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FOR THE RECORD

Characterization of the HslU chaperone affinity for HslV protease

¹ Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany
² H.E.J. Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi-72570, Pakistan
³ School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea
⁴ Molecular & Structural Biology Division, Central Drug Research Institute, Chattar Manzil, Lucknow-226001, India

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 µM) for each other. However, the affinity of HslU for HslV fivefold increased (Kd ~ 0.2 µM) after binding with the MBP~SulA protein indicating the formation of a "ternary complex" of HslV–HslU–MBP~SulA. The molecular interaction studies also revealed that HslU strongly binds to MBP~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 µM) 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~SulA stimulates a conformational change in HslU to a high-affinity form for HslV.

Keywords: ATP-dependent protease; proteasomal homolog; protein–protein interaction; SPR analysis

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04970405.

Reprint requests to: M. Kamran Azim, H.E.J. Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Karachi-72570, Pakistan; e-mail: kamran.azim{at}iccs.edu or mkamranazim{at}yahoo.co.uk; fax: +9221-9243190.

⁵ Present addresses: International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland;

⁶ Max-Planck-Institut for Molecular Cell Biology and Genetics, 01309 Dresden, Germany.

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