Structure of the C-terminal domain of the Prokaryotic Sodium Channel Orthologue NsvBa

Crystallographic and electrophysiological studies have recently provided insight into the structure, function and drug binding of prokaryotic sodium channels. These channels exhibit significant sequence identities, especially in their transmembrane regions, with human voltage-gated sodium channels. However, rather than being single polypeptides with four homologous domains, they are tetramers of single domain polypeptides, with a C-terminal domain (CTD) composed of an inter-subunit four helix coiled-coil. The structures of the CTDs differ between orthologues. In NavBh and NavMs, the C-termini form a disordered region adjacent to the final transmembrane helix, followed by a coiled-coil region, as demonstrated by synchrotron radiation circular dichroism (SRCD) and double electron-electron resonance electron paramagnetic resonance spectroscopic measurements. In contrast, in the crystal structure of the NavAe orthologue, the entire C-terminus is comprised of a helical region followed by a coiled-coil. In this study we have examined the CTD of the NsvBa from Bacillus alcalophilus, which unlike other orthologues is predicted by different methods to have different types of structures: either a disordered adjacent to the transmembrane region, followed by a helical coiled-coil, or a fully helical CTD. To discriminate between the two possible structures we have used SRCD spectroscopy to experimentally determine the secondary structure of the C-terminus of this orthologue and used the results as the basis for modelling the transition between open and closed conformations of the channel

Structure of a bacterial voltage-gated sodium channel pore reveals mechanisms of opening and closing

Voltage-gated sodium channels are vital membrane proteins essential for electrical signalling; in humans, they are key targets for the development of pharmaceutical drugs. Here we report the crystal structure of an open-channel conformation of NavMs, the bacterial channel pore from the marine bacterium Magnetococcus sp. (strain MC-1). It differs from the recently published crystal structure of a closed form of a related bacterial sodium channel (NavAb) by having its internal cavity accessible to the cytoplasmic surface as a result of a bend/rotation about a central residue in the carboxy-terminal transmembrane segment. This produces an open activation gate of sufficient diameter to allow hydrated sodium ions to pass through. Comparison of the open and closed structures provides new insight into the features of the functional states present in the activation cycles of sodium channels and the mechanism of channel opening and closing

A thermostable salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids

Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.This work was supported by the projects FCOMP-01-0124-FEDER-019446, FCOMP-01-0124-FEDER-027462 and PEst-OE/EQB/LA0023/2013 from "Fundacao para a Ciencia e Tecnologia" (FCT), Portugal. The authors thank the Project "BioHealth - Biotechnology and Bioengineering approaches to improve health quality", Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON. 2 - O Novo Norte), QREN, FEDER. Hugo Oliveira acknowledges the FCT grant SFRH/BD/63734/2009. Maarten Walmagh held a PhD scholarship of the IWT Vlaanderen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Membrane protein dynamics: limited lipid control

Correlation of lipid disorder with membrane protein dynamics has been studied with infrared spectroscopy, by combining data characterizing lipid phase, protein structure and, via hydrogen-deuterium (H/D) exchange, protein dynamics. The key element was a new measuring scheme, by which the combined effects of time and temperature on the H/D exchange could be separated. Cyanobacterial and plant thylakoid membranes, mammalian mitochondria membranes, and for comparison, lysozyme were investigated. In dissolved lysozyme, as a function of temperature, H/D exchange involved only reversible movements (the secondary structure did not change considerably); heat-denaturing was a separate event at much higher temperature. Around the low-temperature functioning limit of the biomembranes, lipids affected protein dynamics since changes in fatty acyl chain disorders and H/D exchange exhibited certain correlation. H/D exchange remained low in all membranes over physiological temperatures. Around the high-temperature functioning limit of the membranes, the exchange rates became higher. When temperature was further increased, H/D exchange rates went over a maximum and afterwards decreased (due to full H/D exchange and/or protein denaturing). Maximal H/D exchange rate temperatures correlated neither with the disorder nor with the unsaturation of lipids. In membrane proteins, in contrast to lysozyme, the onsets of sizable H/D exchange rates were the onsets of irreversible denaturing as well. Seemingly, at temperatures where protein self-dynamics allows large-scale H/D exchange, lipid-protein coupling is so weak that proteins prefer aggregating to limit the exposure of their hydrophobic surface regions to water. In all membranes studied, dynamics seemed to be governed by lipids around the low-temperature limit, and by proteins around the high-temperature limit of membrane functionality

