Vaccination with structurally adapted fungal protein fibrils induces immunity to Parkinson's disease.

The pathological misfolding and aggregation of soluble α-synuclein into toxic oligomers and insoluble amyloid fibrils causes Parkinson's disease, a progressive age-related neurodegenerative disease for which there is no cure. HET-s is a soluble fungal protein that can form assembled amyloid fibrils in its prion state. We engineered HET-s(218-298) to form four different fibrillar vaccine candidates, each displaying a specific conformational epitope present on the surface of α-synuclein fibrils. Vaccination with these four vaccine candidates prolonged the survival of immunized TgM83+/- mice challenged with α-synuclein fibrils by 8% when injected into the brain to model brain-first Parkinson's disease or by 21% and 22% when injected into the peritoneum or gut wall, respectively, to model body-first Parkinson's disease. Antibodies from fully immunized mice recognized α-synuclein fibrils and brain homogenates from patients with Parkinson's disease, dementia with Lewy bodies and multiple system atrophy. Conformation-specific vaccines that mimic epitopes present only on the surface of pathological fibrils but not on soluble monomers, hold great promise for protection against Parkinson's disease, related synucleinopathies and other amyloidogenic protein misfolding disorders.

Extracting binding energies and binding modes from biomolecular simulations of fragment binding to endothiapepsin.

Fragment-based drug discovery (FBDD) aims to discover a set of small binding fragments that may be subsequently linked together. Therefore, in-depth knowledge of the individual fragments' structural and energetic binding properties is essential. In addition to experimental techniques, the direct simulation of fragment binding by molecular dynamics (MD) simulations became popular to characterize fragment binding. However, former studies showed that long simulation times and high computational demands per fragment are needed, which limits applicability in FBDD. Here, we performed short, unbiased MD simulations of direct fragment binding to endothiapepsin, a well-characterized model system of pepsin-like aspartic proteases. To evaluate the strengths and limitations of short MD simulations for the structural and energetic characterization of fragment binding, we predicted the fragments' absolute free energies and binding poses based on the direct simulations of fragment binding and compared the predictions to experimental data. The predicted absolute free energies are in fair agreement with the experiment. Combining the MD data with binding mode predictions from molecular docking approaches helped to correctly identify the most promising fragments for further chemical optimization. Importantly, all computations and predictions were done within 5 days, suggesting that MD simulations may become a viable tool in FBDD projects.

Cryo-EM structures of lipidic fibrils of amyloid-ß (1-40).

Alzheimer's disease (AD) is a progressive and incurable neurodegenerative disease characterized by the extracellular deposition of amyloid plaques. Investigation into the composition of these plaques revealed a high amount of amyloid-ß (Aß) fibrils and a high concentration of lipids, suggesting that fibril-lipid interactions may also be relevant for the pathogenesis of AD. Therefore, we grew Aß40 fibrils in the presence of lipid vesicles and determined their structure by cryo-electron microscopy (cryo-EM) to high resolution. The fold of the major polymorph is similar to the structure of brain-seeded fibrils reported previously. The majority of the lipids are bound to the fibrils, as we show by cryo-EM and NMR spectroscopy. This apparent lipid extraction from vesicles observed here in vitro provides structural insights into potentially disease-relevant fibril-lipid interactions.

Cryo-EM of Aß fibrils from mouse models find tg-APPArcSwe fibrils resemble those found in patients with sporadic Alzheimer's disease.

The use of transgenic mice displaying amyloid-ß (Aß) brain pathology has been essential for the preclinical assessment of new treatment strategies for Alzheimer's disease. However, the properties of Aß in such mice have not been systematically compared to Aß in the brains of patients with Alzheimer's disease. Here, we determined the structures of nine ex vivo Aß fibrils from six different mouse models by cryogenic-electron microscopy. We found novel Aß fibril structures in the APP/PS1, ARTE10 and tg-SwDI models, whereas the human type II filament fold was found in the ARTE10, tg-APPSwe and APP23 models. The tg-APPArcSwe mice showed an Aß fibril whose structure resembles the human type I filament found in patients with sporadic Alzheimer's disease. A detailed assessment of the Aß fibril structure is key to the selection of adequate mouse models for the preclinical development of novel plaque-targeting therapeutics and positron emission tomography imaging tracers in Alzheimer's disease.

Atomic Resolution Insights into pH Shift Induced Deprotonation Events in LS-Shaped Aß(1-42) Amyloid Fibrils.

