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Prion diseases are associated with the conversion of the cellular prion protein (PrP) into a pathogenic conformer (PrPSc). A proposed therapeutic approach to avoid the pathogenic transformation is to develop antibodies that bind to PrP and stabilize its structure. POM1 and POM6 are two monoclonal antibodies that bind the globular domain of PrP and have different biological responses, i.e., trigger neurotoxicity mimicking prion infections (POM1) or prevent neurotoxicity (POM6). The crystal structures of PrP in complex with the two antibodies show similar epitopes which seems inconsistent with the opposite phenotypes. Here, we investigate the influence of the POM1 and POM6 antibodies on the flexibility of the mouse PrP by molecular dynamics simulations. The simulations reveal that the POM6/PrP interface is less stable than the POM1/PrP interface, ascribable to localized polar mismatches at the interface, despite the former complex having a larger epitope than the latter. In the presence of any of the two antibodies, the flexibility of the globular domain increases everywhere except for the β 1 - α 1 loop in the POM1/PrP complex which suggests the involvement of this loop in the pathological conversion. The secondary structure of PrP is preserved whereas the polar interactions involving residues Glu146, Arg156 and Arg208 are modified upon antibody binding. • The POM6/prion protein interface is less stable than the POM1/prion protein interface • Antibody binding increases the flexibility of the PrP globular domain, except in the β 1 -α 1 loop which is part of the epitope • The network of intramolecular contacts is perturbed differently upon antibody binding • The binding of the antibodies stabilizes the 3 10 -helix at the C-terminus of α 1 , with POM6 having a more pronounced effect. [ABSTRACT FROM AUTHOR]

The self-perpetuating conversion of cellular prion proteins (PrPC) into an aggregated β-sheet rich conformation is associated with transmissible spongiform encephalopathies (TSE). The loop 166-175 (L1) in PrPC, which displays sequence and structural variation among species, has been suggested to play a role in species barrier, in particular against transmission of TSE from cervids to domestic and laboratory animals. L1 is ordered in elk PrP, as well as in a mouse/elk hybrid (in which L1 of mouse is replaced by elk) but not in other species such as mice, humans, and bovine. To investigate the source and significance of L1 dynamics, we carried out explicit solvent molecular dynamics simulations (≈0.5 µs in total) of the mouse prion protein, the mouse/elk hybrid, and control simulations, in which the mouse sequence is reintroduced into the structure of the mouse/elk hybrid. We found that the flexibility of L1 correlates with the backbone dynamics of Serl70. Furthermore, L1 mobility promotes a substantial displacement of Tyr169, rupture of the Asp178-Tyr128 and Asp178-Tyr169 side chain hydrogen bonds, as well as disruption of Tyr169-Phe175 ~r-stacking interaction. The simulation results go beyond the available experimental data because they highlight the dependence of this network of interactions on residue 170 and L1 plasticity. [ABSTRACT FROM AUTHOR]

In amyloid fibrils, β-strand conformations of polypeptide chains, or segments thereof, are perpendicular to the fibril axis, but knowledge of their three dimensional structure at atomic level of detail is scarce. Two types of computational approaches have been developed recently for investigating the aggregation propensity of peptides and proteins and identifying the segments most prone to form fibrils (hot spots). The physicochemical properties of the natural amino acids (e.g. β-propensity, hydrophobicity, aromatic content and charge) have been used to derive phenomenological models able to predict changes in aggregation rate upon mutation, as well as absolute rates and hot spots. Applications of these models to entire proteomes have provided evidence that intrinsically disordered proteins are less amyloidogenic than globular proteins. In the second type of approach, amyloidogenic polypeptides have been decomposed into overlapping segments, and atomistic simulations of three or more copies of each segment have been performed to obtain insights into aggregation propensity and structural details of the ordered aggregates (e.g. turn regions). [Copyright &y& Elsevier]

