Zinc binding in bovine alpha-lactalbumin: sequence homology may not be a predictor of subtle functional features.
alpha-Lactalbumin (alpha-LA), a calcium-binding protein, also possesses zinc-binding sites comprising a single strong site and several weaker secondary sites. The only site found by X-ray crystallography (Ren et. al., J. Biol. Chem. 1993;268:19292) was Glu 49 of human alpha-LA, but zinc binding had never been measured in solution for human alpha-LA. This residue was genetically substituted by Ala in bovine alpha-LA and the metal-binding properties of the resulting desMetE49A protein were compared with those for native alpha-LA by fluorescence methods. Surprisingly, desMetE49A alpha-LA and the native bovine protein had similar affinities for both Zn(2+) and Ca(2+). Genetic substitution of other possible candidates for Zn(2+) chelating residues, which included Glu 25, did not alter the affinity of bovine alpha-LA to Zn2+; however, substitution of Glu 1 by Met resulted in the disappearance of strong Zn(2+) binding. A proposed site involves Glu 1, Glu 7, Asp 11, and Asp 37, which would participate in strong Zn(2+) binding based on their propinquity to Glu 1. Human alpha-LA, which has a Lys at position 1 rather than Glu, binds zinc with a reduced affinity compared with native bovine alpha-LA, suggesting that the site identified from the X-ray structure did not correspond to strong zinc binding in solution.
Permyakov
S E
SE
Institute for Biological Instrumentation, Russian Academy of Science, Pushchino, Russia.
Veprintsev
D B
DB
Brooks
C L
CL
Permyakov
E A
EA
Berliner
L J
LJ
D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D006801:Humans; D007768:Lactalbumin / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
D007768:Lactalbumin; D015032:Zinc