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Activation of the extracellular signal-regulated kinase-2 (ERK2) by phosphorylation has been shown to involve changes in protein dynamics, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS) and NMR relaxation dispersion measurements. These can be described by a global exchange between two conformational states of the active kinase, named ‘L’ and ‘R,’ where R is associated with a catalytically productive ATP-binding mode. An ATP-competitive ERK1/2 inhibitor, Vertex-11e, has properties of conformation selection for the R-state, revealing movements of the activation loop that are allosterically coupled to the kinase active site. However, the features of inhibitors important for R-state selection are unknown. Here, we survey a panel of ATP-competitive ERK inhibitors using HDX-MS and NMR and identify 14 new molecules with properties of R-state selection. They reveal effects propagated to distal regions in the P +1 and helix αF segments surrounding the activation loop, as well as helix αL16. Crystal structures of inhibitor complexes with ERK2 reveal systematic shifts in the Gly loop and helix αC, mediated by a Tyr-Tyr ring stacking interaction and the conserved Lys-Glu salt bridge. The findings suggest a model for the R-state involving small movements in the N-lobe that promote compactness within the kinase active site and alter mobility surrounding the activation loop. Such properties of conformation selection might be exploited to modulate the protein docking interface used by ERK substrates and effectors.

In the living cells, proteins bind small molecules (or “ligands”) through a “conformational selection” mechanism, where a subset of protein structures are capable of binding the small molecules well while most other protein structures are not capable of such binding. The present work uses machine learning approaches to identify, in a very large amount of protein:ligand complexes, what protein properties are associated with their capacity to bind small molecules. In order to do so, we calculate 40 physicochemical properties on about 1.5 millions of protein conformations: ligand and protein conformations. This work describes a machine learning approach to identify the unique physico-chemical descriptors of a protein that maximize the prediction rate of potential protein molecular conformations for the test case proteins ADORA2A (Adenosine A2a Receptor), ADRB2 (Adrenoceptor Beta 2) and OPRK1 (Opioid Receptor Kappa 1). We find adequate machine learning techniques can increase by an order of magnitude the identification of “binding protein conformations” in an otherwise very large ensemble of protein conformations, compared to random selection of protein conformations. This opens the door to the systematic identification of such “binding conformations” for proteins and provides a big data approach to the conformational selection mechanism.

Synthetic macrocycles such as calixarenes and cucurbiturils are increasingly applied as mediators of protein assembly and crystallization. The macrocycle can facilitate assembly by providing a surface on which two or more proteins bind simultaneously. This work explores the capacity of the sulfonato-calix[ n ]arene (sclx n ) series to effect crystallization of PAF, a small, cationic antifungal protein. Co-crystallization with sclx 4 , sclx 6 or sclx 8 led to high-resolution crystal structures. In the absence of sclx n , diffraction-quality crystals of PAF were not obtained. Interestingly, all three sclx n were bound to a similar patch on PAF. The largest and most flexible variant, sclx 8 , yielded a dimer of PAF. Complex formation was evident in solution via NMR and ITC experiments, showing more pronounced effects with increasing macrocycle size. In agreement with the crystal structure, the ITC data suggested that sclx 8 acts as a bidentate ligand. The contributions of calixarene size/conformation to protein recognition and assembly are discussed. Finally, it is suggested that the conserved binding site for anionic calixarenes implicates this region of PAF in membrane binding, which is a prerequisite for antifungal activity.

