M2 Domain Research Articles

Atypical influenza A(H1N1)pdm09 strains caused an influenza virus outbreak in Saudi Arabia during the 2009–2011 pandemic season

BackgroundThe triple assortment influenza A(H1N1) virus emerged in spring 2009 and disseminated worldwide, including Saudi Arabia. This study was carried out to characterize Saudi influenza isolates in relation to the global strains and to evaluate the potential role of mutated residues in transmission, adaptation, and the pathogenicity of the virus. MethodsNasopharyngeal samples (n = 6492) collected between September 2009 to March 2011 from patients with influenza-like illness were screened by PCR for influenza A(H1N1). Phylogenetic and Molecular evolutionary analysis were carried out to place the Saudi strains in relation to the global strains followed by Mutation analysis of surface and internal proteins. ResultsConcatenated whole-genome phylogenetic analysis along with hemagglutinin (HA) signature changes, that is, Aspartic Acid (D) at position 187, P83S, S203T, and R223Q confirmed that the Saudi strains belong to the antigenic category of A/California/07/2009. However, phylogenetic analysis revealed unusual strains of A(H1N1) circulating in Saudi Arabia, not belonging to any of known clades, appearing in five distinct groups well supported by group-specific mutations and novel mutation complexes. These cases had characteristic inter- and intragroup substitution patterns while few of their closest matches showed up as sporadic cases the world over. Specific mutation patterns were detected within the functional domains of internal proteins PB2, PB1, PA, NP, NS1, and M2 having a putative role in viral fitness and virulence. Bayesian coalescent MCMC analysis revealed that Saudi strains belonged to cluster 2 of A(H1N1)pdm09 and spread a month later as compared to other strains of this cluster. ConclusionInfluenza outbreak in Saudi Arabia during 2009–2011 was caused by atypical strains of influenza A(H1N1)pdm09, probably introduced in this community on multiple occasions. To understand the antigenic significance of these novel point mutations and mutation complexes require functional studies, which will be crucial for risk assessment of emergent strains and defining infection control measures.

Force-dependent unfolding and folding dynamics of protein alpha-catenin modulation domains

[Formula: see text]-catenin is an adhesion protein located at the cadherin-based cell–cell adherens junction. [Formula: see text]-catenin cross-links [Formula: see text]-catenin and actin fiber in the adhesion protein complex, and plays an important role in the formation and modulation of cell–cell adhesion. The central modulation domains can be unfolded to expose binding site of vinculin when stretching force is applied. Here, we studied the force-induced unfolding dynamics of [Formula: see text]-catenin modulation domains under different loading rates from which the unfolding distance of M2 and M3 domains is determined to be 5–7[Formula: see text]nm, and an unfolding intermediate state is identified. We also found that the folding process of M1–M3 domains goes through different pathways with cooperativity.

The NMDA receptor intracellular C-terminal domains reciprocally interact with allosteric modulators

N-methyl-d-aspartate receptors (NMDARs) have multiple prominent roles in CNS function but their excessive or insufficient activity contributes to neuropathological/psychiatric disorders. Consequently, a variety of positive and negative allosteric modulators (PAMs and NAMs, respectively) have recently been developed. Although these modulators bind to extracellular domains, in the present report we find that the NMDAR’s intracellular C-terminal domains (CTDs) significantly influence PAM/NAM activity. GluN2 CTD deletion robustly affected NAM and PAM activity with both enhancing and inhibiting effects that were compound-specific and NMDAR subunit-specific. In three cases, individual PAMs became NAMs at specific GluN2-truncated receptors. In contrast to GluN2, GluN1 CTD removal only reduced PAM activity of UBP684 and CIQ, and did not affect NAM activity. Consistent with these findings, agents altering phosphorylation state or intracellular calcium levels displayed receptor-specific and compound-specific effects on PAM activity. It is possible that the GluN2′s M4 domain transmits intracellular modulatory signals from the CTD to the M1/M4 channel gating machinery and that this site is a point of convergence in the direct or indirect actions of several PAMs/NAMs thus rendering them sensitive to CTD status. Thus, allosteric modulators are likely to have a marked and varied sensitivity to post-translational modifications, protein-protein associations, and intracellular ions. The interaction between PAM activity and NMDAR CTDs appears reciprocal. GluN1 CTD-deletion eliminated UBP684, but not pregnenolone sulfate (PS), PAM activity. And, in the absence of agonists, UBP684, but not PS, was able to promote movement of fluorescently-tagged GluN1-CTDs. Thus, it may be possible to pharmacologically target NMDAR metabotropic activity in the absence of channel activation.

