Cloning, expression, purification and preliminary X-ray crystallographic studies of Escherichia coli Hsp100 ClpB...

Escherichia coli Hsp100 ClpB plays critical roles in multi-chaperone systems in cell physiology. After the ATPase activity is stimulated by protein or peptide binding, ClpB disaggregates denatured polypeptides by employing ATP hydrolysis and allows other molecular chaperones such as Hsp70 DnaK and Hsp40 DnaJ to more efficiently refold the non-native polypeptides. The mechanisms by which the ClpB acts as a molecular chaperone to disaggregate non-native polypeptides are unknown. The N-terminal domain of ClpB has been proposed to interact with non-native polypeptides. To investigate whether the N-terminal domain participates in polypeptide recognition and binding or modulates the activity of ClpB, the ClpB N-terminal domain has been cloned, purified and crystallized. The ClpB N-terminal domain crystals diffract to 1.95 A using a synchrotron X-ray source and belong to the space group P1, with unit-cell parameters a = 50.2, b = 52.6, c = 56.8 A, alpha = 90.5, beta = 111.8, gamma = 107.1 degrees. Structure determination by the multiple anomalous dispersion (MAD) method is under way.