title: Probing structure-function relationships and gating mechanisms in the CorA Mg2+ transport system.
abstract: Recent crystal structures of the CorA Mg(2+) transport protein from Thermotoga maritima (TmCorA) revealed an unusually long ion pore putatively gated by hydrophobic residues near the intracellular end and by universally conserved asparagine residues at the periplasmic entrance. A conformational change observed in an isolated funnel domain structure also led to a proposal for the structural basis of gating. Because understanding the molecular mechanisms underlying ion channel and transporter gating remains an important challenge, we have undertaken a structure-guided engineering approach to probe structure-function relationships in TmCorA. The intracellular funnel domain is shown to constitute an allosteric regulatory module that can be engineered to promote an activated or closed state. A periplasmic gate centered about a proline-induced kink of the pore-lining helix is described where "helix-straightening" mutations produce a dramatic gain-of-function. Mutation to the narrowest constriction along the pore demonstrates that a hydrophobic gate is operational within this Mg(2+)-selective transport protein and likely forms an energetic barrier to ion flux. We also provide evidence that highly conserved acidic residues found in the short periplasmic loop are not essential for TmCorA function or Mg(2+) selectivity but may be required for proper protein folding and stability. This work extends our gating model for the CorA-Alr1-Mrs2 superfamily and reveals features that are characteristic of an ion channel. Aspects of these results that have broader implications for a range of channel and transporter families are highlighted.
authors.affiliation: Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7, Canada. payandeh@uhnres.utoronto.ca
authors.forename: Jian
authors.initials: J
authors.lastname: Payandeh
authors.forename: Canhui
authors.initials: C
authors.lastname: Li
authors.forename: Mohabir
authors.initials: M
authors.lastname: Ramjeesingh
authors.forename: Ewa
authors.initials: E
authors.lastname: Poduch
authors.forename: Christine E
authors.initials: CE
authors.lastname: Bear
authors.forename: Emil F
authors.initials: EF
authors.lastname: Pai
mesh_terms: D001216:Asparagine / Q000737:chemistry; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D002412:Cations; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D005816:Genetic Complementation Test; D007477:Ions; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D008968:Molecular Conformation; D015202:Protein Engineering / Q000379:methods; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D020124:Thermotoga maritima / Q000378:metabolism
chemical_list: D027682:Cation Transport Proteins; D002412:Cations; D007477:Ions; D001216:Asparagine; D008274:Magnesium