Making the Right Moves

The structure of the nucleotide-free F1-ATPase from a thermoalkaliphilic bacterium presented in this issue of Structure (Stocker et al., 2007) reveals the structural interactions that prevent the enzyme from operating naturally in the hydrolytic direction. The data provide new insights into the mechanism of the FoF1-ATP synthase

An initial event in insect innate immune response: structural and biological studies of interactions between β-1,3-glucan and the N-terminal domain of β-1,3-glucan recognition protein

In response to invading microorganisms, insect β-1,3-glucan recognition protein (βGRP), a soluble receptor in the hemolymph, binds to the surfaces of bacteria and fungi and activates serine protease cascades that promote destruction of pathogens by means of melanization or expression of antimicrobial peptides. Here we report on the NMR solution structure of the N-terminal domain of βGRP (N-βGRP) from Indian meal moth (Plodia interpunctella), which is sufficient to activate the prophenoloxidase (proPO) pathway resulting in melanin formation. NMR and isothermal calorimetric titrations of N-βGRP with laminarihexaose, a glucose hexamer containing β-1,3 links, suggest a weak binding of the ligand. However, addition of laminarin, a glucose polysaccharide (~ 6 kDa) containing β-1,3 and β-1,6 links that activates the proPO pathway, to N-βGRP results in the loss of NMR cross-peaks from the backbone 15N-1H groups of the protein, suggesting the formation of a large complex. Analytical ultra centrifugation (AUC) studies of formation of N-βGRP:laminarin complex show that ligand-binding induces sel-fassociation of the protein:carbohydrate complex into a macro structure, likely containing six protein and three laminarin molecules (~ 102 kDa). The macro complex is quite stable, as it does not undergo dissociation upon dilution to sub-micromolar concentrations. The structural model thus derived from the present studies for N-βGRP:laminarin complex in solution differs from the one in which a single N-βGRP molecule has been proposed to bind to a triple helical form of laminarin on the basis of an X-ray crystallographic structure of N-βGRP:laminarihexaose complex [Kanagawa, M., Satoh, T., Ikeda, A., Adachi, Y., Ohno, N., and Yamaguchi, Y. (2011) J. Biol. Chem. 286, 29158-29165]. AUC studies and phenoloxidase activation measurements carried out with the designed mutants of N-βGRP indicate that electrostatic interactions involving Asp45, Arg54, and Asp68 between the ligand-bound protein molecules contribute in part to the stability of N-βGRP:laminarin macro complex and that a decreased stability is accompanied by a reduced activation of the proPO pathway. Increased β-1,6 branching in laminarin also results in destabilization of the macro complex. These novel findings suggest that ligand-induced self-association of βGRP:β-1,3-glucan complex may form a platform on a microbial surface for recruitment of downstream proteases, as a means of amplification of the initial signal of pathogen recognition for the activation of the proPO pathway

Structural basis of nSH2 regulation and lipid binding in PI3Kα

We report two crystal structures of the wild-type phosphatidylinositol 3-kinase α (PI3Kα) heterodimer refined to 2.9 Å and 3.4 Å resolution: the first as the free enzyme, the second in complex with the lipid substrate, diC4-PIP2, respectively. The first structure shows key interactions of the N-terminal SH2 domain (nSH2) and iSH2 with the activation loop that suggest a mechanism by which the enzyme is inhibited in its basal state. In the second structure, the lipid substrate binds in a positively charged pocket adjacent to the ATP-binding site, bordered by the P-loop, the activation loop and the iSH2 domain. An additional lipid-binding site was identified at the interface of the ABD, iSH2 and kinase domains. The ability of PI3Kα to bind an additional PIP2 molecule was confirmed in vitro by fluorescence quenching experiments. The crystal structures reveal key differences in the way the nSH2 domain interacts with wild-type p110α and with the oncogenic mutant p110αH1047R. Increased buried surface area and two unique salt-bridges observed only in the wild-type structure suggest tighter inhibition in the wild-type PI3Kα than in the oncogenic mutant. These differences may be partially responsible for the increased basal lipid kinase activity and increased membrane binding of the oncogenic mutant