Salmonella Gallinarum delivering M2eCD40L in protein and DNA formats acts as a bivalent vaccine against fowl typhoid and H9N2 infection in chickens

Fowl typhoid (FT), a septicemic disease caused by Salmonella Gallinarum (SG), and H9N2 influenza infection are two economically important diseases that affect poultry industry worldwide. Herein, we exploited a live attenuated SG mutant (JOL967) to deliver highly conserved extracellular domains of H9N2 M2 (M2e) to induce protective immunity against both H9N2 infection and FT. To increase the immunogenicity of M2e, we physically linked it with CD40L and cloned the fusion gene into either prokaryotic constitutive expression vector pJHL65 or mammalian expression vector pcDNA3.1+. Then pJHL65-M2eCD40L or pcDNA-M2eCD40L recombinant plasmid was electroporated into JOL967 strain and the resultant clones were designated as JOL2074 and JOL2076, respectively. We demonstrated that the chickens vaccinated once orally with a co-mix of JOL2074 and JOL2076 strains elicited significantly (p < 0.05) higher M2e-specific humoral and cell-mediated immunity compared to JOL2074 alone vaccinated group. However, SG-specific immune responses were comparable in both the vaccination groups. On challenge with the virulent H9N2 virus (105 TCID50) at 28th day post-vaccination, chickens that received a co-mix of JOL2074 plus JOL2076 strains exhibited significantly (p < 0.05) lower lung inflammation and viral load in both lungs and cloacal samples than JOL2074 alone vaccinated group. Against challenge with the lethal wild-type SG, both the vaccination groups exhibited only 12.5% mortality compared to 75% mortality observed in the control group. In conclusion, we show that SG delivering M2eCD40L can act as a bivalent vaccine against FT and H9N2 infection and further studies are warranted to develop this SG-M2eCD40L vaccine as a broadly protective vaccine against avian influenza virus subtypes.

Metabolic flux analysis of glucose/ethanol metabolism in Saccharomyces cerevisiae cultures

Microbes living around hydrothermal vents have developed lots of enzymes with specific characteristics that allow them to thrive in the environment. Extracellular enzyme leucine aminopeptidases (LAPs) are all metallopeptidases that catalyze the hydrolysis of leucine residues from N-terminal residues from peptides and proteins. Genomic analysis of Pseudoalteromonas telluritireducens DSM 16098, a strain of deep-sea bacteria isolated from hydrothermal vents fluid, revealed the presence of an LAP gene (designated as PtLAP). The open reading frame of PtLAP encodes a protein of 614 amino acids (GenBank accession no. KY429939), consisting of a signal peptide, a peptidase_M1 domain and a leuk-A4-hydro_C domain. The putative substrate binding site GGMEN, zinc-binding motif HEXXHX18E and the catalytic residues involved in aminopeptidase activity are all conserved, indicating PtLAP is a member of M1 leucine aminopeptidases. The recombinant PtLAP expressed in Escherichia coli Transetta (DE3) (designated as rPtLAP) exhibits a significant LAP activity to hydrolyze L-leucine 7-amido-4-methylcoumarin (MAC-L). The optimal activity of rPtLAP is observed at pH 7.2 and 28 °C with half-saturation coefficient (Km) of 44.56 ± 5.09 μmol L−1. rPtLAP activity is relatively stable at 30–40 °C and exhibits a relatively wide pH range (6.0–12.5) with the maximal activity ranging at pH 7.2–8.0. It can be inhibited by EDTA, protease inhibitors and some metal ions including Cu2+, Zn2+ and Cd2+, but is relatively tolerant to Mn2+ which is enriched in hydrothermal vent fluids. Furthermore, rPtLAP can significantly promote the proliferation of tumor cells, including the cell lines HCT116, BEL-7402 and Hela, while displays no effect on that of normal cell 3T3. These results together suggest that the extracellular enzyme PtLAP executes the degradation of organic matters, contributes to the adaptive survival of microbe in deep-sea environment and may play important roles in marine biogeochemical cycles.

Modelling the details: integrating structure with function.

Modelling the details: integrating structure with function.

