Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall proteins that protect plants from fungal invasion. They interact with endopolygalacturonases secreted by phytopathogenic fungi, inhibit their enzymatic activity, and favor the accumulation of oligogalacturonides, which activate plant defense responses. PGIPs are members of the leucine-rich repeat (LRR) protein family that in plants play crucial roles in development, defense against pathogens, and recognition of beneficial microbes. Here we report the crystal structure at 1.7-Å resolution of a PGIP from Phaseolus vulgaris. The structure is characterized by the presence of two ␤-sheets instead of the single one originally predicted by modeling studies. The structure also reveals a negatively charged surface on the LRR concave face, likely involved in binding polygalacturonases. The structural information on PGIP provides a basis for designing more efficient inhibitors for plant protection.

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-Å resolution. The enzyme folds into a right-handed parallel b-helical structure typical of pectic enzymes. The inhibitor is almost all helical, with four long a-helices aligned in an antiparallel manner in a classical up-and-down fourhelical 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 b-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.

The folding mechanism of many proteins involves the population of partially organized structures en route to the native state. Identification and characterization of these intermediates is particularly difficult, as they are often only transiently populated and may play different mechanistic roles, being either on-pathway productive species or off-pathway kinetic traps. Following different spectroscopic probes, and employing state-of-the-art kinetic analysis, we present evidence that the folding mechanism of the thermostable cytochrome c 552 from Hydrogenobacter thermophilus does involve the presence of an elusive, yet compact, on-pathway intermediate. Characterization of the folding mechanism of this cytochrome c is particularly interesting for the purpose of comparative folding studies, because H. thermophilus cytochrome c 552 shares high sequence identity and structural homology with its homologue from the mesophilic bacterium Pseudomonas aeruginosa cytochrome c 551 , which refolds through a broad energy barrier without the accumulation of intermediates. Analysis of the folding kinetics and correlation with the three-dimensional structure add new evidence for the validity of a consensus folding mechanism in the cytochrome c family.

The flavodiiron proteins (FDP) are widespread among strict or facultative anaerobic prokaryotes, where they are involved in the response to nitrosative and/or oxidative stress. Unexpectedly, FDPs were fairly recently identified in a restricted group of microaerobic protozoa, including Giardia intestinalis, the causative agent of the human infectious disease giardiasis. The FDP from Giardia was expressed, purified, and extensively characterized by x-ray crystallography, stopped-flow spectroscopy, respirometry, and NO amperometry. Contrary to flavorubredoxin, the FDP from Escherichia coli, the enzyme from Giardia has high O 2 -reductase activity (>40 s ؊1 ), but very low NO-reductase activity (ϳ0.2 s ؊1 ); O 2 reacts with the reduced protein quite rapidly (milliseconds) and with high affinity (K m < 2 M), producing H 2 O. The three-dimensional structure of the oxidized protein determined at 1.9 Å resolution shows remarkable similarities with prokaryotic FDPs. Consistent with HPLC analysis, the enzyme is a dimer of dimers with FMN and the nonheme di-iron site topologically close at the monomer-monomer interface. Unlike the FDP from Desulfovibrio gigas, the residue His-90 is a ligand of the di-iron site, in contrast with the proposal that ligation of this histidine is crucial for a preferential specificity for NO. We propose that in G. intestinalis the primary function of FDP is to efficiently scavenge O 2 , allowing this microaerobic parasite to survive in the human small intestine, thus promoting its pathogenicity.The flavodiiron proteins (FDP, 2 originally named A-type flavoproteins (1)) are widespread among Bacteria and Archaea, either strict or facultative anaerobes, where they have been proposed to play a role in the response to nitrosative and/or oxidative stress (2, 3). A few prokaryotic FDPs have been characterized to date, namely those from the bacteria Desulfovibrio gigas (originally named rubredoxin:oxygen oxidoreductase, ROO (4 -7), and hereafter denoted FDP Dg ), Escherichia coli (named flavorubredoxin, FlRd, 3 Refs. 2, 8 -11), Desulfovibrio vulgaris (12), Moorella thermoacetica (FDP Mt , (13, 14)), and the homologous enzyme from the methanogenic archaeon Methanothermobacter marburgensis (FDP Mm , Refs. 15, 16). The FDPs contain two redox centers: a FMN, the electron entry site into the enzyme, and a non-heme Fe-Fe center, the active site (13). They are cyanide-insensitive enzymes able to catalyze the reduction of O 2 (to H 2 O) and/or NO (to N 2 O). Some of these enzymes are almost exclusively reactive toward NO (such as E. coli FlRd, Refs. 2, 9), 4 others toward O 2 (such as the M. marburgensis enzyme, (15)), whereas some FDPs catalyze the reduction of both gases, though with different efficiency (7,12,13). These enzymes are expected to play a protective role in anaerobic or microaerobic microorganisms that need to survive under O 2 and cope with NO produced by the host defense system to counteract infection (17,18).Surprisingly, a few years ago, genes coding for FDPs were identified also in the geno...