Pectin methylesterase (PME) and invertase are key enzymes in plant carbohydrate metabolism. Inhibitors of both enzymes constitute a sequence family of extracellular proteins. Members of this family are selectively targeted toward either PME or invertase. In a comparative structural approach we have studied how this target specificity is implemented on homologous sequences. By extending crystallographic work on the invertase inhibitor Nt-CIF to a pectin methylesterase inhibitor (PMEI) from Arabidopsis thaliana, we show an a-helical hairpin motif to be an independent and mobile structural entity in PMEI. Removal of this hairpin fully inactivates the inhibitor. A chimera composed of the a-hairpin of PMEI and the four-helix bundle of Nt-CIF is still active against PME. By contrast, combining the corresponding segment of Nt-CIF with the four-helix bundle of PMEI renders the protein inactive toward either PME or invertase. Our experiments provide insight in how these homologous inhibitors can make differential use of similar structural modules to achieve distinct functions. Integrating our results with previous findings, we present a model for the PME-PMEI complex with important implications.
INTRODUCTIONAt the posttranslational level, the activity of enzymes is commonly regulated by various mechanisms, including residuedirected protein modifications such as phosphorylation, glycosylation, and interaction with specific inhibitors. The nature of these inhibitors may range from small molecules to entire proteins, as found with the well-studied inhibitors of proteases (Bode and Huber, 1992). In plants, inhibitory proteins are often targeted toward sugar-modifying enzymes that escape cellular control mechanisms upon secretion into the plant cell wall or the vacuole (Juge et al., 2004). We are focusing on the structure-function relationship of an inhibitor family that regulates the activity of plant acid invertase and pectin methylesterase (PME).Invertases convert the transport sugar sucrose into its building blocks, fructose and glucose. In higher plants, invertases exist in compartment specific isoforms, with only extracytosolic species being sensitive to inhibitory proteins. Altered activity of extracellular invertase has been shown to have dramatic effects on growth and development (Cheng et al., 1996;Tang et al., 1999;Goetz et al., 2001). This is consistent with roles of invertase activity in vital cellular processes such as carbohydrate transport (Roitsch et al., 2003), sugar signaling (Koch, 1996(Koch, , 2004Smeekens, 1998;Wobus and Weber, 1999), and stress response (Ehness et al., 1997;Roitsch et al., 2003). Protein inhibitors of invertase (Greiner et al., 1998) affect enzyme activity in a strictly pH-dependent manner (Rausch and Greiner, 2004) and have been proposed as transgenic tools to engineer post-harvest sucrose metabolism in crop plants (Greiner et al., 1999).PMEs catalyze the demethylesterification of the homogalacturonan component of pectins, highly heterogeneous polymers (Vorwerk et al., 2004) that repre...