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bioRxiv2019

Repositioning septins within the core particle

Septins are GTP binding proteins considered to be a novel component of the cytoskeleton. They polymerize into filaments based on hetero-oligomeric core particles which, in humans, are either hexamers or octamers composed of two copies each of either three or four different septins from the 13 available. Not all combinations are possible as it is believed that these must obey substitution rules which determine that different septins must be derived from four distinct and well-established sub-groups. Here, we have purified and characterized one such combinations, SEPT5-SEPT6-SEPT7, and used TEM to derive the first structural information concerning its assembly. The full complex was purified using an affinity tag attached to only one of its components (SEPT7) and was able to bind to and perturb lipid bilayers. Although the complex assembled into elongated hexameric particles, the position of SEPT5 was incompatible with that predicted by the reported structure of SEPT2-SEPT6-SEPT7 based on the substitution rules. MBP-fusion constructs for SEPT5 and SEPT2 and immuno-staining clearly show that these septins occupy the terminal positions of the SEPT5-SEPT6-SEPT7 and SEPT2-SEPT6-SEPT7 hexamers, respectively. In so doing they expose a so-called NC interface which we show to be more susceptible to perturbation at high salt concentrations. Our results show that the true structure of the hexamer is inverted with respect to that described previously and, as such, is more compatible with that reported for yeast. Taken together, our results suggest that the mechanisms involved in spontaneous self-assembly of septin core particles and their filaments deserve further reflection.

biochemistry↗

bioRxiv2019

Determination of glucosinolate contents in Brassica germplasm collections and inter- & intra-leaves distribution pattern using UPLC-MS/MS Multiple Reaction Monitoring scan mode

Intact glucosinolate (GSL) profile (five aliphatic; three aromatic, and one indolic glucosinolate) in the leaves of 50 germplasm collections and commercial cultivars of Brassica rapa, Brassica juncea, and Brassica oleracea collected from six different countries and grown under uniform cultural conditions were compared by UPLC-MS/MS. Total GSLs content ranged from 36.80 to 2383.12 mol/kg DW. Aliphatic GSLs predominated among the entire samples representing from 23.0 to 98.9% of the total GSLs content, where gluconapin and glucobrassicanapin contributed the greatest proportion. Other GSLs such as, progoitrin (PRO), glucotropaeolin (TRO), and glucobarbarin (BAR) were found in relatively low concentrations. Principal Component Analysis (PCA) yielded three principal components with eigenvalue [≥] 1, representing 70.33% of the total variation across the entire data set. Accessions IT260822 & IT32750, and commercial cultivar, \"Hangamssam2\", were well distinguished from other samples in the PCA plot due to their significantly high amount of BAR, glucobrassicin (GBC), and glucoerucin (ERU), respectively. The inter- and intra-leaf variations of GSLs were examined in three kimichi cabbage varieties. The GSLs content varied significantly among leaves in different positions (outer, middle, and inner) and sections within the leaves (top, middle, bottom, green/red, and white). Higher GSL contents were observed in the proximal half & white section of the leaves and inner positions (younger leaves) in most of the samples. GBC, gluconasturtiin (NAS), and glucoberteroin (BER) should be studied profusely in Brassica plants as some of their degradation products of GBC and NAS are useful in cancer chemopreventive functions, whereas BER takes part in the process of suppressing aging of the skin. GSLs are regarded as allelochemicals; hence, the data related to the patterns of GSLs within the leaf and between leaves at different position could be useful to understand the defense mechanism of Brassica plants. The observed variability could be useful for breeders to develop Brassica crops with high GSL content or specific profiles of GSLs as required.

biochemistry↗

bioRxiv2019

KLK4 inhibition by cyclic and acyclic peptides: structural and dynamical insights into standard-mechanism protease inhibitors

