Leonardo De Maria

Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus

Animals and higher plants express endogenous peptide antibiotics called defensins. These small cysteine-rich peptides are active against bacteria, fungi and viruses. Here we describe plectasin—the first defensin to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin has primary, secondary and tertiary structures that closely resemble those of defensins found in spiders, scorpions, dragonflies and mussels. Recombinant plectasin was produced at a very high, and commercially viable, yield and purity. In vitro, the recombinant peptide was especially active against Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin showed extremely low toxicity in mice, and cured them of experimental peritonitis and pneumonia caused by S. pneumoniae as efficaciously as vancomycin and penicillin. These findings identify fungi as a novel source of antimicrobial defensins, and show the therapeutic potential of plectasin. They also suggest that the defensins of insects, molluscs and fungi arose from a common ancestral gene.

Assessment of blind predictions of protein–protein interactions: Current status of docking methods

The current status of docking procedures for predicting protein–protein interactions starting from their three‐dimensional structure is assessed from a first major evaluation of blind predictions. This evaluation was performed as part of a communitywide experiment on Critical Assessment of PRedicted Interactions (CAPRI). Seven newly determined structures of protein–protein complexes were available as targets for this experiment. These were the complexes between a kinase and its protein substrate, between a T‐cell receptor β‐chain and a superantigen, and five antigen‐antibody complexes. For each target, the predictors were given the experimental structures of the free components, or of one free and one bound component in a random orientation. The structure of the complex was revealed only at the time of the evaluation. A total of 465 predictions submitted by 19 groups were evaluated. These groups used a wide range of algorithms and scoring functions, some of which were completely novel. The quality of the predicted interactions was evaluated by comparing residue–residue contacts and interface residues to those in the X‐ray structures and by analyzing the fit of the ligand molecules (the smaller of the two proteins in the complex) or of interface residues only, in the predicted versus target complexes. A total of 14 groups produced predictions, ranking from acceptable to highly accurate for five of the seven targets. The use of available biochemical and biological information, and in one instance structural information, played a key role in achieving this result. It was essential for identifying the native binding modes for the five correctly predicted targets, including the kinase‐substrate complex where the enzyme changes conformation on association. But it was also the cause for missing the correct solution for the two remaining unpredicted targets, which involve unexpected antigen‐antibody binding modes. Overall, this analysis reveals genuine progress in docking procedures but also illustrates the remaining serious limitations and points out the need for better scoring functions and more effective ways for handling conformational flexibility. Proteins 2003;52:51–67.

Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme’s active site yields insights into the mechanism of action of this important class of enzymes.

Understanding the plasticity of the alpha/beta hydrolase fold: lid swapping on the Candida antarctica lipase B results in chimeras with interesting biocatalytic properties.

The best of both worlds. Long molecular dynamics (MD) simulations of Candida antarctica lipase B (CALB) confirmed the function of helix α5 as a lid structure. Replacement of the helix with corresponding lid regions from CALB homologues from Neurospora crassa and Gibberella zeae resulted in new CALB chimeras with novel biocatalytic properties. The figure shows a snapshot from the MD simulation.

Structural and Mutational Analyses of the Interaction between the Barley alpha-Amylase/Subtilisin Inhibitor and the Subtilisin Savinase Reveal a Novel Mode of Inhibition

Subtilisins represent a large class of microbial serine proteases. To date, there are three-dimensional structures of proteinaceous inhibitors from three families in complex with subtilisins in the Protein Data Bank. All interact with subtilisin via an exposed loop covering six interacting residues. Here we present the crystal structure of the complex between the Bacillus lentus subtilisin Savinase and the barley alpha-amylase/subtilisin inhibitor (BASI). This is the first reported structure of a cereal Kunitz-P family inhibitor in complex with a subtilisin. Structural analysis revealed that BASI inhibits Savinase in a novel way, as the interacting loop is shorter than loops previously reported. Mutational analysis showed that Thr88 is crucial for the inhibition, as it stabilises the interacting loop through intramolecular interactions with the BASI backbone.

Between-Species Variation in the Kinetic Stability of TIM Proteins Linked to Solvation-Barrier Free Energies

