Transmembrane signaling between intracellular compartments is often controlled by regulated proteolysis. Escherichia coli respond to misfolded or unfolded outer-membrane porins (OMPs) in the periplasm by inducing sigma(E)-dependent transcription of stress genes in the cytoplasm. This process requires a proteolytic cascade initiated by the DegS protease, which destroys a transmembrane protein (RseA) that normally binds to and inhibits sigma(E). Here, we show that peptides ending with OMP-like C-terminal sequences bind the DegS PDZ domain, activate DegS cleavage of RseA, and induce sigma(E)-dependent transcription. These results suggest that DegS acts as a sensor of envelope stress by binding unassembled OMPs. DegS activation involves relief of inhibitory interactions between its PDZ and protease domains. Peptide binding to inhibitory PDZ domains in proteases related to DegS, including DegP/HtrA, may also regulate the degradation of specific substrates by these enzymes.

ClpX, a molecular chaperone and the regulatory subunit of the ClpXP protease, is shown to contain tandem modular domains that bind to the C-terminal sequences of target proteins in a manner that parallels functional specificity. Nuclear magnetic resonance studies show that these C-terminal sequences are displayed as disordered peptides on the surface of otherwise folded proteins. The ClpX substrate-binding domains are homologous to sequences in other Clp/Hsp100 proteins and are related more distantly to PDZ domains, which also mediate C-terminal specific protein-protein interactions. Conservation of these binding domains indicates that the mode of substrate recognition characterized here for ClpX will be a conserved feature among Clp/Hsp100 family members and a distinguishing characteristic between this chaperone family and the Hsp70 chaperones.

A "switch" mutant of the Arc repressor homodimer was constructed by interchanging the sequence positions of a hydrophobic core residue, leucine 12, and an adjacent surface polar residue, asparagine 11, in each strand of an intersubunit beta sheet. The mutant protein adopts a fold in which each beta strand is replaced by a right-handed helix and side chains in this region undergo significant repacking. The observed structural changes allow the protein to maintain solvent exposure of polar side chains and optimal burial of hydrophobic side chains. These results suggest that new protein folds can evolve from existing folds without drastic or large-scale mutagenesis.

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Protease-activated triggers have been introduced Into a pore-forming protein, staphylococcal a-hemolysin (αHL). The hemolysin was remodeled by genetic engineering to form two-chain constructs with redundant polypeptide sequences at the central loop, the Integrity of which Is crucial for efficient pore formation. The new hemolysins are activated when the polypeptide extensions are removed by proteases. By alterating the protease recognition sequence in the loop, selective activation by specified proteases can be obtained. Protease-triggered pore-forming proteins might be used for the selective destruction of cancer cells that bear tumor-associated proteases. When certain two-chain constructs are treated with proteases, a full-length polypeptide chain forms as the result of a protease-mediated transpeptidation reaction. This reaction might be used to produce chimeric hemolysins that are Inaccessible by conventional routes.