Rapid topology probing using fluorescence spectroscopy in planar lipid bilayer: the pore-forming mechanism of the toxin Cry1Aa of Bacillus thuringiensis

Pore-forming toxins, many of which are pathogenic to humans, are highly dynamic proteins that adopt a different conformation in aqueous solution than in the lipid environment of the host membrane. Consequently, their crystal structures obtained in aqueous environment do not reflect the active conformation in the membrane, making it difficult to deduce the molecular determinants responsible for pore formation. To obtain structural information directly in the membrane, we introduce a fluorescence technique to probe the native topology of pore-forming toxins in planar lipid bilayers and follow their movement during pore formation. Using a Förster resonance energy transfer (FRET) approach between site-directedly labeled proteins and an absorbing compound (dipicrylamine) in the membrane, we simultaneously recorded the electrical current and fluorescence emission in horizontal planar lipid bilayers formed in plastic chips. With this system, we mapped the topology of the pore-forming domain of Cry1Aa, a biological pesticide from Bacillus thuringiensis, by determining the location of the loops between its seven α helices. We found that the majority of the toxins initially traverse from the cis to the trans leaflet of the membrane. Comparing the topologies of Cry1Aa in the active and inactive state in order to identify the pore-forming mechanism, we established that only the α3–α4 hairpin translocates through the membrane from the trans to the cis leaflet, whereas all other positions remained constant. As toxins are highly dynamic proteins, populations that differ in conformation might be present simultaneously. To test the presence of different populations, we designed double-FRET experiments, where a single donor interacts with two acceptors with very different kinetics (dipicrylamine and oxonol). Due to the nonlinear response of FRET and the dynamic change of the acceptor distribution, we can deduce the distribution of the acceptors in the membrane from the time course of the donor fluorescence. We found that Cry1Aa is present on both membrane leaflets.     

Rescue and repair during photoreceptor cell renewal mediated by docosahexaenoic acid-derived neuroprotectin D1

Retinal degenerative diseases result in retinal pigment epithelial (RPE) and photoreceptor cell loss. These cells are continuously exposed to the environment (light) and to potentially pro-oxidative conditions, as the retina''s oxygen consumption is very high. There is also a high flux of docosahexaenoic acid (DHA), a PUFA that moves through the blood stream toward photoreceptors and between them and RPE cells. Photoreceptor outer segment shedding and phagocytosis intermittently renews photoreceptor membranes. DHA is converted through 15-lipoxygenase-1 into neuroprotectin D1 (NPD1), a potent mediator that evokes counteracting cell-protective, anti-inflammatory, pro-survival repair signaling, including the induction of anti-apoptotic proteins and inhibition of pro-apoptotic proteins. Thus, NPD1 triggers activation of signaling pathway/s that modulate/s pro-apoptotic signals, promoting cell survival. This review provides an overview of DHA in photoreceptors and describes the ability of RPE cells to synthesize NPD1 from DHA. It also describes the role of neurotrophins as agonists of NPD1 synthesis and how photoreceptor phagocytosis induces refractoriness to oxidative stress in RPE cells, with concomitant NPD1 synthesis.     

Activation of the Ghrelin Receptor is Described by a Privileged Collective Motion: A Model for Constitutive and Agonist-induced Activation of a Sub-class A G-Protein Coupled Receptor (GPCR)

Three homology models of the human ghrelin receptor (GHS-R1a) have been generated from the available X-ray structures of rhodopsin (RHO model), opsin (OPS model) and beta-2 adrenergic receptor (B2 model). The latter was used as a starting point for combined molecular dynamics simulation (MDS) and full atom normal modes analysis (NMA). A low-frequency normal mode (mode 16) perfectly reproduced the intracellular motions observed between B2 and RHO models; in the opposite direction along the same mode, the generated structures are closer to the OPS model, suggesting a direct link with GHS-R1a activation. This was in agreement with motions of the seven transmembranous segments, increase of the solvent accessibility of the 140-ERY-142 sequence, and flip of the Trp276 (C WLP) residue, some features related to GPCRs activation. According to our model, His280 was proposed to stabilize Trp276 in the active state; this was verified by site-directed mutagenesis and biochemical characterization of the resulting H280A and H280S mutants, which were fully functional but sharing an important decrease of their basal activities. Docking performed with short ghrelin derivatives Gly-Ser-Ser _[octa]-Phe-NH ₂ and Gly-Ser-Ser _[octa]-Phe-Leu-NH ₂ allowed the identification of a robust position of these peptides in the active site of the receptor. This model was refined by MDS and validated by docking experiments performed on a set of 55 ghrelin receptor ligands based on the 1,2,4- triazole scaffold. Finally, NMA performed on the obtained peptide-receptor complex suggested stabilization of the Trp276 residue and of the whole receptor in the active state, preventing the motion observed along mode 16 computed for the unbound receptor. Our results show that NMA offers a powerful approach to study the conformational diversity and the activation mechanism of GPCRs.     