Abstract: Amyloid fibrils are insoluble mainly β-sheet aggregates of proteins or peptides. The multi-step process of amyloid aggregation is one of the major research topics in structural biology and biophysics because of its relevance in protein misfolding diseases like Alzheimer''s, Parkinson''s, Creutzfeld-Jacob''s, and type II diabetes. Yet, the detailed mechanism of oligomer formation and the influence of protein stability on the aggregation kinetics are still matters of debate. Here a coarse-grained model of an amphipathic polypeptide, characterized by a free energy profile with distinct amyloid-competent (i.e. β-prone) and amyloid-protected states, is used to investigate the kinetics of aggregation and the pathways of fibril formation. The simulation results suggest that by simply increasing the relative stability of the β-prone state of the polypeptide, disordered aggregation changes into fibrillogenesis with the presence of oligomeric on-pathway intermediates, and finally without intermediates in the case of a very stable β-prone state. The minimal-size aggregate able to form a fibril is generated by collisions of oligomers or monomers for polypeptides with unstable or stable β-prone state, respectively. The simulation results provide a basis for understanding the wide range of amyloid-aggregation mechanisms observed in peptides and proteins. Moreover, they allow us to interpret at a molecular level the much faster kinetics of assembly of a recently discovered functional amyloid with respect to the very slow pathological aggregation. [Copyright &y& Elsevier]

The conformation space of a 20 residue antiparallel β-sheet peptide, sampled by molecular dynamics simulations, is mapped to a network. Snapshots saved along the trajectory are grouped according to secondary structure into nodes of the network and the transitions between them are links. The conformation space network describes the significant free energy minima and their dynamic connectivity without requiring arbitrarily chosen reaction coordinates. As previously found for the Internet and the World-Wide Web as well as for social and biological networks, the conformation space network is scale-free and contains highly connected hubs like the native state which is the most populated free energy basin. Furthermore, the native basin exhibits a hierarchical organization, which is not found for a random heteropolymer lacking a predominant free-energy minimum. The network topology is used to identify conformations in the folding transition state (TS) ensemble, and provides a basis for understanding the heterogeneity of the TS and denatured state ensemble as well as the existence of multiple pathways. [Copyright &y& Elsevier]

The N-terminal domain of the Tn916 integrase protein (INT-DBD) is responsible for DNA binding in the process of strand cleavage and joining reactions required for transposition of the Tn916 conjugative transposon. Site-specific association is facilitated by numerous protein–DNA contacts from the face of a three-stranded β-sheet inserted into the major groove. The protein undergoes a subtle conformational transition and is slightly unfolded in the protein–DNA complex. The conformation of many charged residues is poorly defined by NMR data but mutational studies have indicated that removal of polar side chains decreases binding affinity, while non-polar contacts are malleable. Based on analysis of the binding enthalpy and binding heat capacity, we have reasoned that dehydration of the protein–DNA interface is incomplete. This study presents results from a molecular dynamics investigation of the INT–DBD–DNA complex aimed at a more detailed understanding of the role of conformational dynamics and hydration in site-specific binding. Comparison of simulations (total of 13 ns) of the free protein and of the bound protein conformation (in isolation or DNA-bound) reveals intrinsic flexibility in certain parts of the molecule. Conformational adaptation linked to partial unfolding appears to be induced by protein–DNA contacts. The protein–DNA hydrogen-bonding network is highly dynamic. The simulation identifies protein–DNA interactions that are poorly resolved or only surmised from the NMR ensemble. Single water molecules and water clusters dynamically optimize the complementarity of polar interactions at the ‘wet’ protein–DNA interface. The simulation results are useful to establish a qualitative link between experimental data on individual residue's contribution to binding affinity and thermodynamic properties of INT–DBD alone and in complex with DNA. Copyright © 2004 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Twenty-eight years after its original publication, the diffusion-collision model has successfully been applied to describe the folding kinetics of two proteins with the same native structure but different sequences (, this issue of Structure). The calculations show the relative importance of the primary and tertiary structure on the sequence of events and folding. For both proteins, the model suggests parallel folding pathways, a finding which has wide implications for the interpretations of experiments. [Copyright &y& Elsevier]

