Stability and interactions of the amino-terminal domain of ClpB from Escherichia coli.
ClpB is a member of a multichaperone system in Escherichia coli (with DnaK, DnaJ, and GrpE) that reactivates aggregated proteins. The sequence of ClpB contains two ATP-binding regions that are enclosed between the N- and C-terminal extensions. Whereas it has been found that the N-terminal region of ClpB is essential for the chaperone activity, the structure of this region is not known, and its biochemical properties have not been studied. We expressed and purified the N-terminal fragment of ClpB (residues 1-147). Circular dichroism of the isolated N-terminal region showed a high content of alpha-helical structure. Differential scanning calorimetry showed that the N-terminal region of ClpB is thermodynamically stable and contains a single folding domain. The N-terminal domain is monomeric, as determined by gel-filtration chromatography, and the elution profile of the N-terminal domain does not change in the presence of the N-terminally truncated ClpB (ClpBDeltaN). This indicates that the N-terminal domain does not form strong contacts with ClpBDeltaN. Consistently, addition of the separated N-terminal domain does not reverse an inhibition of ATPase activity of ClpBDeltaN in the presence of casein. As shown by ELISA measurements, full-length ClpB and ClpBDeltaN bind protein substrates (casein, inactivated luciferase) with similar affinity. We also found that the isolated N-terminal domain of ClpB interacts with heat-inactivated luciferase. Taken together, our results indicate that the N-terminal fragment of ClpB forms a distinct domain that is not strongly associated with the ClpB core and is not required for ClpB interactions with other proteins, but may be involved in recognition of protein substrates.
Tek
Vekalet
V
Department of Biochemistry, 104 Willard Hall, Kansas State University, Manhattan, KS 66506, USA.
Zolkiewski
Michal
M
D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D002942:Circular Dichroism; D049071:Endopeptidase Clp; D004926:Escherichia coli / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D006360:Heat-Shock Proteins; D015800:Protozoan Proteins; D049071:Endopeptidase Clp; C095049:ClpB protein, Leishmania