Characterization and copper binding properties of human COMMD1 (MURR1).
COMMD1 (copper metabolism gene MURR1 (mouse U2af1-rs1 region1) domain) belongs to a family of multifunctional proteins that inhibit nuclear factor NF-kappaB. COMMD1 was implicated as a regulator of copper metabolism by the discovery that a deletion of exon 2 of COMMD1 causes copper toxicosis in Bedlington terriers. Here, we report the detailed characterization and specific copper binding properties of purified recombinant human COMMD1 as well as that of the exon 2 product, COMMD(61-154). By using various techniques including native-PAGE, EPR, UV-visible electronic absorption, intrinsic fluorescence spectroscopies as well as DEPC modification of histidines, we demonstrate that COMMD1 specifically binds copper as Cu(II) in 1:1 stoichiometry and does not bind other divalent metals. Moreover, the exon 2 product, COMMD(61-154), alone was able to bind Cu(II) as well as the wild type protein, with a stoichiometry of 1 mol of Cu(II) per protein monomer. The protection of DEPC modification of COMMD1 by Cu(II) implied that Cu(II) binding involves His residues. Further investigation by DEPC modification of COMMD(61-154) and subsequent MALDI MS mapping and MS/MS sequencing identified the protection of His101 and His134 residues in the presence of Cu(II). Fluorescence studies of single point mutants of the full-length protein revealed the involvement of M110 in addition to H134 in direct Cu(II) binding. Taken together, the data provide insight into the function of COMMD1 and especially COMMD(61-154), a product of exon 2 that is deleted in terriers affected by copper toxicosis, as a regulator of copper homeostasis.
Narindrasorasak
Suree
S
Program in Molecular Structure and Function, The Research Institute of the Hospital for Sick Children, and Department of Biochemistry, University of Toronto, Toronto, Canada M5G 1X8.
Kulkarni
Prasad
P
Deschamps
Patrick
P
She
Yi-Min
YM
Sarkar
Bibudhendra
B
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000737:chemistry; D019281:Dimerization; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008715:Methionine / Q000737:chemistry; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry
D048868:Adaptor Proteins, Signal Transducing; C480519:COMMD1 protein, human; D002352:Carrier Proteins; D006639:Histidine; D003300:Copper; D008715:Methionine; D004047:Diethyl Pyrocarbonate