Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter.
The calcium (Ca2+) uniporter of mitochondria is a holocomplex consisting of the Ca2+-conducting channel, known as mitochondrial calcium uniporter (MCU), and several accessory and regulatory components. A previous electrophysiology study found that the uniporter has high Ca2+selectivity and conductance and this depends critically on the conserved amino acid sequence motif, DXXE (Asp-X-X-Glu) of MCU. A recent NMR structure of the MCU channel fromCaenorhabditis elegansrevealed that the DXXE forms two parallel carboxylate rings at the channel entrance that seem to serve as the ion selectivity filter, although direct ion interaction of this structural motif has not been addressed. Here, we use a paramagnetic probe, manganese (Mn2+), to investigate ion and inhibitor binding of this putative selectivity filter. Our paramagnetic NMR data show that mutants with a single carboxylate ring, NXXE (Asn-X-X-Glu) and DXXQ (Asp-X-X-Gln), each can bind Mn2+specifically, whereas in the WT the two rings bind Mn2+cooperatively, resulting in ∼1,000-fold higher apparent affinity. Ca2+can specifically displace the bound Mn2+at the DXXE site in the channel. Furthermore, titrating the sample with the known channel inhibitor ruthenium 360 (Ru360) can displace Mn2+binding from the solvent-accessible Asp site but not the inner Glu site. The NMR titration data, together with structural analysis of the DXXE motif and molecular dynamics simulation, indicate that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter and that Ru360 directly blocks ion entry into the filter by binding to the outer carboxylate ring.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.|State Key Laboratory of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, China.
Chan
C
Cao
School of Pharmacy, Tianjin Medical University, Tianjin 300070, China.
Shuqing
S
Wang
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.
Tanxing
T
Cui
State Key Laboratory of Elemento-Organic Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, China; xunchengsu@nankai.edu.cn james_chou@hms.harvard.edu.
Xun-Cheng
XC
Su
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; xunchengsu@nankai.edu.cn james_chou@hms.harvard.edu.|State Key Laboratory of Molecular Biology, National Center for Protein Science Shanghai, Shanghai Institute of Biochemistry and Cell Biology, Shanghai Science Research Center, Chinese Academy of Sciences, Shanghai 200031, China.
James J
JJ
Chou
D020816:Amino Acid Motifs; D001665:Binding Sites; D029742:Caenorhabditis elegans Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D056004:Molecular Dynamics Simulation; D009154:Mutation; D017975:Ruthenium Compounds / Q000378:metabolism / Q000494:pharmacology
D029742:Caenorhabditis elegans Proteins; C000594125:Mcu-1 protein, C elegans; D033681:Mitochondrial Membrane Transport Proteins; C112020:Ru 360; D017975:Ruthenium Compounds; D008345:Manganese; D002118:Calcium
MCU; NMR; Ru360 binding; calcium channel; selectivity filter