Sunflower Trypsin Inhibitor (SFTI-1) is a 14-amino acid serine protease inhibitor. The dual anti-parallel {beta}-sheet arrangement of SFTI-1 is stabilized by a N-terminal-C-terminal backbone cyclization and a further disulfide bridge to form a final bicyclic structure. This constrained structure is further rigidified by an extensive network of internal hydrogen bonds. Thus, the structure of SFTI-1 in solution resembles the protease-bound structure, reducing the entropic penalty upon protease binding. When cleaved at the scissile bond, it is thought that the rigidifying features of SFTI-1 maintain its structure, allowing the scissile bond to be reformed. The lack of structural plasticity for SFTI-1 is proposed to favour initial protease binding and continued occupancy in the protease active site, resulting in an equilibrium between cleaved and uncleaved inhibitor in the presence of protease. We have determined, at 1.15 [A] resolution, the x-ray crystal structures of complexes between human kallikrein-related peptidase 4 (KLK4) and SFTI-FCQR(Asn14), and between KLK4 and an acyclic form of the same inhibitor, SFTI-FCQR(Asn14)[1,14], with the latter displaying a cleaved scissile bond. Structural analysis and MD simulations together reveal the roles of altered contact sequence, intramolecular hydrogen bonding network and backbone cyclization, in altering the state of SFTIs scissile bond ligation at the protease active site. Taken together, the data presented reveal insights into the role of dynamics in the standard-mechanism inhibition, and suggest that modifications on the noncontact strand may be a useful, underexplored approach for generating further potent or selective SFTI-based inhibitors against members of the serine protease family.

biochemistry↗

bioRxiv2019

Effect of Saudi and Egyptian pomegranate polyphenols in regulating the activity of PON1, PON2 and lipid profile for preventing coronary heart disease

Saudi and Egyptian pomegranate juice (APJ and EPJ) contains potent polyphenols antioxidants which were found to reduce serum and macrophage oxidative stress. The protective effects of APJ and EPJ against atherogenicity were evaluated by feeding mice with hypercholesterolemic diet supplemented with PJ. PJ consumption reduced mice serum Triglycerides (TG), Total cholesterol (TC) and (low density lipoprotein (LDL-c) and increase in the High-density lipoprotein (HDL-c) compared with mouse with control diet or atherogenic diet. The risk ratio and atherogenic index were significantly lower in either APJ or EPJ supplemented group. Paraoxonase 1 (PON1) which remains associated with HDL showed significant increase in the activity in mice supplemented with PJ compared to mice with atherogenic diet (AD). PJ consumption significantly reduced lipid peroxidation and increased glutathione levels. The reduction in lipid peroxidation varied from 57 to 76%. PJ supplementation reduced peritoneal macrophages (MPM) mediated oxidation of LDL by 68 to 82% and decreased mouse peritoneal macrophages (MPM) Ox-LDL uptake by 31 to 48%. A significant up regulation of macrophage PON2 activity was induced by PJ reducing the macrophage oxidative stress. In conclusion, PJ consumption resulted in anti-oxidative and antiatherogenic effects on serum and macrophages which could prevent atherosclerosis and coronary heart diseases.

biochemistry↗

bioRxiv2019

Long-lasting effects of proanthocyanidins in rat prevent cafeteria diet effects by limiting adipose tissue accrual

A dose of proanthocyanidins with satiating properties proved to be able to limit body weight increase several weeks after administration under exposure to a cafeteria diet. Here we describe the molecular targets and the duration of the effects. We treated rats with 500 mg GSPE/kg BW for ten days. Seven or seventeen weeks after the last GSPE dose, while animals were on cafeteria diet, we used RT-PCR to measure the mRNA of the key energy metabolism enzymes from liver, adipose depots and muscle. We found that a reduction in the expression of adipose Lpl might explain the lower amount of adipose tissue in rats seven weeks after the last GSPE dose. Liver showed increased expression of Cpt1a and Hmgs2 together with a reduction in Fasn and Dgat2. In addition, fatty oxidation (Oxct1 and Cpt1b mRNA) was increased in muscle. However, after seventeen weeks, there was a completely different gene expression pattern. As conclusion, seven weeks after the last GSPE administration there was a limitation in adipose accrual that might be mediated by an inhibition of the gene expression of the adipose tissue Lpl. Concomitantly there was an increase in fatty acid oxidation in liver and muscle.