Expression in Plants of a Recombinant Protein Based on Flagellin Linked to Conservative Fragments of M2 Protein and Hemagglutintin of Influenza Virus

The composition of traditional influenza vaccines require nearly annual updates due to the high variability of the influenza virus. The use of conservative viral antigens, the extracellular domain of the transmembrane protein M2, and fragments of the second subunit of hemagglutinin provides the opportunity to create recombinant broad-spectrum vaccines. Bacterial flagellin was used as a mucosal adjuvant to increase the immunogenicity of these conservative antigens. Recombinant proteins based on flagellin simultaneously containing M2e and a fragment of the hemagglutinin stem region were expressed in Nicotiana benthamiana plants using a self-replicating vector based on the potato virus X genome. Methods for their isolation from plants and purification have been developed. The developed expression system can be used to produce a new candidate influenza vaccine in plants.

CONSTRUCTION OF MOSAIC HBC PARTICLES PRESENTING CONSERVATIVE FRAGMENTS OF M2 PROTEIN AND HEMAGGLUTININ OF INFLUENZA A VIRUS.

Virus-like HBc particles formed as a result of the self-assembly of the nuclear antigen of the hepatitis B virus can be used as a highly immunogenic carrier for the presentation of foreign epitopes when creating recombinant vaccines. We use this vehicle to create influenza vaccines based on the conservative antigens of the influenza virus, the extracellular domain of the transmembrane protein M2 (M2e) and the fragment of the second subunit of hemagglutinin (HA2). Presentation on the surface of HBc particles should improve the immunogenicity of these peptides. Using genetic engineering techniques, we obtained a fusion protein in which the HA2 sequence is attached to the N-terminus of the HBc antigen, and the M2e peptide is included in the immunodominant loop region exposed on the surface of HBc particle. The hybrid protein expressed in Escherichia coli and purified under denaturing conditions formed virus-like HBc particles after refolding in vitro. Refolding of this protein in the presence of a previously denatured HBc antigen carrying no inserts resulted in formation of mosaic virus-like particles. The developed method will allow construction of mosaic HBc particles carrying different target epitopes of the influenza virus by combining the corresponding modified HBc proteins, which opens the possibility of creating vaccines with a wider spectrum of protection.

Cloning and characterization of a leucine aminopeptidase from Pseudoalteromonas telluritireducens DSM 16098, a strain isolated from hydrothermal vents fluid

Microbes living around hydrothermal vents have developed lots of enzymes with specific characteristics that allow them to thrive in the environment. Extracellular enzyme leucine aminopeptidases (LAPs) are all metallopeptidases that catalyze the hydrolysis of leucine residues from N-terminal residues from peptides and proteins. Genomic analysis of Pseudoalteromonas telluritireducens DSM 16098, a strain of deep-sea bacteria isolated from hydrothermal vents fluid, revealed the presence of an LAP gene (designated as PtLAP). The open reading frame of PtLAP encodes a protein of 614 amino acids (GenBank accession no. KY429939), consisting of a signal peptide, a peptidase_M1 domain and a leuk-A4-hydro_C domain. The putative substrate binding site GGMEN, zinc-binding motif HEXXHX18E and the catalytic residues involved in aminopeptidase activity are all conserved, indicating PtLAP is a member of M1 leucine aminopeptidases. The recombinant PtLAP expressed in Escherichia coli Transetta (DE3) (designated as rPtLAP) exhibits a significant LAP activity to hydrolyze L-leucine 7-amido-4-methylcoumarin (MAC-L). The optimal activity of rPtLAP is observed at pH 7.2 and 28 °C with half-saturation coefficient (Km) of 44.56 ± 5.09 μmol L−1. rPtLAP activity is relatively stable at 30–40 °C and exhibits a relatively wide pH range (6.0–12.5) with the maximal activity ranging at pH 7.2–8.0. It can be inhibited by EDTA, protease inhibitors and some metal ions including Cu2+, Zn2+ and Cd2+, but is relatively tolerant to Mn2+ which is enriched in hydrothermal vent fluids. Furthermore, rPtLAP can significantly promote the proliferation of tumor cells, including the cell lines HCT116, BEL-7402 and Hela, while displays no effect on that of normal cell 3T3. These results together suggest that the extracellular enzyme PtLAP executes the degradation of organic matters, contributes to the adaptive survival of microbe in deep-sea environment and may play important roles in marine biogeochemical cycles.

