of the glutamic residues deep within the pocket. In contrast, the positive charge of tegoprazan is located in the benzimidazole ring, a rigid and bulky structure, which moves away from the center of the cavity when Glu343 or Glu795 are protonated. The inspection of the ionization states of amino acids reveals the conformational flexibility of the protonated group of PCABs as pivotal for binding affinity and highlights the importance of considering the protonation state of protein residues before performing any conventional molecular dynamics simulations for drug design.
P-type ATPases are active transporter enzymes that maintain ion electrochemical gradients for functions such as muscle contraction, kidney function, digestion, and nerve signalling. The dysfunction of these ATPases has been linked to several neurological, cardiovascular, and metabolic disorders. An electrostatic switch mechanism (ESM) has been hypothesised to be involved in the regulation of the sodium-potassium pump (Na+,K+-ATPase) and the gastric proton pump (H+,K+-ATPase). An analysis of primary and tertiary protein structures suggests a potentially higher prevalence of the ESM in human P-type ATPases than previously thought. Evidence for the ESM was found on the N-terminus for the P1B, and P2C subfamilies of P-type ATPases, and on the C-terminus for the P2A and P5 subfamilies. In the case of the P4 family, evidence for the ESM was found on the N-terminus for some subfamilies and on the C-terminus for others. Evidence for the ESM has been identified in P-type ATPase subfamilies distributed in separate lineages of the phylogenetic tree, suggesting that the ESM evolved multiple times independently.
The Na+,K+-ATPase is an integral membrane protein present in all animal plasma membranes. It uses the energy of ATP hydrolysis to pump 3 Na+ ions per ATP hydrolysed from the cell cytoplasm into the extracellular fluid in exchange for 2 K+ ions. The Na+ electrochemical potential gradient it produces is used as an energy source to drive all animal secondary transporters, for example, in nutrient reabsorption in the kidney. In this synthesis of new and previously published results, it is shown that the rate of the mammalian enzyme’s rate-determining E2 → E1 conformational change, during which K+ ions are released to the cytoplasm, is strongly dependent on an electrostatic interaction in the E2 state. This electrostatic interaction must be broken to allow the enzyme to convert to the E1 state and allow Na+ pumping. The strength of the interaction depends on ionic strength and, even more strongly, on the concentration of divalent metal ions, i.e., Ca2+ or Mg2+. A likely candidate for the interaction is between the protein’s positively-charged lysine-rich N-terminus and the negatively-charged membrane cytoplasmic surface. Comparison of the Ca2+ and Mg2+ dissociation constants measured with the physiological levels of the two ions suggests that Mg2+, but not Ca2+, could play a regulatory role for the Na+,K+-ATPase.
Antimicrobial peptides (AMPs) represent a promising class of alternatives to conventional antibiotics. In this study, we compared the structural and functional properties of three synthetic undecapeptides: the anionic Cn-AMP2 and its N-terminally acetylated derivative (Ac-CnAMP2), versus the cationic α-helical peptide BP52. While BP52 exhibited strong antibacterial activity against both Gram-positive and Gram-negative bacteria, Cn-AMP2 and Ac-CnAMP2 showed no measurable antibacterial effects up to 128µM. Hemolysis assays revealed that BP52 caused mild to moderate lysis of red blood cells, whereas the anionic peptides were non-hemolytic. Molecular dynamics simulations confirmed the enhanced membrane insertion, clustering behavior, and bilayer disruption induced by BP52, in contrast to the limited interaction profiles of Cn-AMP2. These findings underscore the importance of positive charge, helical conformation, and amphipathic topology in driving membrane action and offer insights for designing optimized AMP-based therapeutics.
We recently developed a time-correlated single photon counting (TCSPC) spectroscopy approach to investigate the activation mechanisms of the calcium pump SERCA (sarcoplasmic reticulum Ca2+-ATPase). Here, we apply this approach to characterize the effects of two chemically distinct SERCA inhibitors, thapsigargin (TG) and cyclopiazonic acid (CPA), and to determine how they differentially modulate SERCA conformational dynamics. Although TG and CPA appear to stabilize similar SERCA states in structural studies, TCSPC reveals fundamentally distinct mechanisms of inhibition. TG stabilizes an inactive conformation that prevents Ca2+- and ATP-dependent transitions, effectively trapping SERCA in a nonproductive ‘dead-end’ state. In contrast, CPA attenuates, rather than abolishes, Ca2+- and nucleotide-dependent structural transitions, producing graded, concentration-dependent effects that reduce the population of the closed, activation-associated state. Notably, CPA decreases apparent Ca2+ affinity only in the presence of a non-hydrolyzable ATP analog, consistent with an ATP-dependent, allosteric mechanism that redistributes conformational populations rather than directly occluding Ca2+ binding sites. These findings demonstrate that TCSPC resolves mechanistic differences between inhibitors that appear structurally similar and provide a framework for understanding how distinct allosteric ligands modulate SERCA function.
