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Journal of Bacteriology, November 2007, p. 8130-8138, Vol. 189, No. 22
0021-9193/07/$08.00+0     doi:10.1128/JB.00820-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Probing the Antiprotease Activity of CIII, an Inhibitor of the Escherichia coli Metalloprotease HflB (FtsH)

Sabyasachi Halder, Ajit Bikram Datta, and Pradeep Parrack^*

Department of Biochemistry, Bose Institute, P-1/12, CIT Scheme VIIM, Kolkata 700054, India

Received 25 May 2007/ Accepted 7 September 2007

The CIII protein encoded by the temperate coliphage lambda acts as an inhibitor of the ubiquitous Escherichia coli metalloprotease HflB (FtsH). This inhibition results in the stabilization of transcription factor CII, thereby helping the phage to lysogenize the host bacterium. CIII, a small (54-residue) protein of unknown structure, also protects ³², another specific substrate of HflB. In order to understand the details of the inhibitory mechanism of CIII, we cloned and expressed the protein with an N-terminal six-histidine tag. We also synthesized and studied a 28-amino-acid peptide, CIIIC, encompassing the central 14 to 41 residues of CIII that exhibited antiproteolytic activity. Our studies show that CIII exists as a dimer under native conditions, aided by an intersubunit disulfide bond, which is dispensable for dimerization. Unlike CIII, CIIIC resists digestion by HflB. While CIII binds to HflB, it does not bind to CII. On the basis of these results, we discuss various mechanisms for the antiproteolytic activity of CIII.

Published ahead of print on 21 September 2007.

Present address: Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205.