Probing the antiprotease activity of lambda CIII, an inhibitor of the Escherichia coli metalloprotease HflB (FtsH)

Abstract

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 lambda CII, thereby helping the phage to lysogenize the host bacterium. lambda CIII, a small (54-residue) protein of unknown structure, also protects sigma(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, CHIC, 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, CHIC 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.