Differential lipid dependence of the function of bacterial sodium channels

The lipid bilayer is important for maintaining the integrity of cellular compartments and plays a vital role in providing the hydrophobic and charged interactions necessary for membrane protein structure, conformational flexibility and function. To directly assess the lipid dependence of activity for voltage-gated sodium channels, we compared the activity of three bacterial sodium channel homologues (NaChBac, NavMs, and NavSp) by cumulative 22Na+ uptake into proteoliposomes containing a 3:1 ratio of 1-palmitoyl 2-oleoyl phosphatidylethanolamine and different “guest” glycerophospholipids. We observed a unique lipid profile for each channel tested. NavMs and NavSp showed strong preference for different negatively-charged lipids (phosphatidylinositol and phosphatidylglycerol, respectively), whilst NaChBac exhibited a more modest variation with lipid type. To investigate the molecular bases of these differences we used synchrotron radiation circular dichroism spectroscopy to compare structures in liposomes of different composition, and molecular modeling and electrostatics calculations to rationalize the functional differences seen. We then examined pore-only constructs (with voltage sensor subdomains removed) and found that in these channels the lipid specificity was drastically reduced, suggesting that the specific lipid influences on voltage-gated sodium channels arise primarily from their abilities to interact with the voltage-sensing subdomains

Targeting of the MYCN protein with small molecule c-MYC inhibitors

This study was funded by grants from the Swedish Research Council and the Swedish Cancer Society. IM and HZ were recipients of graduate student grants from KI (KID), MAH was recipient of a Senior Investigator Award from the Swedish Cancer Society, and NJW was a Royal Society University Research Fellow when this work began.Members of the MYC family are the most frequently deregulated oncogenes in human cancer and are often correlated with aggressive disease and/or poorly differentiated tumors. Since patients with MYCN-amplified neuroblastoma have a poor prognosis, targeting MYCN using small molecule inhibitors could represent a promising therapeutic approach. We have previously demonstrated that the small molecule 10058-F4, known to bind to the c-MYC bHLHZip dimerization domain and inhibiting the c-MYC/MAX interaction, also interferes with the MYCN/MAX dimerization in vitro and imparts anti-tumorigenic effects in neuroblastoma tumor models with MYCN overexpression. Our previous work also revealed that MYCN-inhibition leads to mitochondrial dysfunction resulting in accumulation of lipid droplets in neuroblastoma cells. To expand our understanding of how small molecules interfere with MYCN, we have now analyzed the direct binding of 10058-F4, as well as three of its analogs; #474, #764 and 10058-F4(7RH), one metabolite C-m/z 232, and a structurally unrelated c-MYC inhibitor 10074-G5, to the bHLHZip domain of MYCN. We also assessed their ability to induce apoptosis, neurite outgrowth and lipid accumulation in neuroblastoma cells. Interestingly, all c-MYC binding molecules tested also bind MYCN as assayed by surface plasmon resonance. Using a proximity ligation assay, we found reduced interaction between MYCN and MAX after treatment with all molecules except for the 10058-F4 metabolite C-m/z 232 and the non-binder 10058-F4(7RH). Importantly, 10074-G5 and 10058-F4 were the most efficient in inducing neuronal differentiation and lipid accumulation in MYCN-amplified neuroblastoma cells. Together our data demonstrate MYCN-binding properties for a selection of small molecules, and provide functional information that could be of importance for future development of targeted therapies against MYCN-amplified neuroblastoma.Publisher PDFPeer reviewe

Origin of pronounced differences in 77 K fluorescence of the green alga Chlamydomonas reinhardtii in state 1 and 2

In response to changes in the reduction state of the plastoquinone pool in its thylakoid membrane, the green alga Chlamydomonas reinhardtti is performing state transitions: remodelling of its thylakoid membrane leads to a redistribution of excitations over photosystems I and II (PSI and PSII). These transitions are accompanied by marked changes in the 77 K fluorescence spectrum, which form the accepted signature of state transitions. The changes are generally thought to reflect a redistribution of light-harvesting complexes (LHCs) over PSII (fluorescing below 700 nm) and PSI (fluorescing above 700 nm). Here we studied the picosecond fluorescence properties of C. reinhardtti over a broad range of wavelengths with very low excitation intensities (0.2 nJ per laser pulse). Cells were directly used for time-resolved fluorescence measurements at 77 K without further treatment, such as medium exchange with glycerol. It is observed that upon going from state 1 (relatively more fluorescence below 700 nm) to state 2 (relatively more fluorescence above 700 nm), a large part of the fluorescence of LHC/PSII becomes substantially quenched in concurrence with LHC detachment from PSII, whereas the absolute amount of PSI fluorescence hardly changes. These results are in agreement with the recent proposal that the amount of LHC moving from PSII to PSI upon going from state 1 to state 2 is rather limited (Unlu et al. Proc Natl Acad Sci USA 111 (9):3460–3465, 2014)