Polygalacturonase-inhibiting protein (PGIP) in plant defence: a structural view.

Polygalacturonase-inhibiting proteins are plant extracellular leucine-rich repeat proteins that specifically bind and inhibit fungal polygalacturonases. The interaction with PGIP limits the destructive potential of polygalacturonases and might trigger the plant defence responses induced by oligogalacturonides. A high degree of polymorphism is found both in PGs and PGIPs, accounting for the specificity of different plant inhibitors for PGs from different fungi. Here, we review the structural features and our current understanding of the PG-PGIP interaction.

Polygalacturonase inhibiting proteins: players in plant innate immunity?

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular leucine-rich repeat (LRR) proteins that recognize and inhibit fungal polygalacturonases (PGs). The PG-PGIP interaction favours the accumulation of elicitor-active oligogalacturonides and causes the activation of defence responses. Small gene families encode PGIP isoforms that differ in affinity and specificity for PGs secreted by different pathogens. The consensus motif within the LRR structure of PGIPs is the same as that of the extracellular receptors of the plant innate immune system. Structural and functional evidence suggest that PGIPs are versatile proteins involved in innate immunity and that they are capable of recognizing different surface motifs of functionally related but structurally variable PGs.

The polygalacturonase-inhibiting protein PGIP2 of Phaseolus vulgaris has evolved a mixed mode of inhibition of endopolygalacturonase PG1 of Botrytis cinerea.

Botrytis cinerea is a phytopathogenic fungus that causes gray mold in >1,000 plant species. During infection, it secretes several endopolygalacturonases (PGs) to degrade cell wall pectin, and among them, BcPG1 is constitutively expressed and is an important virulence factor. To counteract the action of PGs, plants express polygalacturonase-inhibiting proteins (PGIPs) that have been shown to inhibit a variety of PGs with different inhibition kinetics, both competitive and noncompetitive. The PG-PGIP interaction promotes the accumulation of oligogalacturonides, fragments of the plant cell wall that are general elicitors of plant defense responses. Here, we characterize the enzymatic activity of BcPG1 and investigate its interaction with PGIP isoform 2 from Phaseolus vulgaris (PvPGIP2) by means of inhibition assays, homology modeling, and molecular docking simulations. Our results indicate a mixed mode of inhibition. This is compatible with a model for the interaction where PvPGIP2 binds the N-terminal portion of BcPG1, partially covering its active site and decreasing the enzyme affinity for the substrate. The structural framework provided by the docking model is confirmed by site-directed mutagenesis of the residues that distinguish PvPGIP2 from the isoform PvPGIP1. The finding that PvPGIP2 inhibits BcPG1 with a mixed-type kinetics further indicates the versatility of PGIPs to evolve different recognition specificities.

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

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.

Insights into DNA replication: the crystal structure of DNA polymerase B1 from the archaeon Sulfolobus solfataricus.

To minimize the large number of mispairs during genome duplication owing to the large amount of DNA to be synthesized, many replicative polymerases have accessory domains with complementary functions. We describe the crystal structure of replicative DNA polymerase B1 from the archaeon Sulfolobus solfataricus. Comparison between other known structures indicates that although the protein is folded into the typical N-terminal, editing 3'-5'exonuclease, and C-terminal right-handed polymerase domains, it is characterized by the unusual presence of two extra alpha helices in the N-terminal domain interacting with the fingers helices to form an extended fingers subdomain, a structural feature that can account for some functional features of the protein. We explore the structural basis of specific lesion recognition, the initial step in DNA repair, describing how the N-terminal subdomain pocket of archaeal DNA polymerases could allow specific recognition of deaminated bases such as uracil and hypoxanthine in addition to the typical DNA bases.

The crystal structure of the sorcin calcium binding domain provides a model of Ca2+-dependent processes in the full-length protein.

Sorcin is a 21.6 kDa calcium binding protein, expressed in a number of mammalian tissues that belongs to the small, recently identified penta-EF-hand (PEF) family. Like all members of this family, sorcin undergoes a Ca2+-dependent translocation from cytosol to membranes where it binds to target proteins. For sorcin, the targets differ in different tissues, indicating that it takes part in a number of Ca2+-regulated processes. The sorcin monomer is organized in two domains like in all PEF proteins: a flexible, hydrophobic, glycine-rich N-terminal region and a calcium binding C-terminal domain. In vitro, the PEF proteins are dimeric in their Ca2+-free form, but have a marked tendency to precipitate when bound to calcium. Stabilization of the dimeric structure is achieved by pairing of the uneven EF-hand, EF5. Sorcin can also form tetramers at acid pH. The sorcin calcium binding domain (SCBD, residues 33-198) expressed in Escherichia coli was crystallized in the Ca2+-free form. The structure was solved by molecular replacement and was refined to 2.2 A with a crystallographic R-factor of 22.4 %. Interestingly, the asymmetric unit contains two dimers. The structure of the SCBD leads to a model that explains the solution properties and describes the Ca2+-induced conformational changes. Phosphorylation studies show that the N-terminal domain hinders phosphorylation of SCBD, i.e. the rate of phosphorylation increased twofold in the absence of the N-terminal region. In addition, previous fluorescence studies indicated that hydrophobic residues are exposed to solvent upon Ca2+ binding to full-length sorcin. The model accounts for these data by proposing that Ca2+ binding weakens the interactions between the two domains and leads to their reorientation, which exposes hydrophobic regions facilitating the Ca2+-dependent binding to target proteins at or near membranes.