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Orlando, Benjamin

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Orlando, Benjamin

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    Structure and Mechanism of the Cation–chloride Cotransporter NKCC1
    (Springer Science and Business Media LLC, 2019-07-31) Wang, Amy; Liao, Maofu; Liang, Feng; Chew, Thomas; Orlando, Benjamin; Latorraca, Naomi; Zhang, Jinru; Hollingsworth, Scott; Chen, Dong-Hua; Dror, Ron
    Cation-chloride cotransporters (CCCs) mediate the electroneutral transport of chloride, potassium, and/or sodium across the membrane. They play critical roles in regulating cell volume, controlling ion absorption and secretion across epithelia, and maintaining intracellular chloride homeostasis. These transporters are the primary targets for some of the most commonly prescribed drugs in clinic. Here, we determined the cryo-EM structure of a Na-K-Cl cotransporter NKCC1, an extensively-studied member of the CCC transporters. The structure defines the architecture of this protein family and reveals how cytosolic and transmembrane domains are strategically positioned for communication. Structural analyses, functional characterizations, and computational studies uncover the ion translocation pathway, ion-binding sites, and key residues for transport activity. These results provide insights into ion selectivity, coupling, and translocation, and establish a framework for understanding the physiological functions of CCC transporters and interpreting disease-related mutations.
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    Structural Basis of Lipopolysaccharide Extraction by the LptB2FGC Complex
    (Springer Science and Business Media LLC, 2019-03) Liao, Maofu; Orlando, Benjamin; Li, Yanyan
    Lipopolysaccharide (LPS) in Gram-negative bacteria is essential for outer membrane formation and antibiotic resistance. The LPS transport proteins A-G (LptA-G) move LPS from the inner to outer membrane. The ATP-binding cassette (ABC) transporter LptB2FG, tightly associated with LptC, extracts LPS out of the inner membrane. The mechanism of the entire LptB2FGC complex and the role of LptC are poorly understood. Here, we used single-particle cryo-EM to characterize the structures of LptB2FG and LptB2FGC in the nucleotide-free and vanadate-trapped states. These cryo-EM structures resolve the bound LPS, reveal the transporter-LPS interactions with side-chain details, and uncover the basis of coupling LPS capture and extrusion to conformational rearrangements of LptB2FGC. LptC inserts its transmembrane helix between the two transmembrane domains of LptB2FG, representing an unprecedented regulatory mechanism for ABC transporters. Our results suggest a role of LptC in coordinating LptB2FG action and the periplasmic Lpt protein interactions to achieve efficient LPS transport.