On the origin of the stereoselective affinity of Nutlin-3 geometrical isomers for the MDM2 protein

The stereoselective affinity of small-molecule binding to proteins is typically broadly explained in terms of the thermodynamics of the final bound complex. Using Brownian dynamics simulations, we show that the preferential binding of the MDM2 protein to the geometrical isomers of Nutlin-3, an effective anticancer lead that works by inhibiting the interaction between the proteins p53 and MDM2, can be explained by kinetic arguments related to the formation of the MDM2:Nutlin-3 encounter complex. This is a diffusively bound state that forms prior to the final bound complex. We find that the MDM2 protein stereoselectivity for the Nutlin-3a enantiomer stems largely from the destabilization of the encounter complex of its mirror image enantiomer Nutlin-3b, by the K70 residue that is located away from the binding site. On the other hand, the trans-Nutlin-3a diastereoisomer exhibits a shorter residence time in the vicinity of MDM2 compared with Nutlin-3a due to destabilization of its encounter complex by the collective interaction of pairs of charged residues on either side of the binding site: Glu25 and Lys51 on one side, and Lys94 and Arg97 on the other side. This destabilization is largely due to the electrostatic potential of the trans-Nutlin-3a isomer being largely positive over extended continuous regions around its structure, which are otherwise well-identified into positive and negative regions in the case of the Nutlin-3a isomer. Such rich insight into the binding processes underlying biological selectivity complements the static view derived from the traditional thermodynamic analysis of the final bound complex. This approach, based on an explicit consideration of the dynamics of molecular association, suggests new avenues for kinetics-based anticancer drug development and discovery.     

Rapid Evolution of Virus Sequences in Intrinsically Disordered Protein Regions

Nodamura Virus (NoV) is a nodavirus originally isolated from insects that can replicate in a wide variety of hosts, including mammals. Because of their simplicity and ability to replicate in many diverse hosts, NoV, and the Nodaviridae in general, provide a unique window into the evolution of viruses and host-virus interactions. Here we show that the C-terminus of the viral polymerase exhibits extreme structural and evolutionary flexibility. Indeed, fewer than 10 positively charged residues from the 110 amino acid-long C-terminal region of protein A are required to support RNA1 replication. Strikingly, this region can be replaced by completely unrelated protein sequences, yet still produce a functional replicase. Structure predictions, as well as evolutionary and mutational analyses, indicate that the C-terminal region is structurally disordered and evolves faster than the rest of the viral proteome. Thus, the function of an intrinsically unstructured protein region can be independent of most of its primary sequence, conferring both functional robustness and sequence plasticity on the protein. Our results provide an experimental explanation for rapid evolution of unstructured regions, which enables an effective exploration of the sequence space, and likely function space, available to the virus.     

Effect of boundary type and season on predatory arthropods associated with field margins on New Zealand farmland

Pitfall traps were used to monitor predatory arthropod numbers along two types of field boundary, a post and wire fence line and a Cupressus macrocarpa hedge, along the same paddock margin in Canterbury, New Zealand, over 24 months. The seven most abundant predator groups recorded were: Araneae > Phalangiidae > Staphylinidae > Coccinellidae > Chilopoda > Hemerobiidae > Carabidae. Araneae, Phalangiidae, Staphylinidae, Chilopoda and Hemerobiidae were found in larger numbers at the wire fence than at the hedge site, whereas the numbers of Carabidae and Coccinellidae adults exhibited no field margin preference. However, more species of Araneae and Staphylinidae were caught at the hedge site, whereas species richness of carabid beetles was greatest at the wire fence. Principal component analysis clearly separated the samples collected from the two habitats based on the assemblages of Araneae, Staphylinidae and Carabidae, and certain species in each of these taxonomic groups appeared to be particularly associated with one boundary type or the other. All the main taxonomic groups exhibited clear seasonal patterns, with distinct peaks in abundance occurring at certain times of the year. The results of the study reinforce the idea that management of field boundaries can be used to manipulate the type and abundance of particular groups of predatory arthropods, and that seasonal patterns should be taken into account in schemes of integrated pest management so that any adverse effects of biocide application on these beneficial species may be minimised.     

