Remodeling of the AB site of rat parvalbumin and oncomodulin into a canonical EF-hand.
Parvalbumin (PV) and the homologous protein oncomodulin (OM) contain three EF-hand motifs, but the first site (AB) cannot bind Ca2+. Here 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 (nH = 2) and moderate affinity ([Ca2+]0.5 = 2 microM), compared with [Ca2+]0.5 = 37 nM and nH = 1 for the wild-type protein. Increasing Mg2+ concentrations changed nH 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 alpha 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 microM 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 core 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.
Cox
J A
JA
Department of Biochemistry, University of Geneva, Switzerland. jos.cox@biochem.unige.ch
Durussel
I
I
Scott
D J
DJ
Berchtold
M W
MW
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D066298:In Vitro Techniques; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010320:Parvalbumins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D002135:Calcium-Binding Proteins; D010320:Parvalbumins; D011994:Recombinant Proteins; C034733:oncomodulin; D008274:Magnesium; D002118:Calcium