Research on the auxiliary subunits β (CaVβ) and α2δ (CaVα2δ) of voltage-gated calcium channels has gained increasing interest as novel and unexpected functional interactions for these proteins are discovered. Beyond their classic role as regulators of the channel complex, these subunits participate in the spatiotemporal fine-tuning of the channels and in diverse cellular processes. Currently, multiple studies are investigating the interactions between CaVβ and CaVα2δ with other proteins outside the channel complex, and how these associations affect relevant cellular functions such as cell growth, differentiation, and gene expression beyond their effects on channel activity. The auxiliary subunits have also been observed to associate with components involved in calcium channel biogenesis, a process independent of the direct modulation of channel activity. Furthermore, their involvement in the intracellular transport of other channels and receptors, as well as in nuclear signaling, has been demonstrated. The expression of different variants or isoforms may fulfill specific functions in diverse tissues and developmental stages, even outside the channel complex. Undoubtedly, the study of these proteins as scaffolding or regulatory molecules for other proteins has significantly enriched our understanding of their influence on cell signaling and excitability.
Cell-penetrating peptides (CPPs) are increasingly used for delivering cargo into cells, but the mechanisms of their membrane crossing remain poorly understood, even for shorter cationic hydrophobic peptides. This study explores how cationic hydrophobic peptides with various combinations of cationic (K and R) and hydrophobic (W and I) amino acids interact with lipid bilayers made of 90% neutral lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (PC) and 10% anionic lipids (either 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (PG) or 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (PS)). Specifically, it was found that changing the hydrophobic segment from W to I significantly influences turbidity during the pretransition for the PC/PG mixture. Conversely, in the PC/PS mixture, the interaction with I-peptides causes changes in turbidity during the main phase transition, regardless of whether the cationic segment is R or K. Molecular features analyzed through FTIR spectroscopy revealed significant differences in the vibrations of methylene groups within hydrocarbon chains. In the presence of I-peptides, regardless of whether they are R or K derivatives, a notably higher number of kink conformers were observed compared to W-peptides. Although W-peptides tend to form aggregates due to the insertion of their hydrophobic segments into the lipid bilayer, they appear to maintain the membrane's integrity and the organization of lipids. In contrast, the branched side chains of the I-segment induce out-of-plane movements in the hydrocarbon chains. As the initial interaction between the peptide and the lipid membrane is crucial for its translocation, these findings provide insights into the molecular events occurring before translocation and emphasize the specifics that make it unique to CPPs.
Defensins function as critical effectors of innate immunity, displaying broad-spectrum antimicrobial activity against bacteria, fungi, and viruses. While experimental studies have extensively characterized these peptides, the molecular mechanisms governing their membrane interactions remain poorly understood. This investigation employed comprehensive molecular dynamics simulations to analyze six defensins (human and rabbit α-defensins HNP1, HNP3, rabbit NP4 and human β-defensins HBD1, HBD2, HBD3) using the IMM1 implicit membrane model for studying interactions with anionic bacterial membranes. Both monomeric and dimeric forms were examined to elucidate oligomerization effects on membrane binding and penetration mechanisms. Transfer energy calculations revealed distinct binding patterns and orientations among different defensin subtypes, with β-defensins demonstrating superior membrane affinity compared to α-defensins. HBD3 exhibited the most favorable membrane interactions, correlating with its exceptional antimicrobial potency. Binding orientation analysis demonstrated that defensins adopt specific membrane-bound configurations that optimize electrostatic interactions with anionic membrane surfaces while strategically positioning hydrophobic regions for effective membrane insertion. These findings provide molecular level insights into defensin selectivity mechanisms for bacterial membranes and establish a foundation for defensin-based therapeutic development. The results validate that computational approaches effectively complement experimental studies in elucidating complex antimicrobial peptide mechanisms. This modeling framework supports rational design of next generation defensin therapeutics with optimized antimicrobial activity and pathogen selectivity.
