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Abstract:
HsIVU is a bacterial homolog of the proteasome, where HsIV is the protease that is activated by HsIU, an ATPase and chaperone. Structures of singly and doubly capped HsIVU particles have been reported, and different binding modes have been observed. Even among HsIVU structures with I-domains distal to HsIV, no consensus mode of activation has emerged. A feature in the Haemophilus influenzae HsIVU structure, insertion of the C termini of HsIU into pockets in HsIV, was not seen in all other structures of the enzyme. Here we report site-directed mutagenesis, peptide activation, and fluorescence experiments that strongly support the functional relevance of the C terminus insertion mechanism: we find that mutations in HsIV that disrupt the interaction with the C termini of HslU invariably lead to inactive enzyme. Conversely, synthetic peptides derived from the C terminus of HslU bind to HsIV with 10(-5) M affinity and can functionally replace full HslU particles for both peptide and casein degradation but fail to support degradation of a folded substrate. Thus, the data can be taken as evidence for separate substrate unfoldase and protease stimulation activities in HsIU. Enhanced HsIV proteolysis could be due to the opening of a gated channel or allosteric activation of the active sites. To distinguish between these possibilities, we have mutated a series of residues that line the entrance channel into the HsIV particle. Our mutational and fluorescence experiments demonstrate that allosteric activation of the catalytic sites is required in HsIV, but they do not exclude the possibility of channel opening taking place as well. The present data support the conclusion that the H. influenzae structure with I-domains distal to HsIV captures the active species and point to significant differences in the activation mechanism of HsIV, ClpP, and the proteasome.
