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.
Highlights
Mg2ϩ is a required cofactor in hundreds of enzymes and is indispensable for maintaining genomic stability
Two transmembrane (TM) helices are connected by a short periplasmic loop, with the universally conserved GMN motif found at the C-terminal end of the pore-lining helix, TM1
A conformational change that was observed in the crystal structure of an isolated funnel domain (FD) from Archaeoglobus fulgidus CorA (AfCorA) led us to propose a model for the structural basis of gating (Fig. 1A)
Summary
Protein Purification—TmCorA was cloned into pET-15b [26], and site-directed mutants were generated using the QuikChange protocol (Stratagene). The final protein buffer contained 20 mM Tris, pH 8.0, 100 mM NaCl, 0.026% DDM, 1 mM TCEP. TmCorA Reconstitution and Fluorescence Assay—Reconstitution buffer containing 150 mM KCl, 8 mM HEPES, pH 7.2, was added to dried phosphatidylcholine (PC, Avanti Polar Lipids) to produce a final 10 mg/ml liposome preparation by sonication. Protein and MgCl2 solutions were mixed and equilibrated at 4 °C for 20 min; trypsin was added, and reactions were incubated at 4 °C for 15 h. A 500 M solution of wild-type (WT) TmCorA was placed in the sample cell and titrated with 1 mM MgCl2 prepared in the same buffer (20 mM Tris, pH 8.0, 100 mM NaCl, 0.026% DDM, 1 mM TCEP). Data sets representing the titration of each protein by 1 or 20 mM MgCl2 were fitted separately to estimate binding parameters.
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