Structures of intermediate transport states of ZneA, a Zn(II)/proton antiporter.

Efflux pumps belonging to the ubiquitous resistance-nodulation-cell division (RND) superfamily transport substrates out of cells by coupling proton conduction across the membrane to a conformationally driven pumping cycle. The heavy metal-resistant bacteria Cupriavidus metallidurans CH34 relies notably on as many as 12 heavy metal efflux pumps of the RND superfamily. Here we show that C. metallidurans CH34 ZneA is a proton driven efflux pump specific for Zn(II), and that transport of substrates through the transmembrane domain may be electrogenic. We report two X-ray crystal structures of ZneA in intermediate transport conformations, at 3.0 and 3.7 Å resolution. The trimeric ZneA structures capture protomer conformations that differ in the spatial arrangement and Zn(II) occupancies at a proximal and a distal substrate binding site. Structural comparison shows that transport of substrates through a tunnel that links the two binding sites, toward an exit portal, is mediated by the conformation of a short 14-aa loop. Taken together, the ZneA structures presented here provide mechanistic insights into the conformational changes required for substrate efflux by RND superfamily transporters

Structures of intermediate transport states of ZneA, a Zn(II)/proton antiporter.

Efflux pumps belonging to the ubiquitous resistance-nodulation-cell division (RND) superfamily transport substrates out of cells by coupling proton conduction across the membrane to a conformationally driven pumping cycle. The heavy metal-resistant bacteria Cupriavidus metallidurans CH34 relies notably on as many as 12 heavy metal efflux pumps of the RND superfamily. Here we show that C. metallidurans CH34 ZneA is a proton driven efflux pump specific for Zn(II), and that transport of substrates through the transmembrane domain may be electrogenic. We report two X-ray crystal structures of ZneA in intermediate transport conformations, at 3.0 and 3.7 Å resolution. The trimeric ZneA structures capture protomer conformations that differ in the spatial arrangement and Zn(II) occupancies at a proximal and a distal substrate binding site. Structural comparison shows that transport of substrates through a tunnel that links the two binding sites, toward an exit portal, is mediated by the conformation of a short 14-aa loop. Taken together, the ZneA structures presented here provide mechanistic insights into the conformational changes required for substrate efflux by RND superfamily transporters.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe

De bouche à oreille

Ce premier dossier aborde le thème de la transmission de la musique, processus souvent ébranlé dans ses fondements mêmes par les transformations radicales de l'environnement socio-culturel. Dans la multiplicité de ses modalités, cette transmission met en évidence des méthodes d'apprentissage qui sont en soi révélatrices des structures sociales propres à chaque culture. Dans ce premier volume également, un hommage est rendu à l'ethnomusicologue français Pierre Sallée, récemment décédé. Les deux entretiens qui lui font suite présentent deux personnalités marquantes de l'ethnomusicologie: l'africaniste Gilbert Rouget et le musicien japonais Tomiyama Seikin, maître du shamisen. Enfin, une rubrique permanente est réserevée aux comptes rendus de publications récentes

The Net Orientation of Nicotinic Receptor Transmembrane α-Helices in the Resting and Desensitized States

The net orientation of nicotinic acetylcholine receptor transmembrane α-helices has been probed in both the activatable resting and nonactivatable desensitized states using linear dichroism Fourier-transform infrared spectroscopy. Infrared spectra recorded from reconstituted nicotinic acetylcholine receptor membranes after 72 h exposure to (2)H(2)O exhibit an intense amide I component band near 1655 cm(−1) that is due predominantly to hydrogen-exchange-resistant transmembrane peptides in an α-helical conformation. The measured dichroism of this band is 2.37, suggesting a net tilt of the transmembrane α-helices of roughly 40° from the bilayer normal, although this value overestimates the tilt angle because the measured dichroism at 1655 cm(−1) also reflects the dichroism of overlapping amide I component bands. Significantly, no change in the net orientation of the transmembrane α-helices is observed upon agonist binding. In fact, the main changes in structure and orientation detected upon desensitization involve highly solvent accessible regions of the polypeptide backbone. Our data are consistent with a capping of the ligand binding site by the solvent accessible C-loop with little change in the structure of the transmembrane domain in the desensitized state. Changes in structure at the interface between the ligand-binding and transmembrane domains may uncouple binding from gating

Improved stability of a protein vaccine through elimination of a partially unfolded state

Ricin is a potent toxin presenting a threat as a biological weapon. The holotoxin consists of two disulfide-linked polypeptides: an enzymatically active A chain (RTA) and a galactose/N-acetylgalactosamine-binding B chain. Efforts to develop an inactivated version of the A chain as a vaccine have been hampered by limitations of stability and solubility. Previously, recombinant truncated versions of the 267-amino-acid A chain consisting of residues 1–33/44–198 or 1–198 were designed by protein engineering to overcome these limits and were shown to be effective and nontoxic as vaccines in mice. Herein we used CD, dynamic light scattering, fluorescence, and Fourier-transform infrared spectroscopy to examine the biophysical properties of these proteins. Although others have found that recombinant RTA (rRTA) adopts a partially unfolded, molten globule–like state at 45°C, rRTA 1–33/44–198 and 1–198 are significantly more thermostable, remaining completely folded at temperatures up to 53°C and 51°C, respectively. Deleting both an exposed loop region (amino acids 34–43) and the C-terminal domain (199–267) contributed to increased thermostability. We found that chemically induced denaturation of rRTA, but not the truncated variants, proceeds through at least a three-state mechanism. The intermediate state in rRTA unfolding has a hydrophobic core accessible to ANS and an unfolded C-terminal domain. Removing the C-terminal domain changed the mechanism of rRTA unfolding, eliminating a tendency to adopt a partially unfolded state. Our results support the conclusion that these derivatives are superior candidates for development as vaccines against ricin and suggest an approach of reduction to minimum essential domains for design of more thermostable recombinant antigens