In silico epitope mapping and experimental evaluation of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) as a malaria vaccine candidate

BackgroundTechnical limitations for culturing the human malaria parasite Plasmodium vivax have impaired the discovery of vaccine candidates, challenging the malaria eradication agenda. The immunogenicity of the M2 domain of the Merozoite Adhesive Erythrocytic Binding Protein (MAEBL) antigen cloned from the Plasmodium yoelii murine parasite, has been previously demonstrated.ResultsDetailed epitope mapping of MAEBL through immunoinformatics identified several MHCI, MHCII and B cell epitopes throughout the peptide, with several of these lying in the M2 domain and being conserved between P. vivax, P. yoelii and Plasmodium falciparum, hinting that the M2-MAEBL is pan-reactive. This hypothesis was tested through functional assays, showing that P. yoelii M2-MAEBL antisera are able to recognize and inhibit erythrocyte invasion from both P. falciparum and P. vivax parasites isolated from Thai patients, in ex vivo assays. Moreover, the sequence of the M2-MAEBL is shown to be highly conserved between P. vivax isolates from the Amazon and Thailand, indicating that the MAEBL antigen may constitute a vaccine candidate outwitting strain-specific immunity.ConclusionsThe MAEBL antigen is promising candidate towards the development of a malaria vaccine.

Influenza virus Matrix Protein M1 preserves its conformation with pH, changing multimerization state at the priming stage due to electrostatics

Influenza A virus matrix protein M1 plays an essential role in the virus lifecycle, but its functional and structural properties are not entirely defined. Here we employed small-angle X-ray scattering, atomic force microscopy and zeta-potential measurements to characterize the overall structure and association behavior of the full-length M1 at different pH conditions. We demonstrate that the protein consists of a globular N-terminal domain and a flexible C-terminal extension. The globular N-terminal domain of M1 monomers appears preserved in the range of pH from 4.0 to 6.8, while the C-terminal domain remains flexible and the tendency to form multimers changes dramatically. We found that the protein multimerization process is reversible, whereby the binding between M1 molecules starts to break around pH 6. A predicted electrostatic model of M1 self-assembly at different pH revealed a good agreement with zeta-potential measurements, allowing one to assess the role of M1 domains in M1-M1 and M1-lipid interactions. Together with the protein sequence analysis, these results provide insights into the mechanism of M1 scaffold formation and the major role of the flexible and disordered C-terminal domain in this process.

Group A streptococcal M protein activates the NLRP3 inflammasome

Group A Streptococcus (GAS) is among the top ten causes of infection-related mortality in humans. M protein is the most abundant GAS surface protein, and M1 serotype GAS strains are associated with invasive infections, including necrotizing fasciitis and toxic shock syndrome. Here, we report that released, soluble M1 protein triggers programmed cell death in macrophages (Mϕ). M1 served as a second signal for caspase-1-dependent NLRP3 inflammasome activation, inducing maturation and release of proinflammatory cytokine interleukin-1β (IL-1β) and macrophage pyroptosis. The structurally dynamic B-repeat domain of M1 was critical for inflammasome activation, which involved K+ efflux and M1 protein internalization by clathrin-mediated endocytosis. Mouse intraperitoneal challenge showed that soluble M1 was sufficient and specific for IL-1β activation, which may represent an early warning to activate host immunity against the pathogen. Conversely, in systemic infection, hyperinflammation associated with M1-mediated pyroptosis and IL-1β release could aggravate tissue injury.

Decoupled side chain and backbone dynamics for proton translocation - M2 of influenza A.

The 97 amino acid bitopic membrane protein M2 of influenza A forms a tetrameric bundle in which two of the monomers are covalently linked via a cysteine bridge. In its tetrameric assembly the protein conducts protons across the viral envelope and within intracellular compartments during the infectivity cycle of the virus. A key residue in the translocation of the protons is His-37 which forms a planar tetrad in the configuration of the bundle accepting and translocating the incoming protons from the N terminal side, exterior of the virus, to the C terminal side, inside the virus. With experimentally available data from NMR spectroscopy of the transmembrane domains of the tetrameric M2 bundle classical MD simulations are conducted with the protein bundle in different protonation stages in respect to His-37. A full correlation analysis (FCA) of the data sets with the His-37 tetrad either in a fully four times unprotonated or protonated state, assumed to mimic high and low pH in vivo, respectively, in both cases reveal asymmetric backbone dynamics. His-37 side chain rotation dynamics is increased at full protonation of the tetrad compared to the dynamics in the fully unprotonated state. The data suggest that proton translocation can be achieved by decoupled side chain or backbone dynamics. Graphical abstract Visualization of the tetrameric bundle of the transmembrane domains of M2 of influenza A after 200 ns of MD simulations (upper left). The four histidine residues 37 are either not protonated as in M20 or fully protonated is in M24+. The asymmetric dynamics of the backbones are shown after a full correlation analysis (FCA) in blue (lower left). The rotational dynamics of the χ2 dihedral angles of the histidines in M20 (upper right) are less than those in M24+ (lower right).