biochemistry↗

bioRxiv2019

Nicotinamide Deficiency In Primary Open-Angle Glaucoma

PurposeTo investigate the plasma concentration of nicotinamide in primary open-angle glaucoma (POAG).\n\nMethodsPlasma of 34 POAG individuals were compared to that of 30 age-and sex-matched controls using a semi-quantitative method based on liquid chromatography coupled to high-resolution mass spectrometry. Subsequently, an independent quantitative method, based on liquid chromatography coupled to mass spectrometry, was used to assess nicotinamide concentration in the plasma from the same initial cohort and from a replicative cohort of 20 POAG individuals and 15 controls.\n\nResultsUsing the semi-quantitative method, the plasma nicotinamide concentration was significantly lower in the initial cohort of POAG individuals compared to and further confirmed in the same cohort, using the targeted quantitative method, with mean concentrations of 0.14 {micro}M (median: 0.12 {micro}M; range: 0.06-0.28 {micro}M) in the POAG group (-30 %; p = 0.022), and 0.19 {micro}M (median: 0.18 {micro}M; range: 0.08-0.47 {micro}M) in the control group. The quantitative dosage also disclosed a significantly lower plasma nicotinamide concentration (-33 %; p = 0.011) in the replicative cohort with mean concentrations of 0.14 {micro}M (median: 0.14 {micro}M; range: 0.09-0.25 {micro}M) in the POAG group, and 0.19 {micro}M (median: 0.21 {micro}M; range: 0.09-0.26 {micro}M) in the control group.\n\nConclusionsGlaucoma is associated with lower plasmatic nicotinamide levels, compared to controls, suggesting that nicotinamide supplementation might become a future therapeutic strategy. Further studies are needed, in larger cohorts, to confirm these preliminary findings.

biochemistry↗

bioRxiv2019

In vitro transposon targeting using a catalytically inactive Cas9

Homology directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Catalytically inactive Cas9 is attractive candidate for targeting a transposase fusion-protein because of its high specificity and affinity for its binding site. Here we demonstrate efficient Cas9 targeting of a mariner transposon. Targeted integrations were tightly constrained at two adjacent TA dinucleotides about 20 bp to one side of the gRNA binding site. Biochemical analysis of the nucleoprotein complexes demonstrated that the transposase and Cas9 moieties of the fusion protein can bind their respective substrates independently. In the presence of the Cas9 target DNA, kinetic analysis revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase behaved normally and was proficient for integration in vitro and in vivo.

biochemistry↗

bioRxiv2019

An evolutionary-conserved redox regulatory mechanism in human Ser/Thr protein kinases

Reactive oxygen species (ROS) are products of oxygen metabolism, but are also recognized as endogenous physiological mediators of cellular signaling. Eukaryotic protein kinase (ePK) regulation occurs through reversible phosphorylation events in the flexible activation segment. In this study, we demonstrate that the catalytic phosphotransferase output from the mitotic Ser/Thr kinase Aurora A is also controlled by cysteine (Cys) oxidation. Reversible regulation occurs by direct modification of a conserved residue (Cys 290), which lies adjacent to Thr 288, the activating site of phosphorylation. Strikingly, redox modulation of the Cys 290-equivalent in other ePKs is predicted to be an underappreciated regulatory mechanism, since ~100 human Ser/Thr kinases possess a Cys at this position in the conserved activation loop. Using real-time enzyme assays, we confirm that the presence of the equivalent Cys residue is prognostic for redox-sensitivity amongst a cohort of human CAMK, AGC and AGC-like kinases, including AKT, AMPK, CAMK1, MAPKAP-K2/3 and SIK1-3. Our findings demonstrate that dominant Cys-based redox-switching in the activation segment represents an evolutionary-conserved mode of regulation for a significant subset of the human kinome. This finding has important implications for understanding physiological and pathological signaling responses to ROS, and emphasises the importance of multivalent activation segment regulation in ePKs.\n\nONE-SENTENCE SUMMARYThe catalytic activity of Ser/Thr kinases is regulated through a conserved Cys-based redox mechanism.

biochemistry↗

bioRxiv2019

An equilibrium binding model for CpG DNA-dependent dimerization of Toll-like receptor 9

Microbial nucleic acids in the extracellular milieu are recognized in vertebrates by Toll-like receptors (TLRs), one of the most important families of innate immune receptors. TLR9 recognizes single-stranded unmethylated CpG DNA in endosomes. DNA binding induces dimerization of TLR9 and activation of a potent inflammatory response. To provide insights on how DNA ligands induce TLR9 dimerization, we developed a detailed theoretical equilibrium ligand binding model. Light scattering and fluorescence polarization assays performed with a recombinant TLR9 ectodomain fragment and a panel of agonistic and antagonistic DNA ligands provide data that restrain the binding parameters in our binding model. This work brings us one step closer to establishing a rigorous biochemical understanding of how TLRs are activated by their ligands.

