Hotspot-centric de novo design of protein binders

Protein-protein interactions play critical roles in biology, and computational design of interactions could be useful in a range of applications. We describe in detail a general approach to de novo design of protein interactions based on computed, energetically optimized interaction hotspots, which was recently used to produce high-affinity binders of influenza hemagglutinin. We present several alternative approaches to identify and build the key hotspot interactions within both core secondary structural elements and variable loop regions and evaluate the method's performance in natural-interface recapitulation. We show that the method generates binding surfaces that are more conformationally restricted than previous design methods, reducing opportunities for off-target interactions.     

Quasi-symmetry in the cryo-EM structure of EmrE provides the key to modeling its transmembrane domain

Small multidrug resistance (SMR) transporters contribute to bacterial resistance by coupling the efflux of a wide range of toxic aromatic cations, some of which are commonly used as antibiotics and antiseptics, to proton influx. EmrE is a prototypical small multidrug resistance transporter comprising four transmembrane segments (M1-M4) that forms dimers. It was suggested recently that EmrE molecules in the dimer have different topologies, i.e. monomers have opposite orientations with respect to the membrane plane. A 3-D structure of EmrE acquired by electron cryo-microscopy (cryo-EM) at 7.5 Angstroms resolution in the membrane plane showed that parts of the structure are related by quasi-symmetry. We used this symmetry relationship, combined with sequence conservation data, to assign the transmembrane segments in EmrE to the densities seen in the cryo-EM structure. A C alpha model of the transmembrane region was constructed by considering the evolutionary conservation pattern of each helix. The model is validated by much of the biochemical data on EmrE with most of the positions that were identified as affecting substrate translocation being located around the substrate-binding cavity. A suggested mechanism for proton-coupled substrate translocation in small multidrug resistance antiporters provides a mechanistic rationale to the experimentally observed inverted topology.     

Fluorescent polycyclic ligands for nitric oxide synthase (NOS) inhibition

In recent years polycyclic compounds have been shown to exhibit pharmacological profiles of importance in the symptomatic and proposed curative treatment of neurodegenerative diseases (e.g., Parkinson's and Alzheimer's disease). These structures also show modification and improvement of the pharmacokinetic and pharmacodynamic properties of drugs in current use. Nitric oxide (NO) is a molecular messenger involved in a number of physiological processes in mammals. It is synthesised by nitric oxide synthase (NOS) from L-arginine and its overproduction could lead to a number of neurological disorders. The aim of this study was to synthesise a series of novel indazole, indole and other fluorescent derivatives conjugated to polycyclic structures for evaluation in NOS assays. NOS is a target system where fluorescent techniques and fluorescently labelled NOS inhibitors can be used for detecting the biophysical properties of enzyme-ligand interactions and thus facilitate development of novel inhibitors of neurodegeneration. This could lead to a greater insight into the neuroprotective mechanism and a possible cure/treatment for neurodegenerative diseases. A series of compounds incorporating polycyclic structures such as 3-hydroxy-4-aza-8-oxoheptacyclo[9.4.1.0.(2,10)0.(3,14)0.(4,9)0.(9,13)0(12,15)]tetradecane and amantadine as well as suitable fluorescent moieties were selected for synthesis. In the biological evaluation the oxyhaemoglobin (oxyHb) assay was employed to determine the activity of the novel compounds at an enzymatic level of NOS. IC(50) values of the novel fluorescent compounds were compared to that of aminoguanidine (AG) and 7-nitroindazole (7-NI), two known NOS inhibitors, and showed moderate to high affinity (IC(50) values ranging from 7.73 microM to 0.291 microM) for the NOS enzyme.     

