BNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homoand hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed.Mitochondria hold a crucial role in programmed cell death required to control cell development and to maintain homeostasis in multicellular organisms (1). Mitochondria-mediated cell death is both promoted and suppressed by apoptotic proteins of the Bcl-2 family, most of which contain a C-terminal hydrophobic domain essential for membrane targeting (2). A major function of Bcl-2 family proteins is to regulate the permeability state of the outer mitochondrial membrane by forming homo-and hetero-oligomers inside the membrane that determine cell fate (3-5). The pro-apoptotic protein BNip3 (Bcl-2 Nineteen-kDa interacting protein 3) with a single Bcl-2 homology 3 (BH3) domain is one of the most intensively studied members of the family (6). BNip3 and its homologues belong to an independent monophyletic branch with individual evolutionary history (2) and are essentially different from other BH3-only proteins such as Bid/Bik not only in that they do not require BH3 domain for their function but also because they directly cause changes of mitochondrial potential (7). BNip3-induced cell death is independent of caspases and cytochrome c release; it is believed to represent a novel form of programmed cell death, resembling necrosis rather than classical apoptosis (8).For all cells, loss of nutrient supply represents a potent signal for programmed death. BNip3 plays an important role in hypoxic cell death of normal and malignant cells (9). Hypoxia induces expression and accumulation of cytoplasmic or loosely membrane-bound BNip3; however, in order to activate cell death pathway acidosis is required (10). Transition from respiratory to glycolytic metabolism with increased glucose consumption, lactic acid production, and decrease of cytosolic pH causes redistribution of BNip3 to the outer mitochondrial membrane and integration of homodimeric BNip3 into it, triggering a cell death cascade, which ultimately leads to opening of the mitochondrial permeability tra...

NMR structural studies of membrane proteins (MP) are hampered by complications in MP expression, technical difficulties associated with the slow process of NMR spectral peak assignment, and limited distance information obtainable for transmembrane (TM) helices. To overcome the inherent challenges in the determination of MP structures, we have developed a rapid and cost-efficient strategy that combines cell-free (CF) protein synthesis, optimized combinatorial dual-isotope labeling for nearly instant resonance assignment, and fast acquisition of long-distance information using paramagnetic probes. Here we report three backbone structures for the TM domains of the three classes of Escherichia coli histidine kinase receptors (HKRs). The ArcB and QseC TM domains are both two-helical motifs, whereas the KdpD TM domain comprises a four-helical bundle with shorter second and third helices. The interhelical distances (up to 12 Å) reveal weak interactions within the TM domains of all three receptors. Determined consecutively within 8 months, these structures offer insight into the abundant and underrepresented in the Protein Data Bank class of 2-4 TM crossers and demonstrate the efficiency of our CF combinatorial dual-labeling strategy, which can be applied to solve MP structures in high numbers and at a high speed. Our results greatly expand the current knowledge of HKR structure, opening the doors to studies on their widespread and pharmaceutically important bacterial signaling mechanism.backbone NMR structure | cell-free synthesis | combinatorial selective labeling

G protein-gated inwardly rectifying potassium (GIRK) channels are important gatekeepers of neuronal excitability. The surface expression of neuronal GIRK channels is regulated by the psychostimulant-sensitive sorting nexin 27 (SNX27) protein through a class I (-X-Ser/Thr-X-Φ, where X is any residue and Φ is a hydrophobic amino acid) PDZ-binding interaction. The G protein-insensitive inward rectifier channel (IRK1) contains the same class I PDZ-binding motif but associates with a different synaptic PDZ protein, postsynaptic density protein 95 (PSD95). The mechanism by which SNX27 and PSD95 discriminate these channels was previously unclear. Using high-resolution structures coupled with biochemical and functional analyses, we identified key amino acids upstream of the channel's canonical PDZ-binding motif that associate electrostatically with a unique structural pocket in the SNX27-PDZ domain. Changing specific charged residues in the channel's carboxyl terminus or in the PDZ domain converts the selective association and functional regulation by SNX27. Elucidation of this unique interaction site between ion channels and PDZ-containing proteins could provide a therapeutic target for treating brain diseases.

Although nearly half of today’s major pharmaceutical drugs target human integral membrane proteins (hIMPs), only 30 hIMP structures are currently available in the Protein Data Bank, largely owing to inefficiencies in protein production. Here we describe a strategy for the rapid structure determination of hIMPs, using solution NMR spectroscopy with systematically labeled proteins produced via cell-free expression. We report new backbone structures of six hIMPs, solved in only 18 months from 15 initial targets. Application of our protocols to an additional 135 hIMPs with molecular weight <30 kDa yielded 38 hIMPs suitable for structural characterization by solution NMR spectroscopy without additional optimization.