Use of thallium to identify monovalent cation binding sites in GroEL

Monovalent cation-binding sites in GroEL were identified utilizing the anomalous scattering signal from Tl⁺. GroEL is a bacterial chaperone protein that assembles into a homotetradecameric complex exhibiting D₇ symmetry and utilizes the co-chaperone protein GroES and ATP hydrolysis to assist in the proper folding of a variety of cytosolic proteins. GroEL utilizes two metal cofactors, Mg²⁺ and K⁺, to bind and hydrolyze ATP. A K⁺-binding site has been proposed to be located next to the nucleotide-binding site, but the available structural data do not firmly support this conclusion. Moreover, more than one functionally significant K⁺-binding site may exist within GroEL. Because K⁺ has important and complex effects on GroEL activity and is involved in both positive (intra-ring) and negative (inter-ring) cooperativity for ATP hydrolysis, it is important to determine the exact location of these cation-binding site(s) within GroEL. In this study, the K⁺ mimetic Tl⁺ was incorporated into GroEL crystals, a moderately redundant 3.94 Å resolution X-ray diffraction data set was collected from a single crystal and the strong anomalous scattering signal from the thallium ion was used to identify monovalent cation-binding sites. The results confirmed the previously proposed placement of K⁺ next to the nucleotide-binding site and also identified additional binding sites that may be important for GroEL function and cooperativity. These findings also demonstrate the general usefulness of Tl⁺ for the identification of monovalent cation-binding sites in protein crystal structures, even when the quality and resolution of the diffraction data are relatively low