Premium
What We Know about the Structure of NCX1 and How It Relates to Its Function
Author(s) -
NICOLL DEBORA A.,
REN XIAOYAN,
OTTOLIA MICHELA,
PHILLIPS MARTIN,
PAREDES ALFREDO R.,
ABRAMSON JEFF,
PHILIPSON KENNETH D.
Publication year - 2007
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1196/annals.1387.014
Subject(s) - mutant , biophysics , chemistry , binding site , mutagenesis , fusion protein , transmembrane protein , lipid bilayer fusion , crystallography , membrane , biochemistry , biology , receptor , gene , recombinant dna
NCX1 is modeled to contain nine transmembrane segments (TMS) with a large intracellular loop between TMS 5–6 and two reentrant loops connecting TMS 2–3 and TMS 7–8. NCX1 also contains two regions of internal repeats. The α repeats are composed of TMS 2 and 3 and TMS 7 and 8 and are involved in ion binding and transport. The β repeats are in the large intracellular loop and are involved in binding of regulatory Ca 2+ . Our studies on the structure/function analysis of NCX1 have focused on the α‐ and β‐repeat regions and on how the TMS pack in the membrane. We have examined the α1 repeat by mutagenesis of residues modeled to be in the reentrant loop and TMS 3 and by determination of ion affinities of the mutants. Our results show that TMS 3 and not the reentrant loop is involved in Na + binding. No mutants demonstrated altered affinity for transported Ca 2+ . We have synthesized a fusion protein composed of the β1 repeat. This fusion protein was expressed in Escherichia coli and purified. The fusion protein binds Ca 2+ and shows conformational changes on binding. The crystal structure of the β1 repeat shows that it is composed of a seven‐stranded β‐sandwich with Ca 2+ ‐binding sites located at one end of the sandwich. Four Ca 2+ ions bind to the β1 repeat in a manner reminiscent of Ca 2+ binding to C2 domains. Packing of TMS in the membrane has been studied by cross‐linking induced mobility shifts on SDS‐PAGE. Interactions between TMS 1, 2, 3, 6, 7, and 8 have been identified.