[2014 Fall Life Sciences & IBB Regular Seminar]
    
      
   ▶Subject: CLC-ec1, a uniquely tractable membrane protein by multiple approaches
   
   ▶Speaker: Hyun-Ho Lim, Ph.D. (Korea Brain Research Institute)
           
   ▶Date: 5:00PM/Oct./10(Fri)/2014
          
   ▶Place: Chemistry Bldg. Room 401
          
           *Abctract
         Chloride-proton antiport proteins of the “CLC” superfamily are transmembrane proteins that form homodimers and are used for myriad physiological purposes, all requiring the coordinated movements of Cl? anions and H+ cations in opposite directions across biological membranes. Among CLC proteins, a prokaryotic homolog of CLC Cl?/H+ antiporter, CLC-ec1 is uniquely tractable by multiple approaches including transport measurements, electrophysiological recording, equilibrium ligand-binding studies and X-ray crystallography.
Here, I am going to introduce two examples adapting all those experimental maneuvers to address previously unanswered questions: 1. intracellular H+ access in CLC proteins 2. the oddity of anion selectivity of CLC proteins.
1. X-ray crystal structures of CLC antiporters show the Cl? ion pathway through these proteins, but the H+ pathway is known only inferentially by two conserved glutamate residues that act as way-stations for H+ in its path through the protein. The extracellular-facing H+ transfer glutamate becomes directly exposed to aqueous solution during the transport cycle, but the intracellular glutamate E203, Gluin, is buried within the protein. Two regions, denoted "polar" and "interfacial," at the intracellular surface of the bacterial antiporter CLC-ec1 are examined here as possible pathways by which intracellular aqueous protons gain access to Gluin. Mutations at multiple residues of the polar region have little effect on antiport rates. In contrast, mutation of E202, a conserved glutamate at the protein-water boundary of the interfacial region, leads to severe slowing of the Cl-/H+ antiport rate. An X-ray crystal structure of E202Y, the most strongly inhibited of these substitutions, shows an aqueous portal leading to Gluin physically blocked by cross-subunit interactions; moreover, this mutation has only minimal effect on a monomeric CLC variant, which necessarily lacks such interactions. The several lines of experiments presented argue that E202 acts as a water-organizer that creates a proton conduit connecting intracellular solvent with Gluin.
2 Cl?/H+ antiporters of the CLC superfamily transport anions across biological membranes in varied physiological contexts. These proteins are weakly selective among anions commonly studied, including Cl-, Br-, I-, NO3- and SCN-, but they seem to be very selective against F-. The recent discovery of a new CLC clade of F-/H+ antiporters, which are highly selective for F- over Cl-, led us to investigate the mechanism of Clover-F- selectivity by a CLC Cl-/H+ antiporter, CLC-ec1. It wil be discussed that F- binds in the Cl- transport pathway with affinity similar to Cl- but stalls the transport cycle. Examination of various mutant antiporters implies a 'lock-down' mechanism of F- inhibition, in which F-, by virtue of its unique hydrogen-bonding chemistry, greatly retards a proton-linked conformational change essential for the transport cycle of CLC-ec1.

   ▶Inquiry: Prof.Kim, Kyong Tai(279-2297)
         
     * This seminar will be given in English