The cellular prion protein PrPC mediates the neurotoxicity of prions and other protein aggregates through poorly understood mechanisms. Antibody‐derived ligands against the globular domain of PrPC (GDL) can also initiate neurotoxicity by inducing an intramolecular R208‐H140 hydrogen bond ("H‐latch") between the α2‐α3 and β2‐α2 loops of PrPC. Importantly, GDL that suppresses the H‐latch prolong the life of prion‐infected mice, suggesting that GDL toxicity and prion infections exploit convergent pathways. To define the structural underpinnings of these phenomena, we transduced 19 individual PrPC variants to PrPC‐deficient cerebellar organotypic cultured slices using adenovirus‐associated viral vectors (AAV). We report that GDL toxicity requires a single N‐proximal cationic residue (K27 or R27) within PrPC. Alanine substitution of K27 also prevented the toxicity of PrPC mutants that induce Shmerling syndrome, a neurodegenerative disease that is suppressed by co‐expression of wild‐type PrPC. K27 may represent an actionable target for compounds aimed at preventing prion‐related neurodegeneration. [ABSTRACT FROM AUTHOR]

Designed ankyrin repeat proteins (DARPins) are antibody mimetics with high and mostly unexplored potential in drug development. By using in silico analysis and a rationally guided Ala scanning, we identified position 17 of the N-terminal capping repeat to play a key role in overall protein thermostability. The melting temperature of a DARPin domain with a single full-consensus internal repeat was increased by 8 °C to 10 °C when Asp17 was replaced by Leu, Val, Ile, Met, Ala, or Thr. We then transferred the Asp17Leu mutation to various backgrounds, including clinically validated DARPin domains, such as the vascular endothelial growth factor-binding domain of the DARPin abicipar pegol. In all cases, these proteins showed improvements in the thermostability on the order of 8 °C to 16 °C, suggesting the replacement of Asp17 could be generically applicable to this drug class. Molecular dynamics simulations showed that the Asp17Leu mutation reduces electrostatic repulsion and improves van-der-Waals packing, rendering the DARPin domain less flexible and more stable. Interestingly, this beneficial Asp17Leu mutation is present in the N-terminal caps of three of the five DARPin domains of ensovibep, a SARS-CoV-2 entry inhibitor currently in clinical development, indicating this mutation could be partly responsible for the very high melting temperature (>90 °C) of this promising anti-COVID-19 drug. Overall, such N-terminal capping repeats with increased thermostability seem to be beneficial for the development of innovative drugs based on DARPins. [ABSTRACT FROM AUTHOR]

In S-value analysis, the effects of mutations on the folding kinetics are compared with the corresponding effects on thermodynamic stability to investigate the structure of the protein-folding transition state (TS). Here, molecular dynamics (MD) simulations (totaling 0.65 ms) have been performed for a large set of single-point mutants of a 20-residue three-stranded antiparallel β-sheet peptide. Between 57 and 120 folding events were sampled at near equilibrium for each mutant allowing for accurate estimates of folding/unfolding rates and stability changes. The φ values calculated from folding and unfolding rates extracted from the MD trajectories are reliable if the stability loss upon mutation is larger than ∼0.6 kcal/mol, which is observed for 8 of the 32 single-point mutants. The same heterogeneity of the TS of the wild type was found in the mutated peptides, showing two possible pathways for folding. Single-point mutations can induce significant shifts not always detected by φ-value analysis. Specific nonnative interactions at the TS were observed in most of the peptides studied here. The interpretation of φ values based on the ratio of atomic contacts at the IS over the native state, which has been used in the past in MD and Monte Carlo simulations, is in agreement with the TS structures of wild-type peptide. However, φ values tend to overestimate the nativeness of the TS ensemble, when interpreted neglecting the nonnative interactions. [ABSTRACT FROM AUTHOR]

