Dynamic Diagnosis of Familial Prion Diseases Supports the β2-α2 Loop as a Universal Interference Target

[Background] Mutations in the cellular prion protein associated to familial prion disorders severely increase the likelihood of its misfolding into pathogenic conformers. Despite their postulation as incompatible elements with the native fold, these mutations rarely modify the native state structure. However they variably have impact on the thermodynamic stability and metabolism of PrPC and on the properties of PrPSc aggregates. To investigate whether the pathogenic mutations affect the dynamic properties of the HuPrP(125-229) α-fold and find possible common patterns of effects that could help in prophylaxis we performed a dynamic diagnosis of ten point substitutions.[Methodology/Principal Findings] Using all-atom molecular dynamics simulations and novel analytical tools we have explored the effect of D178N, V180I, T183A, T188K, E196K, F198S, E200K, R208H, V210I and E211Q mutations on the dynamics of HuPrP(125-228) α-fold. We have found that while preserving the native state, all mutations produce dynamic changes which perturb the coordination of the α2-α3 hairpin to the rest of the molecule and cause the reorganization of the patches for intermolecular recognition, as the disappearance of those for conversion inhibitors and the emergence of an interaction site at the β2-α2 loop region.[Conclusions/Significance] Our results suggest that pathogenic mutations share a common pattern of dynamical alterations that converge to the conversion of the β2-α2 loop into an interacting region that can be used as target for interference treatments in genetic diseases.This work was supported in parts by grants BFU2009-07971 from the MICINN (MG), FundaciÃ3n Cien (MG); Fondazione Cariplo (GC) and AIRC (GC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.Peer reviewe

Activation of PKA via asymmetric allosteric coupling of structurally conserved cyclic nucleotide binding domains

Cyclic nucleotide-binding (CNB) domains allosterically regulate the activity of proteins with diverse functions, but the mechanisms that enable the cyclic nucleotide-binding signal to regulate distant domains are not well understood. Here we use optical tweezers and molecular dynamics to dissect changes in folding energy landscape associated with cAMP-binding signals transduced between the two CNB domains of protein kinase A (PKA). We find that the response of the energy landscape upon cAMP binding is domain specific, resulting in unique but mutually coordinated tasks: one CNB domain initiates cAMP binding and cooperativity, whereas the other triggers inter-domain interactions that promote the active conformation. Inter-domain interactions occur in a stepwise manner, beginning in intermediate-liganded states between apo and cAMP-bound domains. Moreover, we identify a cAMP-responsive switch, the N3A motif, whose conformation and stability depend on cAMP occupancy. This switch serves as a signaling hub, amplifying cAMP-binding signals during PKA activation

Residual structures, conformational fluctuations, and electrostatic interactions in the synergistic folding of two intrinsically disordered proteins

To understand the interplay of residual structures and conformational fluctuations in the interaction of intrinsically disordered proteins (IDPs), we first combined implicit solvent and replica exchange sampling to calculate atomistic disordered ensembles of the nuclear co-activator binding domain (NCBD) of transcription coactivator CBP and the activation domain of the p160 steroid receptor coactivator ACTR. The calculated ensembles are in quantitative agreement with NMRderived residue helicity and recapitulate the experimental observation that, while free ACTR largely lacks residual secondary structures, free NCBD is a molten globule with a helical content similar to that in the folded complex. Detailed conformational analysis reveals that free NCBD has an inherent ability to substantially sample all the helix configurations that have been previously observed either unbound or in complexes. Intriguingly, further high-temperature unbinding and unfolding simulations in implicit and explicit solvents emphasize the importance of conformational fluctuations in synergistic folding of NCBD with ACTR. A balance between preformed elements and conformational fluctuations appears necessary to allow NCBD to interact with different targets and fold into alternative conformations. Together with previous topology-based modeling and existing experimental data, the current simulations strongly support an ‘‘extended conformational selection’’ synergistic folding mechanism that involves a key intermediate state stabilized by interaction between the C-terminal helices of NCBD and ACTR. In addition, the atomistic simulations reveal the role of long-range as well as short-range electrostatic interactions in cooperating with readily fluctuating residual structures, which might enhance the encounter rate and promote efficient folding upon encounter for facile binding and folding interactions of IDPs. Thus, the current study not only provides a consistent mechanistic understanding of the NCBD/ACTR interaction, but also helps establish a multi-scale molecular modeling framework for understanding the structure, interaction, and regulation of IDPs in general

Lone mothers in Belgium : labor force attachment and risk factors

In Belgium, lone parent families represent almost one fourth of the households with children, aside from the existence of regional differences in the phenomenon’s prevalence. Zooming in on Flanders, the poverty risk for this type of family is significantly higher compared to couple-with-children households and the general population; thus, labour market participation represents a crucial resource for individuals heading such households to cope with the economic needs of the family and to avoid long-lasting poverty or to rely extensively on social assistance. We use data from the Crossroads Bank of Social Security (CBSS Datawarehouse) to study Flemish lone mothers’ patterns of labour market participation and test the association between employment trajectories after lone parenthood and both individual and household characteristics. Flanders represents an interesting case because of (i) the relatively high diffusion of lone motherhood, (ii) the presence of welfare measures supporting a number of different types of recipients (even to different extents and not necessarily so generous to keep them out of poverty), and (iii) the availability of longitudinal data to observe lone mothers’ employment trajectories over time. We find that differences exist among lone mothers, who thus experience different risk of social exclusion driven by family and labour market arrangements set up to resolve the potentially contradictory trade-off between the needs for care and for income. The age at which mothers have children is crucial in understanding their future exclusion from the labour market: selection into early lone motherhood is associated with lower employment opportunities. Furthermore, it is the number of children below 17 in the household rather than the presence of very small children that defines a lower probability of having a strong labour market attachment through full-time jobs, and that increases the likelihood of being unemployed/inactive and receiving welfare benefits

Lone Mothers in Belgium: Labor Force Attachment and Risk Factors

In Belgium, lone parent families represent almost one fourth of the households with children, aside from the existence of regional differences in the phenomenon’s prevalence. Zooming in on Flanders, the poverty risk for this type of family is significantly higher compared to couple-with-children households and the general population; thus, labour market participation represents a crucial resource for individuals heading such households to cope with the economic needs of the family and to avoid long-lasting poverty or to rely extensively on social assistance. We use data from the Crossroads Bank of Social Security (CBSS Datawarehouse) to study Flemish lone mothers’ patterns of labour market participation and test the association between employment trajectories after lone parenthood and both individual and household characteristics. Flanders represents an interesting case because of (i) the relatively high diffusion of lone motherhood, (ii) the presence of welfare measures supporting a number of different types of recipients (even to different extents and not necessarily so generous to keep them out of poverty), and (iii) the availability of longitudinal data to observe lone mothers’ employment trajectories over time. We find that differences exist among lone mothers, who thus experience different risk of social exclusion driven by family and labour market arrangements set up to resolve the potentially contradictory trade-off between the needs for care and for income. The age at which mothers have children is crucial in understanding their future exclusion from the labour market: selection into early lone motherhood is associated with lower employment opportunities. Furthermore, it is the number of children below 17 in the household rather than the presence of very small children that defines a lower probability of having a strong labour market attachment through full-time jobs, and that increases the likelihood of being unemployed/inactive and receiving welfare benefits