Structural basis for the interaction between pectin methylesterase and a specific inhibitor protein

Abstract

Pectin, one of the main components of the plant cell wall, is secreted in a highly methyl-esterified form and subsequently deesterified in muro by pectin methylesterases (PMEs). In many developmental processes, PMEs are regulated by either differential expression or posttranslational control by protein inhibitors (PMEIs). PMEIs are typically active against plant PMEs and ineffective against microbial enzymes. Here, we describe the three-dimensional structure of the complex between the most abundant PME isoform from tomato fruit (Lycopersicon esculentum) and PMEI from kiwi (Actinidia deliciosa) at 1.9-A resolution. The enzyme folds into a right-handed parallel beta-helical structure typical of pectic enzymes. The inhibitor is almost all helical, with four long alpha-helices aligned in an antiparallel manner in a classical up-and-down four-helical bundle. The two proteins form a stoichiometric 1:1 complex in which the inhibitor covers the shallow cleft of the enzyme where the putative active site is located. The four-helix bundle of the inhibitor packs roughly perpendicular to the main axis of the parallel beta-helix of PME, and three helices of the bundle interact with the enzyme. The interaction interface displays a polar character, typical of nonobligate complexes formed by soluble proteins. The structure of the complex gives an insight into the specificity of the inhibitor toward plant PMEs and the mechanism of regulation of these enzymes.

Figure 1.

Structure of the PME-PMEI complex. Ribbon representation illustrating the relative positions of PMEI and PME in the complex. The enzyme is shown in green–blue on the left side. The inhibitor is represented in yellow–red on the right side; the α-helices of the four-helix bundle are indicated as α1 to α4, whereas helices of the N-terminal region are named αa, αb, and αc. The inhibitor binds the active site region of the enzyme, hampering its access to the substrate.

Figure 2.

Close-Up View of the Tomato PME Active Site. (A) Structure of tomato PME in which residues involved in catalysis (violet), in stabilization of the catalytic intermediate (orange), and in substrate binding (blue) are shown in ball and stick representation. (B) Further close-up view representation of amino acid residues and a water molecule (blue ball) putatively involved in catalysis; H-bond pattern is highlighted.

Figure 3.

Comparison of the Known Structures of PMEs. (A) Overlay of the Cα trace of PME from tomato (green) and PME from carrot (orange). Structures are almost completely superimposable, with a RMSD value of 0.7 Å, calculated on all Cα. (B) Superimposition of PME from tomato (green) and PME from E. chrysanthemi (violet). The RMSD value, calculated on 284 out of 317 Cα, is 1.8 Å. Although the β-helices are completely superimposable, main differences are located in the length of the turns protruding out from the β-helix in proximity of the putative active site cleft.

Figure 4.

Structural Superimposition between PMEI and Nt-CIF. PMEI (red) and Nt-CIF (blue) are superimposable, with a RMSD of 1.7 Å, calculated on 144 out of 151 Cα. Main differences are located in the loops connecting the helices of the bundle and in the N-terminal region; a distortion in the α2 helix of Nt-CIF is indicated by the arrow. Conserved disulphide bridges are represented in yellow.

Figure 5.

Molecular Surface of the PME-PMEI Complex. (A) Representation of the molecular surface of the enzyme (violet) and the inhibitor (yellow) in the complex. (B) Same view of the complex as in (A), showing the molecular surface of PME and a ribbon diagram of PMEI. The α-helices α2, α3, and α4 of the inhibitor fit into the substrate binding cleft of the enzyme.

Figure 6.

Representation of the Interacting Surface of PME and PMEI. To open up the complex, PMEI has been shifted along its major axis and rotated by 180° around the vertical axis indicated. The molecular surfaces contributed by carbon atoms are in green, and those contributed by oxygen and nitrogen are in red and blue, respectively. Water molecules involved in water-mediated hydrogen bonds are represented as violet spheres.

Figure 7.

Sequence Comparison of PMEs from Tomato (PME_LYCES), Carrot (PME_DAUCA), and A. aculeatus (PME_ASPAC). Residues involved in H-bonds (violet), Van der Waals contacts (green), and water-mediated H-bonds (yellow) with the inhibitor are shown. Conserved residues are in blue.

Figure 8.

Sequence Comparison of PMEIs form Kiwi (AcPMEI), PMEIs from Arabidopsis (AthPMEI-1, Accession Number NP_175236; AthPMEI-2, Accession Number NP_188348), and Invertase Inhibitor from Tobacco (NtCIF). Residues of the kiwi inhibitor involved in H-bonds (violet), Van der Waals contacts (green), and water-mediated H-bonds (yellow) with tomato PME are shown. Conserved residues are in blue.