Combining Electron Microscopy (EM) and Cross-Linking Mass Spectrometry (XL-MS) for Structural Characterization of Protein Complexes.

Structural biology has recently witnessed the benefits of the combined use of two complementary techniques: electron microscopy (EM) and cross-linking mass spectrometry (XL-MS). EM (especially its cryogenic variant cryo-EM) has proven to be a very powerful tool for the structural determination of proteins and protein complexes, even at an atomic level. In a complementary way, XL-MS allows the precise characterization of particular interactions when residues are located in close proximity. When working from low-resolution, negative-staining images and less-defined regions of flexible domains (whose mapping is made possible by cryo-EM), XL-MS can provide critical information on specific amino acids, thus identifying interacting regions and helping to deduce the overall protein structure. The protocol described here is particularly well suited for the study of protein complexes whose intrinsically flexible or transient nature prevents their high-resolution characterization by any structural technique itself.

The Molecular Chaperone CCT Sequesters Gelsolin and Protects it from Cleavage by Caspase-3.

The actin filament severing and capping protein gelsolin plays an important role in modulation of actin filament dynamics by influencing the number of actin filament ends. During apoptosis, gelsolin becomes constitutively active due to cleavage by caspase-3. In non-apoptotic cells gelsolin is activated by the binding of Ca2+. This activated form of gelsolin binds to, but is not a folding substrate of the molecular chaperone CCT/TRiC. Here we demonstrate that in vitro, gelsolin is protected from cleavage by caspase-3 in the presence of CCT. Cryoelectron microscopy and single particle 3D reconstruction of the CCT:gelsolin complex reveals that gelsolin is located in the interior of the chaperonin cavity, with a placement distinct from that of the obligate CCT folding substrates actin and tubulin. In cultured mouse melanoma B16F1 cells, gelsolin co-localises with CCT upon stimulation of actin dynamics at peripheral regions during lamellipodia formation. These data indicate that localised sequestration of gelsolin by CCT may provide spatial control of actin filament dynamics.

Structure of Fungal α Mating Pheromone in Membrane Mimetics Suggests a Possible Role for Regulation at the Water-Membrane Interface.

Fusarium oxysporum is a highly destructive plant pathogen and an emerging pathogen of humans. Like other ascomycete fungi, F. oxysporum secretes α-pheromone, a small peptide that functions both as a chemoattractant and as a quorum-sensing signal. Three of the ten amino acid residues of α-pheromone are tryptophan, an amino acid whose sidechain has high affinity for lipid bilayers, suggesting a possible interaction with biological membranes. Here we tested the effect of different lipid environments on α-pheromone structure and function. Using spectroscopic and calorimetric approaches, we show that this peptide interacts with negatively charged model phospholipid vesicles. Fluorescence emission spectroscopy and nuclear magnetic resonance (NMR) measurements revealed a key role of the positively charged groups and Trp residues. Furthermore, NMR-based calculation of the 3D structure in the presence of micelles, formed by lipid surfactants, suggests that α-pheromone can establish an intramolecular disulfide bond between the two cysteine residues during interaction with membranes, but not in the absence of lipid mimetics. Remarkably, this oxidized version of α-pheromone lacks biological activity as a chemoattractant and quorum-sensing molecule. These results suggest the presence of a previously unidentified redox regulated control of α-pheromone activity at the surface of the plasma membrane that could influence the interaction with its cognate G-protein coupled receptor.

Minimized natural versions of fungal ribotoxins show improved active site plasticity.

Fungal ribotoxins are highly specific extracellular RNases which cleave a single phosphodiester bond at the ribosomal sarcin-ricin loop, inhibiting protein biosynthesis by interfering with elongation factors. Most ribotoxins show high degree of conservation, with similar sizes and amino acid sequence identities above 85%. Only two exceptions are known: hirsutellin A and anisoplin, produced by the entomopathogenic fungi Hirsutella thompsonii and Metarhizium anisopliae, respectively. Both proteins are similar but smaller than the other known ribotoxins (130 vs 150 amino acids), displaying only about 25% sequence identity with them. They can be considered minimized natural versions of their larger counterparts, best represented by α-sarcin. The conserved α-sarcin active site residue Tyr48 has been replaced by the geometrically equivalent Asp, present in the minimized ribotoxins, to produce and characterize the corresponding mutant. As a control, the inverse anisoplin mutant (D43Y) has been also studied. The results show how the smaller versions of ribotoxins represent an optimum compromise among conformational freedom, stability, specificity, and active-site plasticity which allow these toxic proteins to accommodate the characteristic abilities of ribotoxins into a shorter amino acid sequence and more stable structure of intermediate size between that of other nontoxic fungal RNases and previously known larger ribotoxins.

Fungal Ribotoxins: A Review of Potential Biotechnological Applications.

Fungi establish a complex network of biological interactions with other organisms in nature. In many cases, these involve the production of toxins for survival or colonization purposes. Among these toxins, ribotoxins stand out as promising candidates for their use in biotechnological applications. They constitute a group of highly specific extracellular ribonucleases that target a universally conserved sequence of RNA in the ribosome, the sarcin-ricin loop. The detailed molecular study of this family of toxic proteins over the past decades has highlighted their potential in applied research. Remarkable examples would be the recent studies in the field of cancer research with promising results involving ribotoxin-based immunotoxins. On the other hand, some ribotoxin-producer fungi have already been studied in the control of insect pests. The recent role of ribotoxins as insecticides could allow their employment in formulas and even as baculovirus-based biopesticides. Moreover, considering the important role of their target in the ribosome, they can be used as tools to study how ribosome biogenesis is regulated and, eventually, may contribute to a better understanding of some ribosomopathies.