Role of the Ion Channel Extracellular Collar in AMPA Receptor Gating

AMPA subtype ionotropic glutamate receptors mediate fast excitatory neurotransmission and are implicated in numerous neurological diseases. Ionic currents through AMPA receptor channels can be allosterically regulated via different sites on the receptor protein. We used site-directed mutagenesis and patch-clamp recordings to probe the ion channel extracellular collar, the binding region for noncompetitive allosteric inhibitors. We found position and substitution-dependent effects for introduced mutations at this region on AMPA receptor gating. The results of mutagenesis suggested that the transmembrane domains M1, M3 and M4, which contribute to the ion channel extracellular collar, undergo significant relative displacement during gating. We used molecular dynamics simulations to predict an AMPA receptor open state structure and rationalize the results of mutagenesis. We conclude that the ion channel extracellular collar plays a distinct role in gating and represents a hub for powerful allosteric modulation of AMPA receptor function that can be used for developing novel therapeutics.

Activation pH and Gating Dynamics of Influenza A M2 Proton Channel Revealed by Single‐Molecule Spectroscopy

AbstractBecause of its importance in viral replication, the M2 proton channel of the influenza A virus has been the focus of many studies. Although we now know a great deal about the structural architecture underlying its proton conduction function, we know little about its conformational dynamics, especially those controlling the rate of this action. Herein, we employ a single‐molecule fluorescence method to assess the dynamics of the inter‐helical channel motion of both full‐length M2 and the transmembrane domain of M2. The rate of this motion depends not only on the identity of the channel and membrane composition but also on the pH in a sigmoidal manner. For the full‐length M2 channel, the rate is increased from approximately 190 μs−1 at high pH to approximately 80 μs−1 at low pH, with a transition midpoint at pH 6.1. Because the latter value is within the range reported for the conducting pKa value of the His37 tetrad, we believe that this inter‐helical motion accompanies proton conduction.

Activation pH and Gating Dynamics of Influenza A M2 Proton Channel Revealed by Single-Molecule Spectroscopy.

Because of its importance in viral replication, the M2 proton channel of the influenza A virus has been the focus of many studies. Although we now know a great deal about the structural architecture underlying its proton conduction function, we know little about its conformational dynamics, especially those controlling the rate of this action. Herein, we employ a single-molecule fluorescence method to assess the dynamics of the inter-helical channel motion of both full-length M2 and the transmembrane domain of M2. The rate of this motion depends not only on the identity of the channel and membrane composition but also on the pH in a sigmoidal manner. For the full-length M2 channel, the rate is increased from approximately 190 μs-1 at high pH to approximately 80 μs-1 at low pH, with a transition midpoint at pH 6.1. Because the latter value is within the range reported for the conducting pKa value of the His37 tetrad, we believe that this inter-helical motion accompanies proton conduction.

Molecular Mechanism of AMPA Receptor Modulation by TARP/Stargazin

AMPA receptors (AMPARs) mediate the majority of fast excitatory transmission in the brain and critically contribute to synaptic plasticity and pathology. AMPAR trafficking and gating are tightly controlled by auxiliary transmembrane AMPAR regulatory proteins (TARPs). Here, using systematic domain swaps with the TARP-insensitive kainate receptor GluK2, we show that AMPAR interaction with the prototypical TARP stargazin/γ2 primarily involves the AMPAR membrane domains M1 and M4 of neighboring subunits, initiated or stabilized by the AMPAR C-tail, and that these interactions are sufficient to enable full receptor modulation. Moreover, employing TARP chimeras disclosed a key role in this process also for the TARP transmembrane domains TM3 and TM4 and extracellular loop 2. Mechanistically, our data support a two-step action in which binding of TARP to the AMPAR membrane domains destabilizes the channel closed state, thereby enabling an efficient opening upon agonist binding, which then stabilizes the open state via subsequent interactions.