Crystal Structure of Yeast FAD Synthetase (Fad1) in Complex with FAD

Flavin adenine dinucleotide (FAD) synthetase is an essential enzyme responsible for the synthesis of FAD by adenylation of riboflavin monophosphate (FMN). We have solved the 1.9 Å resolution structure of Fad1, the yeast FAD synthetase, in complex with the FAD product in the active site. The structure of Fad1 shows it to be a member of the PP-ATPase superfamily. Important conformational differences in the two motifs involved in binding the phosphate moieties of FAD compared to the Candida glabrata FMNT ortholog suggests that this loop is dynamic and undergoes substantial conformational changes during its catalytic cycle.     

THE DISTINCTIVE FOOTPRINTS OF LOCAL HITCHHIKING IN A VARIED ENVIRONMENT AND GLOBAL HITCHHIKING IN A SUBDIVIDED POPULATION

Loci with higher levels of population differentiation than the neutral expectation are traditionally interpreted as evidence of ongoing selection that varies in space. This article emphasizes an alternative explanation that has been largely overlooked to date: in species subdivided into large subpopulations, enhanced differentiation can also be the signature left by the fixation of an unconditionally favorable mutation on its chromosomal neighborhood. This is because the hitchhiking effect is expected to diminish as the favorable mutation spreads from the deme in which it originated to other demes. To discriminate among the two alternative scenarios one needs to investigate how genetic structure varies along the chromosomal region of the locus. Local hitchhiking is shown to generate a single sharp peak of differentiation centered on the adaptive polymorphism and the standard signature of a selective sweep only in those subpopulations in which the allele is favored. Global hitchhiking produces two domes of differentiation on either side of the fixed advantageous mutation and signatures of a selective sweep in every subpopulation, albeit of different magnitude. Investigating population differentiation around a locus that strongly differentiates two otherwise genetically similar populations of the marine mussel Mytilus edulis, plausible evidence for the global hitchhiking hypothesis has been obtained. Global hitchhiking is a neglected phenomenon that might prove to be important in species with large population sizes such as many marine invertebrates.     

KIN‐BASED RECOGNITION AND SOCIAL AGGREGATION IN A CILIATE

Aggregative groups entail costs that must be overcome for the evolution of complex social interactions. Understanding the mechanisms that allow aggregations to form and restrict costs of cheating can provide a resolution to the instability of social evolution. Aggregation in Tetrahymena thermophila is associated with costs of reduced growth and benefits of improved survival through “growth factor” exchange. We investigated what mechanisms contribute to stable cooperative aggregation in the face of potential exploitation by less‐cooperative lines using experimental microcosms. We found that kin recognition modulates aggregative behavior to exclude cheaters from social interactions. Long‐distance kin recognition across patches modulates social structure by allowing recruitment of kin in aggregative lines and repulsion in asocial lines. Although previous studies have shown a clear benefit to social aggregation at low population densities, we found that social aggregation has very different effects at higher densities. Lower growth rates are a cost of aggregation, but also present potential benefits when restricted to kin aggregations: slow growth and crowd tolerance allow aggregations to form and permit longer persistence on ephemeral resources. Thus in highly dynamic metapopulations, kin recognition plays an important role in the formation and stability of social groups that increase persistence through cooperative consumptive restraint.     

HETEROZYGOSITY‐FITNESS CORRELATIONS: A TIME FOR REAPPRAISAL

Owing to the remarkable progress of molecular techniques, heterozygosity‐fitness correlations (HFCs) have become a popular tool to study the impact of inbreeding in natural populations. However, their underlying mechanisms are often hotly debated. Here we argue that these “debates” rely on verbal arguments with no basis in existing theory and inappropriate statistical testing, and that it is time to reconcile HFC with its historical and theoretical fundaments. We show that available data are quantitatively and qualitatively consistent with inbreeding‐based theory. HFC can be used to estimate the impact of inbreeding in populations, although such estimates are bound to be imprecise, especially when inbreeding is weak. Contrary to common belief, linkage disequilibrium is not an alternative to inbreeding, but rather comes with some forms of inbreeding, and is not restricted to closely linked loci. Finally, the contribution of local chromosomal effects to HFC, while predicted by inbreeding theory, is expected to be small, and has rarely if ever proven statistically significant using adequate tests. We provide guidelines to safely interpret and quantify HFCs, and present how HFCs can be used to quantify inbreeding load and unravel the structure of natural populations.