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A conformational landscape for alginate secretion across the outer Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycanaureus peptidoglycan

DOI: 10.1107/S1399004714001850 DOI Help
PMID: 25084326 PMID Help

Authors: Jingquan Tan (Trinity College, Dublin) , Sarah L. Rouse (Department of Biochemistry, University of Oxford, U.K.) , Dianfan Li (Trinity College, Dublin) , Valerie Pye (Trinity College Dublin) , Lutz Vogeley (Trinity College, Dublin) , Alette Brinth (Trinity College, Dublin) , Toufic El Arnaout (Trinity College, Dublin) , John C. Whitney (Program in Molecular Structure and Function, The Hospital for Sick Children, Toronto, Canada) , P. Lynne Howell (Program in Molecular Structure and Function, The Hospital for Sick Children, Toronto, Canada) , Mark S. P. Sansom , Martin Caffrey (Trinity College, Dublin)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section D Biological Crystallography , VOL 70 , PAGES 2054 - 2068

State: Published (Approved)
Published: August 2014
Diamond Proposal Number(s): 8224

Open Access Open Access

Abstract: The exopolysaccharide alginate is an important component of biofilms produced by Pseudomonas aeruginosa, a major pathogen that contributes to the demise of cystic fibrosis patients. Alginate exits the cell via the outer membrane porin AlgE. X-ray structures of several AlgE crystal forms are reported here. Whilst all share a common β-barrel constitution, they differ in the degree to which loops L2 and T8 are ordered. L2 and T8 have been identified as an extracellular gate (E-gate) and a periplasmic gate (P-gate), respectively, that reside on either side of an alginate-selectivity pore located midway through AlgE. Passage of alginate across the membrane is proposed to be regulated by the sequential opening and closing of the two gates. In one crystal form, the selectivity pore contains a bound citrate. Because citrate mimics the uronate monomers of alginate, its location is taken to highlight a route through AlgE taken by alginate as it crosses the pore. Docking and molecular-dynamics simulations support and extend the proposed transport mechanism. Specifically, the P-gate and E-gate are flexible and move between open and closed states. Citrate can leave the selectivity pore bidirectionally. Alginate docks stably in a linear conformation through the open pore. To translate across the pore, a force is required that presumably is provided by the alginate-synthesis machinery. Accessing the open pore is facilitated by complex formation between AlgE and the periplasmic protein AlgK. Alginate can thread through a continuous pore in the complex, suggesting that AlgK pre-orients newly synthesized exopolysaccharide for delivery to AlgE.

Subject Areas: Biology and Bio-materials

Instruments: I24-Microfocus Macromolecular Crystallography

Other Facilities: APS