Membrane channel proteins of the aquaporin family are highly selective for permeation of specific small molecules, with absolute exclusion of ions and charged solutes and without dissipation of the electrochemical potential across the cell membrane. We report the crystal structure of the Escherichia coli glycerol facilitator (GlpF) with its primary permeant substrate glycerol at 2.2 angstrom resolution. Glycerol molecules line up in an amphipathic channel in single file. In the narrow selectivity filter of the channel the glycerol alkyl backbone is wedged against a hydrophobic corner, and successive hydroxyl groups form hydrogen bonds with a pair of acceptor, and donor atoms. Two conserved aspartic acid-proline-alanine motifs form a key interface between two gene-duplicated segments that each encode three-and-one-half membrane-spanning helices around the channel. This structure elucidates the mechanism of selective permeability for linear carbohydrates and suggests how ions and water are excluded.

YiiP is a membrane transporter that catalyzes Zn2+/H+ exchange across the inner membrane of Escherichia coli. Mammalian homologs of YiiP play critical roles in zinc homeostasis and cell signaling. Here, we report the x-ray structure of YiiP in complex with zinc at 3.8 angstrom resolution. YiiP is a homodimer held together in a parallel orientation through four Zn2+ ions at the interface of the cytoplasmic domains, whereas the two transmembrane domains swing out to yield a Y-shaped structure. In each protomer, the cytoplasmic domain adopts a metallochaperone-like protein fold; the transmembrane domain features a bundle of six transmembrane helices and a tetrahedral Zn2+ binding site located in a cavity that is open to both the membrane outer leaflet and the periplasm.

Distinguishing tumor recurrence from radiation necrosis following brain tumor therapy remains a major clinical challenge. Here we demonstrate the ability to distinguish these lesions using the amide proton transfer (APT) MRI signals of endogenous cellular proteins and peptides as an imaging biomarker. When comparing two orthotopic glioma models (SF188/V+ glioma and 9L gliosarcoma) with a radiation necrosis model in rats, viable glioma (hyperintense) and radiation necrosis (hypointense to isointense) could be clearly differentiated using APT MRI. When irradiating rats with U87MG gliomas, the APT signals in the irradiated tumors decreased significantly at 3 days and 6 days post-radiation. The amide protons detected by APT provide a unique and non-invasive MRI biomarker for assessing viable malignancy versus radiation necrosis and predicting tumor response to therapy.

Zinc transporters play critical roles in cellular zinc homeostatic control. The 2.9-Å resolution structure of the zinc transporter YiiP from Escherichia coli reveals a richly charged dimer-interface stabilized by zinc binding. Site-directed fluorescent resonance energy transfer (FRET) measurements and mutation-activity analysis suggest that zinc binding triggers hinge movements of two electrically repulsive cytoplasmic domains pivoting around four salt-bridges situated at the juncture of the cytoplasmic and transmembrane domains. These highly conserved salt-bridges interlock transmembrane helices at the dimer-interface, well positioned to transmit zinc-induced inter-domain movements to reorient transmembrane helices, thereby modulating coordination geometry of the active-site for zinc transport. The cytoplasmic domain of YiiP is a structural mimic of metal trafficking proteins and the metal-binding domains of metal-transporting P-type ATPases. The use of this common structural module to regulate metal coordination chemistry may enable a tunable transport activity in response to cytoplasmic metal fluctuations.

ZitB is a member of the cation diffusion facilitator (CDF) family that mediates efflux of zinc across the plasma membrane of Escherichia coli. We describe the first kinetic study of the purified and reconstituted ZitB by stopped-flow measurements of transmembrane fluxes of metal ions using a metal-sensitive fluorescent indicator encapsulated in proteoliposomes. Metal ion filling experiments showed that the initial rate of Zn 2؉ influx was a linear function of the molar ratio of ZitB to lipid and was related to the concentration of Zn 2؉ or Cd 2؉ by a hyperbola with a Michaelis-Menten constant (K m ) of 104.9 ؎ 5.4 M and 90.1 ؎ 3.7 M, respectively. Depletion of proton stalled Cd 2؉ transport down its diffusion gradient, whereas tetraethylammonium ion substitution for K ؉ did not affect Cd 2؉ transport, indicating that Cd 2؉ transport is coupled to H ؉ rather than to K ؉ . H ؉ transport was inferred by the H ؉ dependence of Cd 2؉ transport, showing a hyperbolic relationship with a K m of 19.9 nM for H ؉ . Applying H ؉ diffusion gradients across the membrane caused Cd 2؉ fluxes both into and out of proteoliposomes against the imposed H ؉ gradients. Likewise, applying outwardly oriented membrane electrical potential resulted in Cd 2؉ efflux, demonstrating the electrogenic effect of ZitB transport. Taken together, these results indicate that ZitB is an antiporter catalyzing the obligatory exchange of Zn 2؉ or Cd 2؉ for H ؉ . The exchange stoichiometry of metal ion for proton is likely to be 1:1.