Dairy cattle breeding has historically focused on relatively small numbers of elite bulls as sires of sons. In recent years, even if generation intervals were reduced and more diverse sires of sons could have been selected, genomic selection has not fundamentally changed the fact that a large number of individuals are being analyzed. However, a relatively small number of elite bulls are still siring those animals. Therefore inbreeding-derived negative consequences in the gene pool have brought concern. The detrimental effects of non-additive genetic changes such as inbreeding depression and dominance have been widely disseminated while seriously affecting bioeconomically important parameters because of an antagonistic relationship between dairy production and reproductive traits. Therefore, the estimation of benefits and limitations of inbreeding and variance of the selection response deserves to be evaluated and discussed to preserve genetic variability, a significant concern in the selection of individuals for reproduction and production. Short-term strategies for genetic merit improvement through modern breeding programs have severely lowered high-producing dairy cattle fertility potential. Since the current selection programs potentially increase long-term costs, genetic diversity has decreased globally as a consequence. Therefore, a greater understanding of the potential that selection programs have for supporting long-term genetic sustainability and genetic diversity among dairy cattle populations should be prioritized in managing farm profitability. The present review provides a broad approach to current inbreeding-derived problems, identifying critical points to be solved and possible alternative strategies to control selection against homozygous haplotypes while maintaining sustained selection pressure. Moreover, this manuscript explores future perspectives, emphasizing theoretical applications and critical points, and strategies to avoid the adverse effects of inbreeding in dairy cattle. Finally, this review provides an overview of challenges that will soon require multidisciplinary approaches to managing dairy cattle populations, intending to combine increases in productive trait phenotypes with improvements in reproductive, health, welfare, linear conformation, and adaptability traits into the foreseeable future.

Schizophrenia is a debilitating psychiatric condition often associated with poor quality of life and decreased life expectancy. Lack of progress in improving treatment outcomes has been attributed to limited knowledge of the underlying biology, although large-scale genomic studies have begun to provide insights. We report a new genome-wide association study of schizophrenia (11,260 cases and 24,542 controls), and through meta-analysis with existing data we identify 50 novel associated loci and 145 loci in total. Through integrating genomic fine-mapping with brain expression and chromosome conformation data, we identify candidate causal genes within 33 loci. We also show for the first time that the common variant association signal is highly enriched among genes that are under strong selective pressures. These findings provide new insights into the biology and genetic architecture of schizophrenia, highlight the importance of mutation-intolerant genes and suggest a mechanism by which common risk variants persist in the population. A new GWAS of schizophrenia (11,260 cases and 24,542 controls) and meta-analysis identifies 50 new associated loci and 145 loci in total. The common variant association signal is highly enriched in mutation-intolerant genes and in regions under strong background selection.

The average productive lifespan is approximately 3 to 4 years in countries with high-producing dairy cows. This is much shorter than the natural life expectancy of dairy cattle. Dairy farmers continue to cull cows primarily for reasons related to poor health, failure to conceive or conformation problems prior to culling. These reasons may indicate reduced welfare leading up to culling. Improvements in health care, housing and nutrition will reduce forced culling related to these welfare reasons. However, productive lifespan has remained similar in decades, despite large improvements in cow comfort and genetic selection for the ability to avoid culling. On the other hand, genetic progress for economically important traits is accelerating within the last decade, which should slightly shorten the average economically optimal productive lifespan. A major driver of productive lifespan is the availability of replacement heifers that force cows out when they calve. The average productive lifespan could be extended by reducing the supply of dairy heifers, which would also have benefits for environmental sustainability. Improvements in culling decision support tools would strengthen economically optimal replacement decisions. In conclusion, major factors of the relatively short productive lifespan of dairy cows are welfare-related, but other economic factors like supply of heifers, genetic progress and non-optimal decision-making also play important roles.

Soluble oligomeric aggregates of the amyloid-β peptide (Aβ) have been implicated in the pathogenesis of Alzheimer's disease (AD). Although the conformation adopted by Aβ within these aggregates is not known, a β-hairpin conformation is known to be accessible to monomeric Aβ. Here we show that this β-hairpin is a building block of toxic Aβ oligomers by engineering a double-cysteine mutant (called AβCC) in which the β-hairpin is stabilized by an intramolecular disulfide bond. Aβ₄₀CC and Aβ₄₂CC both spontaneously form stable oligomeric species with distinct molecular weights and secondary-structure content, but both are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Aβ aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in AβCC oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that β-sheet-containing Aβ₄₂CC oligomers or protofibrillar species formed by these oligomers are 50 times more potent inducers of neuronal apoptosis than amyloid fibrils or samples of monomeric wild-type Aβ₄₂, in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Aβ oligomer preparations will facilitate studies of their structure and role in the pathogenesis of AD. For example, here we show how kinetic partitioning into different aggregation pathways can explain why Aβ₄₂ is more toxic than the shorter Aβ₄₀, and why certain inherited mutations are linked to protofibril formation and early-onset AD.