Alzheimer's disease is a neurodegenerative disorder associated with the deposition of misfolded aggregates of the amyloid-ß protein (Aß). Aß(1-42) is one of the most aggregation-prone components in senile plaques of AD patients. We demonstrated that relatively homogeneous Aß(1-42) fibrils with one predominant fold visible in solid-state NMR spectra can be obtained at acidic pH. The structure of these fibrils differs remarkably from some other polymorphs obtained at neutral pH. In particular, the entire N-terminal region is part of the rigid fibril core. Here, we investigate the effects of a pH shift on the stability and the fold of these fibrils at higher pH values. Fibril bundling at neutral pH values renders cryo-EM studies impractical, but solid-state NMR spectroscopy, molecular dynamics simulations, and biophysical methods provide residue-specific structural information under these conditions. The LS-fold of the Aß(1-42) fibrils does not change over the complete pH range from pH 2 to pH 7; in particular, the N-terminus remains part of the fibril core. We observe changes in the protonation state of charged residues starting from pH 5 on a residue-specific level. The deprotonation of the C-terminal carboxyl group of A42 in the intermolecular salt bridge with D1 and K28 is slow on the NMR time scale, with a local pKa of 5.4, and local conformations of the involved residues are affected by deprotonation of A42. Thus, we demonstrate that this fibril form is stable at physiological pH values.

The 3D structure of lipidic fibrils of α-synuclein.

α-synuclein misfolding and aggregation into fibrils is a common feature of α-synucleinopathies, such as Parkinson's disease, in which α-synuclein fibrils are a characteristic hallmark of neuronal inclusions called Lewy bodies. Studies on the composition of Lewy bodies extracted postmortem from brain tissue of Parkinson's patients revealed that lipids and membranous organelles are also a significant component. Interactions between α-synuclein and lipids have been previously identified as relevant for Parkinson's disease pathology, however molecular insights into their interactions have remained elusive. Here we present cryo-electron microscopy structures of six α-synuclein fibrils in complex with lipids, revealing specific lipid-fibril interactions. We observe that phospholipids promote an alternative protofilament fold, mediate an unusual arrangement of protofilaments, and fill the central cavities of the fibrils. Together with our previous studies, these structures also indicate a mechanism for fibril-induced lipid extraction, which is likely to be involved in the development of α-synucleinopathies. Specifically, one potential mechanism for the cellular toxicity is the disruption of intracellular vesicles mediated by fibrils and oligomers, and therefore the modulation of these interactions may provide a promising strategy for future therapeutic interventions.

Molecular interactions of FG nucleoporin repeats at high resolution.

Proteins that contain repeat phenylalanine-glycine (FG) residues phase separate into oncogenic transcription factor condensates in malignant leukaemias, form the permeability barrier of the nuclear pore complex and mislocalize in neurodegenerative diseases. Insights into the molecular interactions of FG-repeat nucleoporins have, however, remained largely elusive. Using a combination of NMR spectroscopy and cryoelectron microscopy, we have identified uniformly spaced segments of transient ß-structure and a stable preformed α-helix recognized by messenger RNA export factors in the FG-repeat domain of human nucleoporin 98 (Nup98). In addition, we have determined at high resolution the molecular organization of reversible FG-FG interactions in amyloid fibrils formed by a highly aggregation-prone segment in Nup98. We have further demonstrated that amyloid-like aggregates of the FG-repeat domain of Nup98 have low stability and are reversible. Our results provide critical insights into the molecular interactions underlying the self-association and phase separation of FG-repeat nucleoporins in physiological and pathological cell activities.

Quaternary structure of patient-homogenate amplified α-synuclein fibrils modulates seeding of endogenous α-synuclein.

Parkinson's disease (PD) and Multiple System Atrophy (MSA) are progressive and unremitting neurological diseases that are neuropathologically characterized by α-synuclein inclusions. Increasing evidence supports the aggregation of α-synuclein in specific brain areas early in the disease course, followed by the spreading of α-synuclein pathology to multiple brain regions. However, little is known about how the structure of α-synuclein fibrils influence its ability to seed endogenous α-synuclein in recipient cells. Here, we aggregated α-synuclein by seeding with homogenates of PD- and MSA-confirmed brain tissue, determined the resulting α-synuclein fibril structures by cryo-electron microscopy, and characterized their seeding potential in mouse primary oligodendroglial cultures. The combined analysis shows that the two patient material-amplified α-synuclein fibrils share a similar protofilament fold but differ in their inter-protofilament interface and their ability to recruit endogenous α-synuclein. Our study indicates that the quaternary structure of α-synuclein fibrils modulates the seeding of α-synuclein pathology inside recipient cells. It thus provides an important advance in the quest to understand the connection between the structure of α-synuclein fibrils, cellular seeding/spreading, and ultimately the clinical manifestations of different synucleinopathies.

The clinical drug candidate anle138b binds in a cavity of lipidic α-synuclein fibrils.

