ABC exporters pump substrates across the membrane by coupling ATP-driven movements of nucleotide binding domains (NBDs) to the transmembrane domains (TMDs), which switch between inward- and outward-facing (IF, OF) orientations. DEER measurements on the heterodimeric ABC exporter TM287/288 from Thermotoga maritima, which contains a non-canonical ATP binding site, revealed that in the presence of nucleotides the transporter exists in an IF/OF equilibrium. While ATP binding was sufficient to partially populate the OF state, nucleotide trapping in the pre- or post-hydrolytic state was required for a pronounced conformational shift. At physiologically high temperatures and in the absence of nucleotides, the NBDs disengage asymmetrically while the conformation of the TMDs remains unchanged. Nucleotide binding at the degenerate ATP site prevents complete NBD separation, a molecular feature differentiating heterodimeric from homodimeric ABC exporters. Our data suggest hydrolysis-independent closure of the NBD dimer, which is further stabilized as the consensus site nucleotide is committed to hydrolysis.DOI: http://dx.doi.org/10.7554/eLife.20236.001

Bax is a Bcl-2 protein critical for apoptosis induction. In healthy cells, Bax is mostly a monomeric, cytosolic protein, while upon apoptosis initiation it inserts into the outer mitochondrial membrane, oligomerizes, and forms pores that release proapoptotic factors like Cytochrome c into the cytosol. The structures of active Bax and its homolog Bak are only partially understood and the topology of the proteins with respect to the membrane bilayer is controversially described in the literature. Here, we systematically review and examine the protein-membrane, protein-water, and protein-protein contacts of the nine helices of active Bax and Bak, and add a new set of topology data obtained by fluorescence and EPR methods. We conclude based on the consistent part of the datasets that the core/dimerization domain of Bax (Bak) is water exposed with only helices 4 and 5 in membrane contact, whereas the piercing/latch domain is in peripheral membrane contact, with helix 9 being transmembrane. Among the available structural models, those considering the dimerization/core domain at the rim of a toroidal pore are the most plausible to describe the active state of the proteins, although the structural flexibility of the piercing/latch domain does not allow unambiguous discrimination between the existing models.

Retinal guanylate cyclases (RetGCs) are regulated by a family of guanylate cyclase-activating proteins (called GCAP1−7). GCAPs form dimers that bind to Ca 2+ and confer Ca 2+ sensitive activation of RetGC during visual phototransduction. The GCAP5 homologue from zebrafish contains two nonconserved cysteine residues (Cys15 and Cys17) that bind to ferrous ion, which stabilizes GCAP5 dimerization and diminishes its ability to activate RetGC. Here, we present NMR and EPR-DEER structural analysis of a GCAP5 dimer in the Mg 2+ -bound, Ca 2+ -free, Fe 2+ -free activator state. The NMR-derived structure of GCAP5 is similar to the crystal structure of Ca 2+ -bound GCAP1 (root-mean-square deviation of 2.4 Å), except that the N-terminal helix of GCAP5 is extended by two residues, which allows the sulfhydryl groups of Cys15 and Cys17 to become more solvent exposed in GCAP5 to facilitate Fe 2+ binding. Nitroxide spinlabel probes were covalently attached to particular cysteine residues engineered in GCAP5: C15, C17, T26C, C28, N56C, C69, C105, N139C, E152C, and S159C. The intermolecular distance of each spin-label probe in dimeric GCAP5 (measured by EPR-DEER) defined restraints for calculating the dimer structure by molecular docking. The GCAP5 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H18, Y21, M25, F72, V76, and W93, as well as an intermolecular salt bridge between R22 and D71. The structural model of the GCAP5 dimer was validated by mutations (H18E/Y21E, H18A/Y21A, R22D, R22A, M25E, D71R, F72E, and V76E) at the dimer interface that disrupt dimerization of GCAP5 and affect the activation of RetGC. We propose that GCAP5 dimerization may play a role in the Fe 2+ -dependent regulation of cyclase activity in zebrafish photoreceptors.

The availability of bioresistant spin labels is crucial for the optimization of site‐directed spin labeling protocols for EPR structural studies of biomolecules in a cellular context. As labeling can affect proteins’ fold and/or function, having the possibility to choose between different spin labels will increase the probability to produce spin‐labeled functional proteins. Here, we report the synthesis and characterization of iodoacetamide‐ and maleimide‐functionalized spin labels based on the gem ‐diethyl pyrroline structure. The two nitroxide labels are compared to conventional gem ‐dimethyl analogs by site‐directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy, using two water soluble proteins: T4 lysozyme and Bid. To foster their use for structural studies, we also present rotamer libraries for these labels, compatible with the MMM software. Finally, we investigate the “true” biocompatibility of the gem ‐diethyl probes comparing the resistance towards chemical reduction of the NO group in ascorbate solutions and E. coli cytosol at different spin concentrations.

Highlights d H/D exchange experiments performed on a minimal photosensory module d EPR reveals light-induced changes in side chain dynamics and interspin distances d Coarse-grained model of the domain mechanics d Common light response independent of quaternary interaction and downstream modules

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Invited for this month's cover picture is the group of Professor Enrica Bordignon at the Ruhr University Bochum. The cover picture shows an artistic view of E. coli cells and two spin‐labeled recombinantly produced proteins, which can be inserted into the cells for EPR studies. The primary sequence of the proteins is schematically shown with the one‐letter amino acid code, and cysteine residues are functionalized with the two new gem diethyl nitroxide spin labels designed to better sustain the reducing cellular environment. Read the full text of their Full Paper at 10.1002/open.201900119.

Graphene derivatives have been attracting extensive interest as effective antimicrobial agents. In the present study, ternary nanocomposites are prepared based on graphene oxide quantum dots (GOQD), polyaniline (PANI), and manganese oxides. Because of the hydrophilic GOQD and PANI, the resulting GPM nanocomposites are readily dispersible in water and upon photoirradiation at 365 nm exhibit antimicrobial activity toward both Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus epidermidis (S. epidermidis). Notably, the nanocomposite with a high Mn2+ and Mn4+ content is found to be far more active than that with a predominant Mn3+ component, although both samples feature a similar elemental composition and average Mn valence state. The bactericidal activity is largely ascribed to the photocatalytic production of hydroxy radicals and photogenerated holes; both are known to exert oxidative stress on bacterial cells. Further antimicrobial contributions may arise from the strong affinity of the nanocomposites to the cell surfaces. These results suggest that the metal valence state may be a critical parameter in the design and engineering of high-performance antimicrobial agents based on metal oxide nanocomposites.