biochemistry↗

bioRxiv2019

Endothelial PKA targets ATG16L1 to regulate angiogenesis by limiting autophagy

The cAMP-dependent protein kinase A (PKA) regulates a plethora of cellular functions in health and disease. During angiogenesis, PKA activity in endothelial cells controls the transition from sprouting to vessel maturation and limits tip cell formation independently of Notch signaling. The molecular PKA targets mediating these effects remain unknown. We report a chemical genetics screen identifying endothelial-specific substrates of PKA in human umbilical vein endothelial cells (HUVEC). We identified ATG16L1, a regulator of autophagy, as novel target of PKA. Biochemical validation, mass spectrometry and peptide spot arrays revealed that PKA phosphorylates ATG16L1 at Ser268 and ATG16L1{beta} at Ser269. The phosphorylations drive degradation of ATG16L1 protein. Knocking down PKA or inhibiting its activity increased ATG16L1 protein levels and endothelial autophagy. In vivo genetics and pharmacological experiments demonstrated that autophagy inhibition partially rescues vascular hypersprouting caused by PKA deficiency. We propose that endothelial PKA activity restricts active sprouting by reducing endothelial autophagy through phosphorylation of ATG16L1.

biochemistry↗

bioRxiv2019

Selective labelling of arginine residues engaged in binding sulfatedglycosaminoglycans

IThe activities of hundreds of proteins in the extracellular space are regulated by binding to the glycosaminoglycan heparan sulfate (HS). These interactions are driven by ionic bonds between sulfate and carboxylate groups on the polysaccharide and the side chains of basic residues in the protein. Here we develop a method to selectively label the guanidino side chains of arginine residues in proteins that engage the anionic groups in the sugar. The protein is bound to heparin (a common experimental proxy for HS) on an affinity column. Arginine side chains that are exposed to solvent, and thus involved in binding, are protected by reaction with the dicarbonyl phenylgyoxal (PGO). Elution of the bound proteins then exposes arginine side chains that had directly engaged with anionic groups on the polysaccharide. These are reacted with hydroxyl-phenylglyoxal (HPG). PGO was found to generate three products: a 1:1 product, the 1:1 water condensed product and a 2:1 PGO:arginine product. These three reaction products and that of HPG had distinct masses. Scripts were written to analyse the mass spectra and so identify HPG labelled arginine residues. Proof of principle was acquired on model peptides. The method was then applied to the identification of heparin binding arginine residues in fibroblast growth factors (FGF) 1 and 2. The data demonstrate that four out of eleven arginine residues on FGF2 and five out of six arginine residues of FGF1 engage heparin. Our approach provides a rapid and reliable means to identify arginines involved in functional complexes such as those of proteins with heparin

biochemistry↗

bioRxiv2019

Association between hot flashes severity and oxidative stress among Mexican postmenopausal women: a cross-sectional study

ObjectiveTo assess the association between hot flashes (HFs) severity and oxidative stress (OS) in Mexican postmenopausal women.\n\nMethodsA cross-sectional study was carried out with perimenopausal women aged 40-59 years community-dwelling from Mexico City, Mexico. They participated in Menopause and Oxidative Stress Project. The baseline sample consisted of 476 women recruited to participate; 161 women were excluded due to different reasons. Hence, 315 women were selected to establish two groups, a) 145 premenopausal women (yet with menstrual bleeding), and b) 170 postmenopausal women (without menses). All women were free of cardiovascular, kidney, hepatic or cancer disease, and without antioxidant supplement intake for at least six months prior to the beginning of the study; none had previously received hormone therapy. As OS markers, we measured plasma malondialdehyde using the TBARS assay, erythrocyte superoxide dismutase (SOD) and glutathione peroxidase (GPx), uric acid, and total antioxidant status; also, we calculated SOD/GPx ratio, antioxidant gap and an oxidative stress score ranging from 0 to 7. The HFs were evaluated using the Menopause Rating Scale. The women completed Spanish version of the Athens Insomnia Scale, Zung Self-Rating Anxiety Scale and Zung Self-Rating Depression Scale and a questionnaire of pro-oxidant factors.\n\nResultsStress score increased with HFs severity (mild 2.9{+/-}0.23, moderate 3.1{+/-}0.21 and severe 3.8{+/-}0.18, p<0.01) in postmenopausal women. We observed a positive correlation between HFs severity and stress score, r=0.247 (p=0.001) in postmenopausal women; other test scores were not correlated. Severe HFs were a risk factor for OS (OR=3.37, 95%CI: 1.20-9.51, p<0.05) in an adjusted multivariate analysis by different postmenopausal symptoms and pro-oxidant factors; we did not see any association in premenopausal women.\n\nConclusionOur findings suggest an association between HFs severity and OS in Mexican postmenopausal women.