Plants use different classes of photoreceptors to collect information about their light environment. Cryptochromes are blue light photoreceptors that control deetiolation, entrain the circadian clock, and are involved in flowering time control. Here, we describe the crvl-L407F allele of Arabidopsis (Arabidopsis thaliana), which encodes a hypersensitive cryptochromel (cry1) protein. Plants carrying the cry1-L407F point mutation have elevated expression of CONSTANS and FLOWERING LOCUS under short-day conditions, leading to very early flowering. These results demonstrate that not only the well-studied cry2, with an unequivocal role in flowering promotion, but also cry1 can function as an activator of the floral transition. The cry1- L407F mutants are also hypersensitive toward blue, red, and far-red light in hypocotyl growth inhibition. In addition, cry1-L407F seeds are hypersensitive to germination-inducing red light pulses, but the far-red reversibility of this response is not compromised. This demonstrates that the cry1-L407F photoreceptor can increase the sensitivity of phytochrome signaling cascades. Molecular dynamics simulation of wild-type and mutant cry1 proteins indicated that the L407F mutation considerably reduces the structural flexibility of two solvent-exposed regions of the protein, suggesting that the hypersensitivity might result from a reduced entropic penalty of binding events during downstream signal transduction. Other nonmutually exclusive potential reasons for the cry1-L407F gain of function are the location of phenylalanine-407 close to three conserved tryptophans, which could change cry1's photochemical properties, and stabilization of ATP binding, which could extend the lifetime of the signaling state of cry1. [ABSTRACT FROM AUTHOR]

Polycomb group (PcG) proteins are essential to maintain gene expression patterns during development. Transcriptional repression by PcG proteins involves trimethylation of H3K27 (H3K27me3) by Polycomb Repressive Complex 2 (PRC2) in animals and plants. PRC1 binds to H3K27me3 and is required for transcriptional repression in animals, but in plants PRC1- like activities have remained elusive. One candidate protein that could be involved in PRC1-like functions in plants is LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), because LHP1 associates with genes marked by H3K27me3 in vivo and has a chromodomain that binds H3K27me3 in vitro. Here, we show that disruption of the chromodomain of Arabidopsis thaliana LHP1 abolishes H3K27me3 recognition, releases gene silencing and causes similar phenotypic alterations as transcriptional lhp1 null mutants. Therefore, binding to H3K27me3 is essential for LHP1 protein function. [ABSTRACT FROM AUTHOR]

Biological processes are regulated by intermolecular interactions and chemical modifications that do not affect protein levels, thus escaping detection in classical proteomic screens. We demonstrate here that a global protein structural readout based on limited proteolysis-mass spectrometry (LiP-MS) detects many such functional alterations, simultaneously and in situ, in bacteria undergoing nutrient adaptation and in yeast responding to acute stress. The structural readout, visualized as structural barcodes, captured enzyme activity changes, phosphorylation, protein aggregation, and complex formation, with the resolution of individual regulated functional sites such as binding and active sites. Comparison with prior knowledge, including other 'omics data, showed that LiP-MS detects many known functional alterations within well-studied pathways. It suggested distinct metabolite-protein interactions and enabled identification of a fructose-1,6-bisphosphate-based regulatory mechanism of glucose uptake in E. coli. The structural readout dramatically increases classical proteomics coverage, generates mechanistic hypotheses, and paves the way for in situ structural systems biology. • Dynamic structural proteomic screens detect functional changes at high resolution • Detect enzyme activity, phosphorylation, and molecular interactions in situ • Generate new molecular hypotheses and increase functional proteomics coverage • Enabled discovery of a regulatory mechanism of glucose uptake in E. coli [ABSTRACT FROM AUTHOR]