Dissecting the dimerization motif of Enterococcus hirae’s Zn(II)CopY
Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, PA, 15282, USA.European Journal of Biochemistry (Impact Factor: 2.54). 07/2012; 17(7):1063-70. DOI: 10.1007/s00775-012-0919-7
The regulation of the copper homeostasis pathway in Enterococcus hirae is conducted through activity of the zinc metalloprotein Zn(II)CopY, which is a Cu(I)-responsive dimeric repressor (Cobine et al., Biochemistry 41:5822-5829, 2002). Its dimerization domain contains a C-terminal cysteine-rich metal-binding motif used for Cu(I) sensing adjacent to an aliphatic-rich repeating sequence, but it is unclear as to which regions contribute most to the interaction. To accomplish this, a synthetically produced CopY construct (CDG) was fused with solubility enhancement tags so the key components of the elements of the aliphatic repeat and metal-binding site could be probed for their dimerization activity. The resultant fusion constructs were tested using two independent methods. Isothermal titration calorimetry, an in vitro technique, was employed to determine dimer affinity thermodynamically. Protein fragment complementation, an in vivo technique, made it possible to rapidly screen homodimeric and heterodimeric complexes within live cells. The combination of in vivo and in vitro studies enabled the identification of CDG sequences that dimerize and sequences that do not, in addition to deciphering relative dimer affinity between all constructs screened. The in vivo technique allowed the formation of heterodimers to be tested for their ability to form specific complexes between dissimilar CDG analogs.
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