PurposeApplication of multi-scale modelling workflows to characterise polymorphism in ritonavir with regard to its stability, bioavailability and processing.MethodsMolecular conformation, polarizability and stability are examined using quantum mechanics (QM). Intermolecular synthons, hydrogen bonding, crystal morphology and surface chemistry are modelled using empirical force fields.ResultsThe form I conformation is more stable and polarized with more efficient intermolecular packing, lower void space and higher density, however its shielded hydroxyl is only a hydrogen bond donor. In contrast, the hydroxyl in the more open but less stable and polarized form II conformation is both a donor and acceptor resulting in stronger hydrogen bonding and a more stable crystal structure but one that is less dense. Both forms have strong 1D networks of hydrogen bonds and the differences in packing energies are partially offset in form II by its conformational deformation energy difference with respect to form I. The lattice energies converge at shorter distances for form I, consistent with its preferential crystallization at high supersaturation. Both forms exhibit a needle/lath-like crystal habit with slower growing hydrophobic side and faster growing hydrophilic capping habit faces with aspect ratios increasing from polar-protic, polar-aprotic and non-polar solvents, respectively. Surface energies are higher for form II than form I and increase with solvent polarity. The higher deformation, lattice and surface energies of form II are consistent with its lower solubility and hence bioavailability.ConclusionInter-relationship between molecular, solid-state and surface structures of the polymorphic forms of ritonavir are quantified in relation to their physical-chemical properties.

The energy landscape used by nature over evolutionary timescales to select protein sequences is essentially the same as the one that folds these sequences into functioning proteins, sometimes in microseconds. We show that genomic data, physical coarse-grained free energy functions, and family-specific information theoretic models can be combined to give consistent estimates of energy landscape characteristics of natural proteins. One such characteristic is the effective temperature T ₛₑₗ at which these foldable sequences have been selected in sequence space by evolution. T ₛₑₗ quantifies the importance of folded-state energetics and structural specificity for molecular evolution. Across all protein families studied, our estimates for T ₛₑₗ are well below the experimental folding temperatures, indicating that the energy landscapes of natural foldable proteins are strongly funneled toward the native state.

During circulation in humans and natural selection to escape antibody recognition for decades, A/H3N2 influenza viruses emerged with altered receptor specificities. These viruses lost the ability to agglutinate erythrocytes critical for antigenic characterization and give low yields and acquire adaptive mutations when cultured in eggs and cells, contributing to recent vaccine challenges. Examination of receptor specificities of A/H3N2 viruses reveals that recent viruses compensated for decreased binding of the prototypic human receptor by recognizing α2,6-sialosides on extended LacNAc moieties. Erythrocyte glycomics shows an absence of extended glycans providing a rationale for lack of agglutination by recent A/H3N2 viruses. A glycan remodeling approach installing functional receptors on erythrocytes, allows antigenic characterization of recent A/H3N2 viruses confirming the cocirculation of antigenically different viruses in humans. Computational analysis of HAs in complex with sialosides having extended LacNAc moieties reveals that mutations distal to the RBD reoriented the Y159 side chain resulting in an extended receptor binding site. Here, Broszeit et al. show that circulating A/H3N2 viruses have evolved binding specificity to α2,6-sialosides on extended LacNAc moieties and therefore cannot agglutinate erythrocytes. Applying glycan remodeling allows to install functional receptors on erythrocytes and promotes identification of newly circulating variants to facilitate vaccine design.