Structural insights into the target specificity of plant invertase and pectin methylesterase inhibitory proteins

Abstract

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 alpha-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 alpha-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.

Figure 1.

Structure of PMEI and Comparison with the Invertase Inhibitor CIF. (A) Ribbon representation of the PMEI dimer with the respective molecules shown in green and yellow. (B) CIF shown in the same orientation as the green molecule in (A). (C) The linker region (residues 25^PMEI to 29^PMEI) interconnecting the dimer as well as a C-terminal extension shown in bonds representation and including the final 2 |F_obs-F_calc| electron density map (contoured at 1.2 σ). (D) A 280-nm absorbance trace of an analytical size-exclusion chromatography reveals the presence of PMEI (shown in red) dimers (peak 1) and monomers (peak 2). The invertase inhibitor CIF (shown in blue) appears to be exclusively monomeric. PMEI mutant P28A (dashed red line) does not resemble the dimeric state. Void (V₀) and total (V_t) volume are shown for the column together with the elution volumes of molecular weight standards (A, BSA; B, ovalbumin; C, chymotrypsinogen A; D, ribonuclease A). The estimated molecular weight values of the At-PMEI1 monomer and dimer are 19,600 and 37,000, respectively. The calculated monomer molecular weight is 16,400.

Figure 2.

The α-Helical Hairpin Module in PMEI and CIF. (A) Stereo close-up view of the bundle-hairpin interface in PMEI with invariant (blue) and conserved residues (green) contributing to interface stabilization included. The small helix-α3 connecting hairpin and bundle in CIF (blue) is unwound in PMEI (red). (B) Sequence comparison of representative inhibitors with secondary structure assignment according to DSSP (Kabsch and Sander, 1983) and invariant Cys residues shown in yellow. Residues contributing to the bundle-hairpin interface are highlighted, dependent on their properties, in green and red. Conserved residues shown in (A) are denoted with a colored dot. The linker region discussed in the text is highlighted in gray; the linker Pro in PMEI is shown in blue.

Figure 3.

Removal of the N-terminal α-Helical Hairpin Inactivates PMEI. Dose-dependent inhibition effect of the wild-type inhibitor (solid circle) and the Δ1-28 truncation on a preparation of PME from Arabidopsis flowers, prepared as described (Wolf et al., 2003).

Figure 4.

Structural Determinants of Flexibility within the N-terminal Hairpin Module. (A) Stereo view of the three PMEI molecules in the asymmetric unit, superimposed with respect to the four-helix bundle. The relative displacement indicates conformational variability. Note that the difference in orientation between molA and molC is almost 90°. (B) Decreased inhibitory power of the PMEI P28A mutant in comparison with the wild-type inhibitor (solid circle) in plant PME inhibition assays. (C) Structural superposition of the wild-type PMEI dimer (green) and two P28A mutant structures shown in dark (form A) and light blue (form B) highlight conformational flexibility of the PMEI hairpin. The flipped-out state (2) might resemble an intermediate in transition between dimer (1) and monomer (3).

Figure 5.

Determinants of Target Specificity. (A) Dose-dependent inhibition of Arabidopsis PME by protein chimera between PMEI and the invertase inhibitor CIF. (B) Schematic representation of protein chimera. In X^PMEI-CIF, the α-hairpin of PMEI has been connected to the bundle core of CIF. X^CIF-PMEI combines the corresponding hairpin segment of CIF with the four-helix bundle of PMEI. The color coding follows the structural representations in Figure 1. (C) Proposed model for the inactivation of plant PME by PMEI. Schematic view, in which the four-helix bundle covers the enzyme's pectin binding cleft, which measures ∼40 Å in length. The helical hairpin anchors the inhibitor to its target enzyme and mediates specificity. (D) Manual docking of PMEI onto the pectin binding cleft of plant PME. Carrot PME (PDB ID 1gq8) is shown in both ribbon and molecular surface representation in a view along the cleft region. The inhibitor (in green) covers the entire cleft. Active-site residues and residues W227/252 discussed in the text are highlighted in red and blue, respectively. The longer loop regions of the bacterial enzyme (PDB ID 1qjv) are indicated in magenta.