Atp7a determines a hierarchy of copper metabolism essential for notochord development

The critical developmental and genetic requirements of copper metabolism during embryogenesis are unknown. Utilizing a chemical genetic screen in zebrafish, we identified small molecules that perturb copper homeostasis. Our findings reveal a role for copper in notochord formation and demonstrate a hierarchy of copper metabolism within the embryo. To elucidate these observations, we interrogated a genetic screen for embryos phenocopied by copper deficiency, identifying calamity, a mutant defective in the zebrafish ortholog of the Menkes disease gene (atp7a). Copper metabolism in calamity is restored by human ATP7A, and transplantation experiments reveal that atp7a functions cell autonomously, findings with important therapeutic implications. The gene dosage of atp7a determines the sensitivity to copper deprivation, revealing that the observed developmental hierarchy of copper metabolism is informed by specific genetic factors. Our data provide insight into the developmental pathophysiology of copper metabolism and suggest that suboptimal copper metabolism may contribute to birth defects.     

Isolation of two populations of sperm cells and microelectrophoresis of pairs of sperm cells from pollen tubes of tobacco (Nicotiana tabacum)

Prior research has indicated that the two sperm cells of Nicotiana tabacum are dimorphic, suggesting that they may participate in preferential fertilization during in vivo fusion with the egg and central cells. To probe the mechanism of potential preferential fertilization in this plant, it will be necessary to use modern sensitive molecular techniques. For this purpose, two individual populations of two sperm cells, constituting the Svn (associated with the vegetative nucleus) and Sua (unassociated with the vegetative nucleus), were isolated in the thousands from tobacco pollen tubes with a micromanipulator as a preliminary step toward research on gametic recognition using molecular techniques. Microelectrophoresis of paired sperm cells from a single pollen tube was conducted at different developmental stages. Sperm cells isolated from 1-, 2-, 3- and 4-cm stylar lengths migrated to the negative pole, with the Sua displaying significantly greater electrophoretic mobility than the Svn, reflecting a more positively charged cell surface on the Sua. The sperm cells isolated from 1-cm style are very sensitive to electron potential in an electrophoretic field, presumably reflecting that they are still in a young state. Differences in cell surface charge between the Sua and Svn may be related with cell fate during fertilization. Supported by National Natural Science Foundation of CHINA (30170060)     

MDA-5 recognition of a murine norovirus

Noroviruses are important human pathogens responsible for most cases of viral epidemic gastroenteritis worldwide. Murine norovirus-1 (MNV-1) is one of several murine noroviruses isolated from research mouse facilities and has been used as a model of human norovirus infection. MNV-1 infection has been shown to require components of innate and adaptive immunity for clearance; however, the initial host protein that recognizes MNV-1 infection is unknown. Because noroviruses are RNA viruses, we investigated whether MDA5 and TLR3, cellular sensors that recognize dsRNA, are important for the host response to MNV-1. We demonstrate that MDA5-/- dendritic cells(DC) have a defect in cytokine response to MNV-1. In addition, MNV-1 replicates to higher levels in MDA5-/- DCs as well as in MDA5-/- mice in vivo. Interestingly, TLR3-/- DCs do not have a defect in vitro, but TLR3-/- mice have a slight increase in viral titers. This is the first demonstration of an innate immune sensor for norovirus and shows that MDA5 is required for the control of MNV-1 infection. Knowledge of the host response to MNV-1 may provide keys for prevention and treatment of the human disease.     

Studies on the biotin-binding sites of avidin and streptavidin. A chemically induced dynamic nuclear polarization investigation of the status of tyrosine residues.

We applied the protein photochemically induced dynamic nuclear polarization (photo-c.i.d.n.p.) method to explore the conformation of the side chains of tyrosine, tryptophan and histidine residues in three biotin-binding proteins. The c.i.d.n.p. spectra of avidin, streptavidin and 'core' streptavidin were compared with those of their complexes with biotin and its derivatives. The data indicate that the single tyrosine residue (Tyr-33) of avidin is clearly inaccessible to the triplet flavin photo-c.i.d.n.p. probe. The same holds for all tryptophan and histidine side chains. Although the analogous Tyr-43 residue of streptavidin is also buried, at least three of the other tyrosine residues of this protein are exposed. The same conclusions apply to the truncated form of the protein, core streptavidin. As judged by the photo-c.i.d.n.p. results, complexing of avidin and streptavidin with biotin, N-epsilon-biotinyl-L-lysine (biocytin) or biotinyltyrosine has little or no effect on tyrosine accessibility in these proteins. Biotinyltyrosine can be used to probe the depth of the corresponding binding site. The accessibility of the tyrosine side chain of biotinyltyrosine in the complex demonstrates the exquisite fit of the biotin-binding cleft of avidin: only the biotin moiety appears to be accommodated, leaving the tyrosine side chain exposed.