biochemistry↗

bioRxiv2019

Btbd6-dependent Plzf recruitment to Cul3 E3 ligase complexes through BTB domain heterodimerization

The Cul3 adaptor Btbd6 plays crucial roles in neural development by driving the ubiquitin-dependent degradation of promyelocytic zinc finger transcription factor (Plzf). Btbd6 has conserved motifs, BTB-BACK-PHR, and by analogy with other BTB-BACK adaptors, might be expected to bind to Cul3 through the BTB-BACK domain, and to substrate through the PHR domain. However, we now present a mode of adaptor-substrate interaction through heterodimerisation between the normally homodimeric BTB domains of Btbd6 and Plzf. This heterodimerization appears to occur through monomer exchange that is detected only at or near physiological concentrations. The Btbd6-Plzf heterodimer thus formed assembles into a ternary complex with Cul3. In addition we show that the BTB and PHR domains of Btbd6 promote localisation in the nucleus and that the BACK domain contains a nuclear export signal. Our findings support a model whereby Btbd6 moves into and out of the nucleus, iteratively sweeping Plzf into the cytoplasm and enabling complex formation with Cul3 that presents Plzf for ubiquitination.\n\nHighlightsO_LIA general mechanism for recruitment of BTB domain-containing substrates by BTBdomain adaptors for the Cul3 E3 ligase complex\nC_LIO_LINuclear export of the Plzf/Btbd6 complex mediated by a NES within the Btbd6 BACK domain\nC_LIO_LICul3-dependent Plzf ubiquitylation through heterodimerisation of BTB domains on adaptor and substrate by monomer exchang\nC_LI

biochemistry↗

bioRxiv2019

Conformational resolution of nucleotide cycling and effector interactions for multiple small GTPases in parallel

Small GTPase proteins alternatively bind GDP/GTP guanine nucleotides to gate signaling pathways that direct most cellular processes. Numerous GTPases are implicated in oncogenesis, particularly three RAS isoforms HRAS, KRAS and NRAS, and the RHO family GTPase RAC1. Signaling networks comprising small GTPases are highly connected, and there is evidence of direct biochemical crosstalk between the functional G-domains of these proteins. The activation potential of a given GTPase is contingent on a co-dependent interaction with nucleotide and a Mg2+ ion, which bind to individual variants via distinct affinities coordinated by residues in the nucleotide binding pocket. Here, we utilize a selective-labelling strategy coupled with real-time nuclear magnetic resonance (NMR) spectroscopy to monitor nucleotide exchange, GTP hydrolysis and effector interactions of multiple small GTPases in a single complex system. We provide new insight on nucleotide preference and the role of Mg2+ in activating both wild-type and oncogenic mutant enzymes. Multiplexing reveals GEF, GAP and effector binding specificity in mixtures of GTPases and establishes the complete biochemical equivalence of the three related RAS isoforms. This work establishes that direct quantitation of the nucleotide-bound conformation is required to accurately resolve GTPase activation potential, as GTPases such as RALA or the G12C mutant of KRAS demonstrate fast exchange kinetics but have a high affinity for GDP. Further, we propose that the G-domains of small GTPases behave autonomously in solution and nucleotide cycling proceeds independent of protein concentration but is highly impacted by Mg2+ abundance.

biochemistry↗

bioRxiv2019

Cryo-EM structure of the human L-type amino acid transporter 1 in complex with glycoprotein CD98hc

