Induction of thermal and chemical stability of O6-methylguanine-DNA methyltransferase by Ca2+.
Divalent cations stabilized rat recombinant O6-methylguanine-DNA methyltransferase (rMGMT) protein against heat treatment. Activity of rMGMT was completely abolished by incubating at 45 degrees C for 30 min, however, addition of 1.0 mM Mg2+, Ca2+ or Mn2+ significantly protected heat-induced inactivation of MGMT activity (50-60% vs. 97% inactivation). Protective effect of Ca2+ on the MGMT activity was concentration-dependent up to 3 mM, and the thermal protection was effective up to 45 degrees C. In order to investigate Ca2+ binding site in rMGMT protein, truncated GST-rMGMT proteins containing N-terminal 39 amino acids (GST-rMGMT39), 70 amino acids (GST-rMGMT70) and full-length protein (GST-rMGMT) were prepared. Radiolabeled calcium ion [45Ca2+] was bound only to the GST-rMGMT70 and GST-rMGMT, but not to the GST-rMGMT39, indicating that divalent cations could bind the residues between 40th and 70th of the rMGMT protein. Calcium binding was not observed in the site-directed mutant rMGMT proteins (rMGMT(D42A) and rMGMT(E45A)), confirmed by autoradiography using [45Ca2+] after nondenaturing gel electrophoresis; however, the above two mutants had the same catalytic activity as well as proteolytic sensitivity as the wild MGMT protein. Analysis by equilibrium dialysis revealed stoichiometric binding of one molecule of Ca2+ to one molecule of the protein. Since circular dichroism (CD) spectra indicated no discernible difference before and after Ca2+ binding, the above results suggested that neutralization of two negative charges of Asp42 and Glu45 by Ca2+ resulted in thermal stabilization of the protein with minimum perturbation of its tertiary structure.
Park
Tae Jun
TJ
Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon 442-721, Gyunggi-do, South Korea.
Paik
Woon Ki
WK
Lim
In Kyoung
IK
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D002942:Circular Dichroism; D004795:Enzyme Stability; D006801:Humans; D019216:Metals, Heavy; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors
D002413:Cations, Divalent; D019216:Metals, Heavy; D019853:O(6)-Methylguanine-DNA Methyltransferase; D002118:Calcium