It's a well-known fact that proteases facilitate digestion. However, nature has also evolved proteases whose primary function is not degradation, but specific cleavage of proteins to activate their functions. Frequently, the targets are themselves proteases that cleave other ones. This yields cascades of successive proteolytic reactions that rapidly amplify those signals that initiated the first cleaving step. Such proteolytic cascades are used in signalling and regulate important physiological functions.

Insects have perfected a network of extracellular serine proteases that control embryonic development and both blood clotting and the innate immune response. Many of the proteases involved in these functions belong to the family of Clip-domain serine proteases that have one or two so-called Clip-domains attached before the serine protease domain. The early stages of melanization, one component of the innate immune system, are catalyzed by a phenoloxidase, which is produced as an inactive precursor, the prophenoloxidase. Prophenoloxidase is activated by prophenoloxidase-activating factors (ppAFs), However, as part of the signalling cascade, ppAFs are also synthesised as pro-proteins that require activation before they can in turn activate prophenoloxidase. The ppAFs include Clip-domain serine proteases (ppAF-I/III), and Clip-domain serine protease homologues (ppAF-II), which contain a serine protease domain that has lost catalytic activity.

To uncover the functions of Clip-domains, a Korean–Japanese team led by Bok Leul Lee, Byung-Ha Oh and Nam-Chul Ha have crystallized the inactive Clip-domain serine protease homologue ppAF-II from the beetle Holotrichia diomphalia and determined the protein's structure. Analysing the structure of the Clip-domain yielded a novel protein fold with a central four-stranded, irregular β-sheet entwined by numerous loop-like structures that are interlinked by three disulfide bonds. The Clip-domain is tightly connected to the serine protease homologue domain and contains a central cleft that could bind hydrophobic protein regions or other hydrophobic patches.

As ppAFs are activated by upstream proteases, the team asked whether cleavage and activation changes the proteins' quaternary structure. After cleaving ppAF-II with a recombinant upstream protease and analysing its structure by electron microscopy, they found that the activated ppAF-II is no longer a monomer but a dodecamer composed of two hexameric rings. The additional finding that the activated ppAF-II can interact with phenoloxidases thereby forming supramolecular, ball-like structures is interesting as this would allow phenoloxidases to cluster, facilitating subsequent enzyme reactions. To map ppAF-II's phenoloxidase binding site, the scientists produced recombinant ppAF-IIs with altered amino acids and tested them for phenoloxidase binding and oligomerization. It turned out that the central cleft of the Clip-domain binds the phenoloxidase but does not mediate ppAF oligomerization.

Wondering how the Clip-domain functions in ppAFs that retain a serine protease activity, the team analysed the properties of ppAF-I and found that the Clip-domains are not tightly associated with the protein's serine protease domain, possibly allowing the Clip-domain to bind to the surfaces of invading pathogens, leaving the serine protease exposed to interact with other components of the signalling cascade. This step could be crucial for the local amplification of signals that initialise the immune response, suggesting that Clip-domain serine proteases serve multiple functions in insect innate immunity and beyond. Both ppAFs, with and without serine protease activity, evidently play different roles in melanization. What we learn from this system is that slight variations in the arrangement of different protein domains allows diversification and tuning of regulatory mechanisms, a process that may be an essential building block in an insect's survival strategy.