Remodeling of the AB site of rat parvalbumin and oncomodulin into a canonical EF-hand

被引:17
作者
Cox, JA [1 ]
Durussel, I
Scott, DJ
Berchtold, MW
机构
[1] Univ Geneva, Dept Biochem, CH-1211 Geneva, Switzerland
[2] Univ York, Dept Biol, York YO1 5DD, N Yorkshire, England
[3] Univ Copenhagen, Dept Mol & Cell Biol, DK-1168 Copenhagen, Denmark
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1999年 / 264卷 / 03期
基金
英国惠康基金;
关键词
calcium binding; conformational changes; EF-hand calcium binding proteins; oncomodulin; parvalbumin;
D O I
10.1046/j.1432-1327.1999.00650.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Parvalbumin (PV) and the homologous protein oncomodulin (OM) contain three EF-hand motifs, but the first site (AE) cannot bind Ca2+. Were we aimed to recreate the putative ancestral proteins [D19-28E]PV and [D19-28E]OM by replacing the 10-residue-long nonfunctional loop in the AB site by a 12-residue canonical loop. To create an optical conformational probe we also expressed the homologs with a F102W replacement. Unexpectedly, in none of the proteins did the mutation reactivate the AB site. The AB-remodeled parvalbumins bind two Ca2+ ions with strong positive cooperativity (nx = 2) and moderate affinity ([Ca2+](0.5) = 2 mu M ) compared with [Ca2+](0.5) = 37 nM and n(H) = 1 for the wild-type protein. Increasing Mg2+ concentrations changed nx from 2 to 0.65, but without modification of the [Ca2+](0.5)-value. CD revealed that the Ca2+ and Mg2+ forms of the remodeled parvalbumins lost one-third of their or helix content compared with the Ca2+ form of wild-type parvalbumin. However, the microenvironment of single Trp residues in the hydrophobic cores, monitored using intrinsic fluorescence and difference optical density, is the same. The metal-free remodeled parvalbumins possess unfolded conformations. The AB-remodeled oncomodulins also bind two Ca2+ with [Ca2+](0.5) = 43 mu M and nH = 1.45. Mg2+ does not affect Ca2+ binding. Again the Ca2+ forms display two-thirds of the alpha-helical content in the wild-type, while their fore is still strongly hydrophobic as monitored by Trp and Tyr fluorescence. The metal-free oncomodulins are partially unfolded and seem not to possess a hydrophobic core. Our data indicate that AB-remodeled parvalbumin has the potential to regulate cell functions, whereas it is unlikely that [D19-28E]OM can play a regulatory role in vivo. The predicted evolution of the AB site from a canonical to an abortive EF-hand may have been dictated by the need for stronger interaction with Mg2+ and Ca2+, and a high conformational stability of the metal-free forms.
引用
收藏
页码:790 / 799
页数:10
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