Incorporating an allosteric regulatory site in an antibody through backbone design

Allosteric regulation underlies living cells' ability to sense changes in nutrient and signaling‐molecule concentrations, but the ability to computationally design allosteric regulation into non‐allosteric proteins has been elusive. Allosteric‐site design is complicated by the requirement to encode the relative stabilities of active and inactive conformations of the same protein in the presence and absence of both ligand and effector. To address this challenge, we used Rosetta to design the backbone of the flexible heavy‐chain complementarity‐determining region 3 (HCDR3), and used geometric matching and sequence optimization to place a Zn²⁺‐coordination site in a fluorescein‐binding antibody. We predicted that due to HCDR3's flexibility, the fluorescein‐binding pocket would configure properly only upon Zn²⁺ application. We found that regulation by Zn²⁺ was reversible and sensitive to the divalent ion's identity, and came at the cost of reduced antibody stability and fluorescein‐binding affinity. Fluorescein bound at an order of magnitude higher affinity in the presence of Zn²⁺ than in its absence, and the increase in fluorescein affinity was due almost entirely to faster fluorescein on‐rate, suggesting that Zn²⁺ preorganized the antibody for fluorescein binding. Mutation analysis demonstrated the extreme sensitivity of Zn²⁺ regulation on the atomic details in and around the metal‐coordination site. The designed antibody could serve to study how allosteric regulation evolved from non‐allosteric binding proteins, and suggests a way to designing molecular sensors for environmental and biomedical targets.     

Influence of socioeconomic status on design of Batswana home gardens and associated plant diversity patterns in northern South Africa

Over the last two decades, home garden studies have markedly increased in both developed and developing countries. However, garden design and its influence on the overall biodiversity of the urban green infrastructure remains a neglected aspect of home garden research. Home garden surveys were conducted in the North West and Gauteng Provinces of South Africa to contribute to this research focus. The two questions asked in this paper were: (1) Are Batswana garden designs associated with socioeconomic status (SES)? (2) Are the different garden designs characterized by specific plant species richness patterns? We hypothesized that SES influences garden design and that, as the SES of Batswana residents increases, the garden design changes from tshimo to colonial. Our results indicated that garden design reflected less cultural influences and took on a more Westernized colonial design appearance with improvement of SES of Batswana inhabitants. Tshimo gardens tended to have more native and utilitarian species. In contrast, colonial gardens have more alien ornamental species. In affluent areas, sampled Batswana gardens completely changed from a tshimo to colonial garden design. This change indicates that improved socioeconomic status overrides traditional cultural practices.     

The activity of p-methoxybenzylisothiocyanate against Neisseria gonorrhoeae,Haemophilus ducreyi,and other microorganisms

p-Methoxybenzylisothiocyanate was isolated from Lepidium bonariense and found to be responsible for the plants antimicrobial and STD activity. MIC determinations were conducted for p-methoxybenzylisothiocyanate on Haemophilus ducreyi, Neisseria gonorrheae, Candida albicans, Bacillus subtilus, Micrococcus luteus, Staphylococcus aureus, Enterobacter sp., Escherichia coli, Klebsiella pneumoniae, and Psuedomanas aeruginosa. An in vitro cellular toxicity assay showed that at 100 microM (17,9 microg/mL) p-methoxybenzylisothiocyanate is not toxic to living cells.     

Mast cell costimulation by CD226/CD112 (DNAM-1/Nectin-2): a novel interface in the allergic process

Mast cells have critical effector functions in various immune reactions. In allergic inflammation, mast cells interact with tissue-infiltrating eosinophils, forming a regulatory unit in the late and chronic phases of the allergic process. However, the pathways and molecules within this unit are still largely undefined. Here, we show that human mast cells and eosinophils express DNAX accessory molecule 1 (DNAM-1, CD226) and its ligand Nectin-2 (CD112). CD226 synergizes with FcepsilonRI on mast cells, and its engagement augments degranulation through a pathway involving Fyn, linker of activation of T-cells, phospholipase C gamma2, and CD18. This pathway is subject to negative interference by inhibitory receptors and is completely inhibited by linking IgE with IRp60 (CD300a) using a bispecific antibody. Moreover, blocking CD112 expressed on eosinophils using neutralizing antibodies normalized the hyperactivity resulting from IgE-dependent activation of mast cells co-cultured with eosinophils. Our findings demonstrate a novel interface between these two effector cells, implicating relevance for in vivo allergic states. Moreover, costimulatory responses might be a critical component in allergic reactions and may therefore become novel targets for anti-allergic therapy.