Characterization of the HslU chaperone affinity for HslV protease.
The HslVU complex is a bacterial two-component ATP-dependent protease, consisting of HslU chaperone and HslV peptidase. Investigation of protein-protein interactions using SPR in Escherichia coli HslVU and the protein substrates demonstrates that HslU and HslV have moderate affinity (Kd = 1 microM) for each other. However, the affinity of HslU for HslV fivefold increased (Kd approximately 0.2 microM) after binding with the MBP approximately SulA protein indicating the formation of a "ternary complex" of HslV-HslU-MBP approximately SulA. The molecular interaction studies also revealed that HslU strongly binds to MBP approximately SulA with 10(-9) M affinity but does not associate with nonstructured casein. Conversely, HslV does not interact with the MBP-SulA whereas it strongly binds with casein (Kd = 0.2 microM) requiring an intact active site of HslV. These findings provide evidence for "substrate-induced" stable HslVU complex formation. Presumably, the binding of HslU to MBP approximately SulA stimulates a conformational change in HslU to a high-affinity form for HslV.
Azim
M Kamran
MK
Max-Planck-Institut für Biochemie, Martinsried, Germany. kamran.azim@iccs.edu
Goehring
Walter
W
Song
Hyun Kyu
HK
Ramachandran
Ravishankar
R
Bochtler
Matthias
M
Goettig
Peter
P
D002364:Caseins / Q000378:metabolism; D049071:Endopeptidase Clp / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
D002364:Caseins; C483566:ClpYQ protease, E coli; D029968:Escherichia coli Proteins; D049071:Endopeptidase Clp