Two adjacent phenylalanines in the NMDA receptor GluN2A subunit M3 domain interactively regulate alcohol sensitivity and ion channel gating

The N-methyl-d-aspartate (NMDA) receptor is a key target of ethanol action in the central nervous system. Alcohol inhibition of NMDA receptor function involves small clusters of residues in the third and fourth membrane-associated (M) domains. Previous results from this laboratory have shown that two adjacent positions in the M3 domain, F636 and F637, can powerfully regulate alcohol sensitivity and ion channel gating. In this study, we report that these positions interact with one another in the regulation of both NMDA receptor gating and alcohol action. Using dual mutant cycle analysis, we detected interactions among various substitution mutants at these positions with respect to regulation of glutamate EC50, steady-state to peak current ratios (Iss:Ip), mean open time, and ethanol IC50. This interaction apparently involves a balancing of forces on the M3 helix, such that the disruption of function due to a substitution at one position can be reversed by a similar substitution at the other position. For example, tryptophan substitution at F636 or F637 increased or decreased channel mean open time, respectively, but tryptophan substitution at both positions did not alter open time. Interestingly, the effects of a number of mutations on receptor kinetics and ethanol sensitivity appeared to depend upon subtle structural differences, such as those between the isomeric amino acids leucine and isoleucine, as they could not be explained on the basis of sidechain molecular volume or hydrophilicity.

Evidence for Cleavage of the Metalloprotease Vsm from Vibrio splendidus Strain JZ6 by an M20 Peptidase (PepT-like Protein) at Low Temperature.

Metalloprotease Vsm is a major extracellular virulence factor of Vibrio splendidus. The toxicity of Vsm from V. splendidus strain JZ6 has been characterized, and production of this virulence factor proved to be temperature-regulated. The present study provides evidence that two forms (JZE1 and JZE2) of Vsm protein exist in extracellular products (ECPs) of strain JZ6, and a significant conversion of these two forms was detected by SDS-PAGE and immunoblotting analyses of samples obtained from cells grown at 4, 10, 16, 20, 24, and 28°C. Mass spectroscopy confirmed that JZE1 was composed only of the peptidase_M4 domain of Vsm, and JZE2 contained both the PepSY domain and the peptidase_M4 domain. An M20 peptidase T-like protein (PepTL) was screened from the transcriptome data of strain JZ6, which was considered as a crucial molecule to produce the active Vsm (JZE1) by cleavage of the propeptide. Similar to that of Vsm, PepTL mRNA accumulation was highest at 4°C (836.82-fold of that at 28°C), decreased with increasing of temperature and reached its lowest level at 28°C. Deletion of the gene encoding the PepTL resulted in a mutant strain that did not produce the JZE1 cleavage product. The peptidase activity of PepTL recombinant protein (rPepTL) was confirmed by cleaving the Vsm in ECPs with an in vitro degradation reaction. These results demonstrate that PepTL participates in activating Vsm in strain JZ6 by proteolytic cleavage at low temperature.

Influenza M2 Transmembrane Domain Senses Membrane Heterogeneity and Enhances Membrane Curvature

Targeting host cell membranes by M2 of influenza A virus is important for virus invasion and replication. We study the transmembrane domain of M2 (M2TM) interacting with mica-supported planar bilayers and free-standing giant unilamellar vesicles (GUVs). Using solution atomic force microscopy (AFM), we show that the size of M2TM oligomers is dependent on lipid composition. The addition of M2TM to lipid bilayers containing liquid-ordered (Lo) and liquid-disordered (Ld) phases reveals that M2TM preferentially partitions into the Ld phase; phase-dependent partitioning results in a larger rigidity of the Ld phase. We next use fluorescence microscopy to study the effects of M2TM on phase-coexisting GUVs. In particular, M2TM is found to increase GUVs' miscibility transition temperature Tmix. The augmented thermodynamic stability can be accounted for by considering an enhanced energy barrier of lipid mixing between coexisting phases. Our GUV study also shows that M2TM can elicit an array of vesicle shapes mimicking virus budding. M2TM enhanced membrane curvature is consistent with our AFM data, which show altered membrane rigidity and consequently line tension at domain edges. Together, our results highlight that in addition to conducting protons, M2TM can actively regulate membrane heterogeneity and augment membrane curvature.