The L-type amino acid transporter 1 (LAT1) transports large neutral amino acids and drugs across the plasma membrane and is crucial for nutrient uptake, brain drug delivery and tumor growth. LAT1 is a unique solute carrier that forms a disulfide-linked heterodimer with the cell-surface glycoprotein CD98 heavy chain (CD98hc), but the mechanisms of its molecular assembly and amino acid transport are poorly understood. Here we report the cryo-EM structure of the human LAT1-CD98hc heterodimer at 3.4 [A] resolution, revealing the hitherto unprecedented architecture of a solute carrier-glycoprotein heterocomplex. LAT1 features a canonical LeuT-fold while exhibiting an unusual loop structure on transmembrane helix 6, creating an extended cavity to accommodate bulky hydrophobic amino acids and drugs. CD98hc engages with LAT1 through multiple interactions, not only in the extracellular and transmembrane domains but also in the interdomain linker. The heterodimer interface features multiple sterol molecules, corroborating previous biochemical data on the role of cholesterols in heterodimer stabilization. We also visualized the binding modes of two anti-CD98 antibodies and show that they recognize distinct, multiple epitopes on CD98hc but not its glycans, explaining their robust reactivities despite the glycan heterogeneity. Furthermore, we mapped disease-causing mutations onto the structure and homology models, which rationalized some of the phenotypes of SLC3- and SLC7-related congenital disorders. Together, these results shed light on the principles of the structural assembly between a glycoprotein and a solute carrier, and provide a template for improving preclinical drugs and therapeutic antibodies targeting LAT1 and CD98.

biochemistry↗

bioRxiv2019

Mechanism of the allosteric activation of the ClpP protease machinery by substrates and active-site inhibitors

Coordinated conformational transitions in oligomeric enzymatic complexes modulate function in response to substrates and play a crucial role in enzyme inhibition and activation. ClpP protease is a tetradecameric complex that has emerged as a drug target against multiple pathogenic bacteria. During drug development efforts, the activation of different ClpPs by inhibitors was independently reported, but so far, no rationale for inhibitor-induced activation has been proposed. Using an integrated approach that included X-ray crystallography, solid-and solution-state NMR, MD simulations and ITC we show that the proteasome-inhibitor bortezomib binds to the ClpP active site serine mimicking a peptide substrate and induces the concerted allosteric activation of the complex. The bortezomib activated conformation also displays a higher affinity for its cognate unfoldase ClpX. We propose a universal allosteric mechanism where substrate binding to a single subunit locks ClpP into an active conformation optimized for chaperone association as well as protein processive degradation.

biochemistry↗

bioRxiv2019

Structural basis for the delivery of activated sialic acid into Golgi for sialyation

The decoration of secretory glycoproteins and glycolipids with sialic acid is critical to many physiological and pathological processes. Sialyation is dependent on a continuous supply of sialic acid into Golgi organelles in the form of CMP-sialic acid. Translocation of CMP-sialic acid into Golgi is carried out by the CMP-sialic acid transporter (CST). Mutations in human CST are linked to glycosylation disorders, and CST is important for glycopathway engineering, as it is critical for sialyation efficiency of therapeutic glycoproteins. The mechanism of how CMP-sialic acid is recognized and translocated across Golgi membranes in exchange for CMP is poorly understood. Here we have determined the crystal structure of a eukaryotic CMP-sialic acid transporter in complex with CMP. We conclude that the specificity of CST for CMP-sialic acid is established by the nucleotide CMP to such an extent, they are uniquely able to work both as passive and as (secondary) active antiporters.

biochemistry↗

bioRxiv2019

The structure of Macrophage Expressed Gene-1, a phagolysosome immune effector that is activated upon acidification.

Macrophage Expressed Gene-1 (MPEG-1; also termed Perforin-2) is an endosomal / phagolysosomal perforin-like protein that is conserved across the metazoan kingdom and that functions within the phagolysosome to damage engulfed microbes. Like the Membrane Attack Complex and perforin, MPEG-1 has been postulated to form pores in target membranes, however, its mode of action remains to be established. We used single particle cryo-Electron Microscopy to determine the 2.4 [A] structure of a hexadecameric assembly of MPEG-1 that displays the expected features of a soluble pre-pore complex. We further discovered that the MPEG-1 pre-pore-like assemblies can be induced to perforate membranes through mild acidification, such as would occur within maturing phagolysosomes. We next solved the 3.6 [A] cryo-EM structure of MPEG-1 in complex with liposomes. Remarkably these data revealed that a C-terminal Multi-vesicular body of 12 kDa (MVB12)-associated {beta}-prism (MABP) domain interacts with target membranes in a mode that positions the pore forming machinery of MPEG-1 to point away from the bound membrane. This unexpected mechanism of membrane interaction raises the intriguing possibility that MPEG-1 may be able to remain bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets.

biochemistry↗