Mimicking damaged DNA with a small molecule inhibitor of human UNG2

Human nuclear uracil DNA glycosylase (UNG2) is a cellular DNA repair enzyme that is essential for a number of diverse biological phenomena ranging from antibody diversification to B-cell lymphomas and type-1 human immunodeficiency virus infectivity. During each of these processes, UNG2 recognizes uracilated DNA and excises the uracil base by flipping it into the enzyme active site. We have taken advantage of the extrahelical uracil recognition mechanism to build large small-molecule libraries in which uracil is tethered via flexible alkane linkers to a collection of secondary binding elements. This high-throughput synthesis and screening approach produced two novel uracil-tethered inhibitors of UNG2, the best of which was crystallized with the enzyme. Remarkably, this inhibitor mimics the crucial hydrogen bonding and electrostatic interactions previously observed in UNG2 complexes with damaged uracilated DNA. Thus, the environment of the binding site selects for library ligands that share these DNA features. This is a general approach to rapid discovery of inhibitors of enzymes that recognize extrahelical damaged bases

Knowledge-based energy functions for computational studies of proteins

This chapter discusses theoretical framework and methods for developing knowledge-based potential functions essential for protein structure prediction, protein-protein interaction, and protein sequence design. We discuss in some details about the Miyazawa-Jernigan contact statistical potential, distance-dependent statistical potentials, as well as geometric statistical potentials. We also describe a geometric model for developing both linear and non-linear potential functions by optimization. Applications of knowledge-based potential functions in protein-decoy discrimination, in protein-protein interactions, and in protein design are then described. Several issues of knowledge-based potential functions are finally discussed.Comment: 57 pages, 6 figures. To be published in a book by Springe

Healthcare workers' perspectives and practices regarding the disclosure of HIV status to children in Malawi: A cross-sectional study

Background: In 2011 the World Health Organisation recommended that children with a diagnosis of HIV be gradually informed about their HIV status between the ages of 6 and 12 years. However, to date, literature has focused mainly on primary caregiver and child experiences with HIV disclosure, little is known about healthcare workers' perspectives and practices of HIV status disclosure to children. The aim of this study was to assess healthcare workers' perspectives and practices regarding the disclosure of HIV status to children aged between 6 and 12 years in Malawi. Methods: A cross-sectional survey was used to collect data from 168 healthcare providers working in antiretroviral clinics in all government District and Tertiary Hospitals in Malawi. Participants were asked questions regarding their knowledge, practice, and barriers to HIV disclosure. Data were analysed using binary logistic regression. Results: Almost all healthcare workers (98%) reported that it was important to disclose HIV status to children. A significant proportion (37%) reported that they had never disclosed HIV status to a child and about half estimated that the rate of HIV disclosure at their facility was 25% or less. The main barriers to disclosure were lack of training on disclosure (85%) and lack of a standard tool for disclosure (84%). Female healthcare workers (aOR) 2.4; 95% CI: 1.1-5.5) and lack of training on disclosure (aOR 7.7; 95% CI: 3.4-10.7) were independently associated with never having disclosed HIV status to a child. Conclusions: This study highlights the need for providing appropriate training in HIV disclosure for healthcare workers and the provision of standardised disclosure materials

Primary caregivers, healthcare workers, teachers and community leaders' perceptions and experiences of their involvement, practice and challenges of disclosure of HIV status to children living with HIV in Malawi: A qualitative study

Background: The World Health Organisation has recommended that healthcare workers, teachers and community leaders work with parents to support children living with HIV. The aim of this study was to assess the perceptions and experiences of primary caregivers and other care providers such as healthcare workers, teachers, and community leaders regarding their involvement, practice and challenges of HIV disclosure to children aged between 6 and 12 years living with HIV in Malawi. Methods: Twelve focus group discussions and 19 one-on-one interviews involving a total of 106 participants were conducted in all three administrative regions of Malawi. The interviews and focus group discussions explored perceptions and experiences regarding involvement, practice and challenges of disclosure of HIV status to children. Data were analysed using thematic analysis. Results: Primary caregivers, healthcare workers, teachers, and community leaders all reported that the disclosure of HIV status to children was not well coordinated because each of the groups of participants was working in isolation instead of working as a team. A "working together" model emerged from the data analysis where participants expressed the need for them to work as a team in order to promote safe and effective HIV status disclosure through talking about HIV, sharing responsibility and open communication. Participants reported that by working together, the team members would ensure that the prevalence of HIV disclosure to young children increases and that there would be a reduction in any negative impact of disclosure. Conclusion: Global resources are required to better support children living with HIV and their families. Healthcare workers and teachers would benefit greatly from training in working together with families living with HIV and, specifically, training in the disclosure process. Resources, in the form of books and other educational materials, would help them explain HIV and its effective management to children and families