Overlapping binding sites for typsin and papain on a kunitz-type proteinase inhibitor from prosopis juliflora

Proteinase inhibitors are among the most promising candidates for expression by transgenic plants andconsequent protectionagainst insect predation. However, some insects can respond to the threat of the proteinase inhibitor by the production of enzymes insensitive to inhibition. Inhibitors combiningmore thanone favorable activityarethereforestronglyfavored. Recently,aknown small Kunitz trypsin inhibitor from Prosopis juliflora (PTPKI) has been shown to possess unexpected potent cysteine proteinase inhibitory activity. Hereweshow, byenzymeassayandgel filtration, that, unlike other Kunitz inhibitors withdual activities, this inhibitor is incapable of simultaneous inhibition of trypsin and papain. These data are most readily interpreted by proposing overlapping binding sites for the two enzymes. Molecular modeling and docking experiments favor an interaction mode in which the same inhibitor loop that interacts in a canonical fashion with trypsin can also bind into the papain catalytic site cleft. Unusual residue substitutions at the proposed interface can explain the relative rarity of twin trypsin/papain inhibition. Other changes seem responsible for the relative low affinity of PTPKI for trypsin. The predicted coincidence of trypsin and papain binding sites, once confirmed, would facilitate the search, by phage display for example, for mutants highly active against bothproteinases. Proteins 2002;49:335–341