Theoretical, computational, and experimental studies have suggested the existence of solvation barriers in protein unfolding and denaturation processes. These barriers are related to the finite size of water molecules and can be envisioned as arising from the asynchrony between water penetration and breakup of internal interactions. Solvation barriers have been proposed to play roles in protein cooperativity and kinetic stability; therefore, they may be expected to be subject to natural selection. We study the thermal denaturation, in the presence and in the absence of chemical denaturants, of triosephosphate isomerases (TIMs) from three different species: Trypanosoma cruzi, Trypanosoma brucei, and Leishmania mexicana. In all cases, denaturation was irreversible and kinetically controlled. Surprisingly, however, we found large differences between the kinetic denaturation parameters, with T. cruzi TIM showing a much larger activation energy value (and, consequently, much lower room-temperature, extrapolated denaturation rates). This disparity cannot be accounted for by variations in the degree of exposure to solvent in transition states (as measured by kinetic urea m values) and is, therefore, to be attributed mainly to differences in solvation-barrier contributions. This was supported by structure-energetics analyses of the transition states and by application of a novel procedure to estimate from experimental data the solvation-barrier impact at the entropy and free-energy levels. These analyses were actually performed with an extended protein set (including six small proteins plus seven variants of lipase from Thermomyces lanuginosus and spanning a wide range of activation parameters), allowing us to delineate the general trends of the solvation-barrier contributions. Overall, this work supports that proteins sharing the same structure and function but belonging to different organisms may show widely different solvation barriers, possibly as a result of different levels of the selection pressure associated with cooperativity, kinetic stability, and related factors.

A structural overview of GH61 proteins - fungal cellulose degrading polysaccharide monooxygenases.

Recent years have witnessed a spurt of activities in the elucidation of the molecular function of a class of proteins with great potential in biomass degradation. GH61 proteins are of fungal origin and were originally classified in family 61 of the glycoside hydrolases. From the beginning they were strongly suspected to be involved in cellulose degradation because of their expression profiles, despite very low detectable endoglucanase activities. A major breakthrough came from structure determination of the first members, establishing the presence of a divalent metal binding site and a similarity to bacterial proteins involved in chitin degradation. A second breakthrough came from the identification of cellulase boosting activity dependent on the integrity of the metal binding site. Finally very recently GH61 proteins were demonstrated to oxidatively cleave crystalline cellulose in a Cu and reductant dependant manner. This mini-review in particular focuses on the contribution that structure elucidation has made in the understanding of GH61 molecular function and reviews the currently known structures and the challenges remaining ahead for exploiting this new class of enzymes to the full.

Mechanism of Protein Kinetic Stabilization by Engineered Disulfide Crosslinks

The impact of disulfide bonds on protein stability goes beyond simple equilibrium thermodynamics effects associated with the conformational entropy of the unfolded state. Indeed, disulfide crosslinks may play a role in the prevention of dysfunctional association and strongly affect the rates of irreversible enzyme inactivation, highly relevant in biotechnological applications. While these kinetic-stability effects remain poorly understood, by analogy with proposed mechanisms for processes of protein aggregation and fibrillogenesis, we propose that they may be determined by the properties of sparsely-populated, partially-unfolded intermediates. Here we report the successful design, on the basis of high temperature molecular-dynamics simulations, of six thermodynamically and kinetically stabilized variants of phytase from Citrobacter braakii (a biotechnologically important enzyme) with one, two or three engineered disulfides. Activity measurements and 3D crystal structure determination demonstrate that the engineered crosslinks do not cause dramatic alterations in the native structure. The inactivation kinetics for all the variants displays a strongly non-Arrhenius temperature dependence, with the time-scale for the irreversible denaturation process reaching a minimum at a given temperature within the range of the denaturation transition. We show this striking feature to be a signature of a key role played by a partially unfolded, intermediate state/ensemble. Energetic and mutational analyses confirm that the intermediate is highly unfolded (akin to a proposed critical intermediate in the misfolding of the prion protein), a result that explains the observed kinetic stabilization. Our results provide a rationale for the kinetic-stability consequences of disulfide-crosslink engineering and an experimental methodology to arrive at energetic/structural descriptions of the sparsely populated and elusive intermediates that play key roles in irreversible protein denaturation.

Optimization of a small laccase by active-site redesign.

Small but faster: A small laccase from Streptomyces coelicolor (SLAC) has been engineered by structure-based design and site-directed mutagenesis to improve the activity on commercially relevant substrates. The variants generated showed up to 40-fold increased efficiency on 2,6-dimethoxyphenol and the ability to use mediators with considerably higher redox potentials (methylsyringate and TEMPO).

Beyond Lumry–Eyring: An unexpected pattern of operational reversibility/irreversibility in protein denaturation

We have found that, contrary to naïve intuition, the degree of operational reversibility in the thermal denaturation of lipase from Thermomyces lanuginosa (an important industrial enzyme) in urea solutions is maximum when the protein is heated several degrees above the end of the temperature‐induced denaturation transition. Upon cooling to room temperature, the protein seems to reach a state with enzymatic activity similar to that of the initial native state, but with higher denaturation temperature and radically different behavior in terms of susceptibility to irreversible denaturation. These results show that patterns of operational reversibility/irreversibility in protein denaturation may be more complex than the often‐taken‐for‐granted, two‐situation classification (reversible vs. irreversible). Furthermore, they are consistent with the possibility of existence of different native or native‐like states separated by high kinetic barriers under native conditions and they suggest experimental procedures to reach and study such “alternative” native states. Proteins 2008.