On the evolutionary significance and metal-binding characteristics of a monolobal transferrin from Ciona intestinalis.
Transferrins are a family of proteins that bind and transport Fe(III). Modern transferrins are typically bilobal and are believed to have evolved from an ancient gene duplication of a monolobal form. A novel monolobal transferrin, nicatransferrin (nicaTf), was identified in the primitive ascidian species Ciona intestinalis that possesses the characteristic features of the proposed ancestral Tf protein. In this work, nicaTf was expressed in Pichia pastoris. Extensive solution studies were performed on nicaTf, including UV-vis, fluorescence, CD, EPR and NMR spectroscopies, and electrospray time-of-flight mass spectrometry. The expressed protein is nonglycosylated, unlike the protein isolated from the organism. This property does not affect its ability to bind Fe(III). However, Fe(III)-bound nicaTf displays important spectral differences from other Fe(III)-bound transferrins, which are likely the consequence of differences in metal coordination. Coordination differences could also account for the weaker affinity of nicaTf for Fe(III) (log K = 18.5) compared with bilobal human serum transferrin (HsTf) (log K = 22.5 and 21.4). The Fe-nicaTf complex is not labile, as indicated by slow metal removal kinetics by the high-affinity chelator tiron at pH 7.4. The protein alternatively binds up to one equivalent of Ti(IV) or V(V), which suggests that it may transport nonferric metals. These solution studies provide insight into the structure and function of the primitive monolobal transferrin of C. intestinalis for comparison with higher order bilobal transferrins. They suggest that a major advantage for the evolution of modern transferrins, dominantly of bilobal form, is stronger Fe(III) affinity because of cooperativity.
Tinoco
Arthur D
AD
Department of Chemistry, Yale University, P.O. Box 208107, New Haven, CT 06520-8107, USA.
Peterson
Cynthia W
CW
Lucchese
Baldo
B
Doyle
Robert P
RP
Valentine
Ann M
AM
D000818:Animals; D002938:Ciona intestinalis / Q000737:chemistry*; D003001:Cloning, Molecular; D019143:Evolution, Molecular; D007501:Iron / Q000378:metabolism*; D011485:Protein Binding; D013057:Spectrum Analysis; D014168:Transferrin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D014168:Transferrin; D007501:Iron