Metal-binding properties of a dicysteine-containing motif in protein tyrosine kinases

Studying the structural consequences of the direct binding of arsenite, cadmium, cobalt, nickel, and lead to a number of protein tyrosine kinases led to the discovery of the metal-binding properties of a dicysteine-containing motif in the C-terminal (CT) lobe of the kinases. Of all the synthesized peptides derived from different domains of c-Src and Csk, only peptides based on a dicysteine-containing motif located in the CT lobe of the kinase domain - CPESLHDLMCQC and CPESLHDLMC in c-Src, and CPPA-VYDVMKNC in Csk - exhibited significant conformational changes in the presence of all metals, as shown by circular dichroism (CD) analyses. Furthermore, CD analysis of natural enzymes c-Src, Csk, Fyn, c-Abl, Lck, EGFR, and c-Src domains containing the CT lobe in the presence of metals showed a significant concentration-dependent conformational change. ICP-MS, ¹¹³Cd NMR, ³³S NMR, and/or molecular modeling studies of CPESLHDLMC and CPPA-VYDVMKNC confirmed the binding between the free sulfhydryl groups of the cysteine residues and Cd^II or As^III. UV-titration studies suggested a high-affinity interaction between Cd^II and As^III and the peptides (K_d, values in the range of 0.6-18.3 nm).