The structural dynamics of the flavivirus fusion peptide-membrane interaction.

Membrane fusion is a crucial step in flavivirus infections and a potential target for antiviral strategies. Lipids and proteins play cooperative roles in the fusion process, which is triggered by the acidic pH inside the endosome. This acidic environment induces many changes in glycoprotein conformation and allows the action of a highly conserved hydrophobic sequence, the fusion peptide (FP). Despite the large volume of information available on the virus-triggered fusion process, little is known regarding the mechanisms behind flavivirus-cell membrane fusion. Here, we evaluated the contribution of a natural single amino acid difference on two flavivirus FPs, FLA(G) ((98)DRGWGNGCGLFGK(110)) and FLA(H) ((98)DRGWGNHCGLFGK(110)), and investigated the role of the charge of the target membrane on the fusion process. We used an in silico approach to simulate the interaction of the FPs with a lipid bilayer in a complementary way and used spectroscopic approaches to collect conformation information. We found that both peptides interact with neutral and anionic micelles, and molecular dynamics (MD) simulations showed the interaction of the FPs with the lipid bilayer. The participation of the indole ring of Trp appeared to be important for the anchoring of both peptides in the membrane model, as indicated by MD simulations and spectroscopic analyses. Mild differences between FLA(G) and FLA(H) were observed according to the pH and the charge of the target membrane model. The MD simulations of the membrane showed that both peptides adopted a bend structure, and an interaction between the aromatic residues was strongly suggested, which was also observed by circular dichroism in the presence of micelles. As the FPs of viral fusion proteins play a key role in the mechanism of viral fusion, understanding the interactions between peptides and membranes is crucial for medical science and biology and may contribute to the design of new antiviral drugs.

Moniliophthora perniciosa necrosis- and ethylene-inducing protein 2 (MpNep2) as a metastable dimer in solution: structural and functional implications.

Understanding how Nep-like proteins (NLPs) behave during the cell cycle and disease progression of plant pathogenic oomycetes, fungi and bacteria is crucial in light of compelling evidence that these proteins play a role in Witches` Broom Disease (WBD) of Theobroma cacao, one of the most important phytopathological problems to afflict the Southern Hemisphere. The crystal structure of MpNep2, a member of the NLP family and the causal agent of WBD, revealed the key elements for its activity. This protein has the ability to refold after heating and was believed to act as a monomer in solution, in contrast to the related homologs MpNep1 and NPP from the oomyceteous fungus Phytophthora parasitica. Here, we identify and characterize a metastable MpNep2 dimer upon over-expression in Escherichia coli using different biochemical and structural approaches. We found using ultra-fast liquid chromatography that the MpNep2 dimer can be dissociated by heating but not by dilution, oxidation or high ionic strength. Small-angle X-ray scattering revealed a possible tail-to-tail interaction between monomers, and nuclear magnetic resonance measurements identified perturbed residues involved in the putative interface of interaction. We also explored the ability of the MpNep2 monomer to refold after heating or chemical denaturation. We observed that MpNep2 has a low stability and cooperative fold that could be an explanation for its structure and activity recovery after stress. These results can provide new insights into the mechanism for MpNep2's action in dicot plants during the progression of WBD and may open new avenues for the involvement of NLP- oligomeric species in phytopathological disorders.

The proapoptotic protein Smac/DIABLO dimer has the highest stability as measured by pressure and urea denaturation.

Apoptosis is an essential mechanism of cell death required for normal development and homeostasis of all multicellular organisms. Smac/DIABLO is a dimeric protein important in the control of apoptosis by removing the inhibitory activity of IAPs (inhibitor of apoptosis proteins). In vitro studies reveal that dimerization is required for its function. Here we investigate the structural and thermodynamic features of folding and dimerization of Smac/DIABLO. To disturb the folded, dimeric structure, we used high hydrostatic pressure, low and high temperatures, and chemical denaturing agents. Conformational changes were monitored using spectroscopic techniques such as fluorescence and circular dichroism (CD) as well as gel filtration chromatography. Our data show that Smac/DIABLO is very stable under pressures up to 3.1 kbar, even at subzero temperatures. A complete denaturation/dissociation process is obtained when we use high concentrations of urea, which affect its secondary structure as assessed by CD. The association of pressure and subdenaturing urea concentrations also results in complete denaturation/dissociation of the protein. Under these conditions, unfolding of the protein shows concentration dependence that is in accordance with the dimer-monomer dissociation equilibrium, confirming Smac/DIABLO dissociation. These results suggest that most of the treatments lead to a reversible disruption of the dimeric structure with a dissociation constant ( K d) of 34 x 10 (-21) M (34 zM). This tight dimer is biologically relevant, considering that monomeric mutants bind IAP with low affinity. The extremely high stability of the dimeric form of Smac/DIABLO also implies that once expressed in the cell the protein has a low probability of dissociation and, consequently, loss of function. In addition, the stability in the zeptomolar range is the highest so far measured for a dimeric protein. It also indicates that under most circumstances Smac/DIABLO does not exist as a monomer in the cell and suggests that the dimer-to-monomer equilibrium does not play a regulatory role in the Smac/DIABLO-IAP interaction.

Dissecting the role of protein-protein and protein-nucleic acid interactions in MS2 bacteriophage stability.

To investigate the role of protein-protein and protein-nucleic acid interactions in virus assembly, we compared the stabilities of native bacteriophage MS2, virus-like particles (VLPs) containing nonviral RNAs, and an assembly-defective coat protein mutant (dlFG) and its single-chain variant (sc-dlFG). Physical (high pressure) and chemical (urea and guanidine hydrochloride) agents were used to promote virus disassembly and protein denaturation, and the changes in virus and protein structure were monitored by measuring tryptophan intrinsic fluorescence, bis-ANS probe fluorescence, and light scattering. We found that VLPs dissociate into capsid proteins that remain folded and more stable than the proteins dissociated from authentic particles. The proposed model is that the capsid disassembles but the protein remains bound to the heterologous RNA encased by VLPs. The dlFG dimerizes correctly, but fails to assemble into capsids, because it lacks the 15-amino acid FG loop involved in inter-dimer interactions at the viral fivefold and quasi-sixfold axes. This protein was very unstable and, when compared with the dissociation/denaturation of the VLPs and the wild-type virus, it was much more susceptible to chemical and physical perturbation. Genetic fusion of the two subunits of the dimer in the single-chain dimer sc-dlFG stabilized the protein, as did the presence of 34-bp poly(GC) DNA. These studies reveal mechanisms by which interactions in the capsid lattice can be sufficiently stable and specific to ensure assembly, and they shed light on the processes that lead to the formation of infectious viral particles.