Use of thallium to identify monovalent cation binding sites in GroEL.
GroEL is a bacterial chaperone protein that assembles into a homotetradecameric complex exhibiting D(7) 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, Mg(2+) 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 A 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.
Case Western Reserve University, USA.
Philip D
PD
Kiser
George H
GH
Lorimer
Krzysztof
K
Palczewski
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000187:drug effects; D002414:Cations, Monovalent / Q000378:metabolism*; D018834:Chaperonin 60 / Q000737:chemistry*; D018360:Crystallography, X-Ray; D029968:Escherichia coli Proteins / Q000378:metabolism; D006868:Hydrolysis; D008024:Ligands; D011188:Potassium / Q000378:metabolism*; D013793:Thallium / Q000378:metabolism*
D002414:Cations, Monovalent; D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D008024:Ligands; D000255:Adenosine Triphosphate; D013793:Thallium; D011188:Potassium