Aggregation of amyloidogenic proteins is a characteristic of multiple neurodegenerative diseases. Atomic resolution of small molecule binding to such pathological protein aggregates is of interest for the development of therapeutics and diagnostics. Here we investigate the interaction between α-synuclein fibrils and anle138b, a clinical drug candidate for disease modifying therapy in neurodegeneration and a promising scaffold for positron emission tomography tracer design. We used nuclear magnetic resonance spectroscopy and the cryogenic electron microscopy structure of α-synuclein fibrils grown in the presence of lipids to locate anle138b within a cavity formed between two ß-strands. We explored and quantified multiple binding modes of the compound in detail using molecular dynamics simulations. Our results reveal stable polar interactions between anle138b and backbone moieties inside the tubular cavity of the fibrils. Such cavities are common in other fibril structures as well.

Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects.

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.75 ± 1.22 µM. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

Development of a First-in-Class Small-Molecule Inhibitor of the C-Terminal Hsp90 Dimerization.

Heat shock proteins 90 (Hsp90) are promising therapeutic targets due to their involvement in stabilizing several aberrantly expressed oncoproteins. In cancerous cells, Hsp90 expression is elevated, thereby exerting antiapoptotic effects, which is essential for the malignant transformation and tumor progression. Most of the Hsp90 inhibitors (Hsp90i) under investigation target the ATP binding site in the N-terminal domain of Hsp90. However, adverse effects, including induction of the prosurvival resistance mechanism (heat shock response or HSR) and associated dose-limiting toxicity, have so far precluded their clinical approval. In contrast, modulators that interfere with the C-terminal domain (CTD) of Hsp90 do not inflict HSR. Since the CTD dimerization of Hsp90 is essential for its chaperone activity, interfering with the dimerization process by small-molecule protein-protein interaction inhibitors is a promising strategy for anticancer drug research. We have developed a first-in-class small-molecule inhibitor (5b) targeting the Hsp90 CTD dimerization interface, based on a tripyrimidonamide scaffold through structure-based molecular design, chemical synthesis, binding mode model prediction, assessment of the biochemical affinity, and efficacy against therapy-resistant leukemia cells. 5b reduces xenotransplantation of leukemia cells in zebrafish models and induces apoptosis in BCR-ABL1+ (T315I) tyrosine kinase inhibitor-resistant leukemia cells, without inducing HSR.

Functional and structural characterization of interactions between opposite subunits in HCN pacemaker channels.

Hyperpolarization-activated and cyclic nucleotide (HCN) modulated channels are tetrameric cation channels. In each of the four subunits, the intracellular cyclic nucleotide-binding domain (CNBD) is coupled to the transmembrane domain via a helical structure, the C-linker. High-resolution channel structures suggest that the C-linker enables functionally relevant interactions with the opposite subunit, which might be critical for coupling the conformational changes in the CNBD to the channel pore. We combined mutagenesis, patch-clamp technique, confocal patch-clamp fluorometry, and molecular dynamics (MD) simulations to show that residue K464 of the C-linker is relevant for stabilizing the closed state of the mHCN2 channel by forming interactions with the opposite subunit. MD simulations revealed that in the K464E channel, a rotation of the intracellular domain relative to the channel pore is induced, which is similar to the cAMP-induced rotation, weakening the autoinhibitory effect of the unoccupied CL-CNBD region. We suggest that this CL-CNBD rotation is considerably involved in activation-induced affinity increase but only indirectly involved in gate modulation. The adopted poses shown herein are in excellent agreement with previous structural results.

Conformational heterogeneity coupled with ß-fibril formation of a scaffold protein involved in chronic mental illnesses.

Chronic mental illnesses (CMIs) pose a significant challenge to global health due to their complex and poorly understood etiologies and hence, absence of causal therapies. Research of the past two decades has revealed dysfunction of the disrupted in schizophrenia 1 (DISC1) protein as a predisposing factor involved in several psychiatric disorders. DISC1 is a multifaceted protein that serves myriads of functions in mammalian cells, for instance, influencing neuronal development and synapse maintenance. It serves as a scaffold hub forming complexes with a variety (~300) of partners that constitute its interactome. Herein, using combinations of structural and biophysical tools, we demonstrate that the C-region of the DISC1 protein is highly polymorphic, with important consequences for its physiological role. Results from solid-state NMR spectroscopy and electron microscopy indicate that the protein not only forms symmetric oligomers but also gives rise to fibrils closely resembling those found in certain established amyloid proteinopathies. Furthermore, its aggregation as studied by isothermal titration calorimetry (ITC) is an exergonic process, involving a negative enthalpy change that drives the formation of oligomeric (presumably tetrameric) species as well as ß-fibrils. We have been able to narrow down the ß-core region participating in fibrillization to residues 716-761 of full-length human DISC1. This region is absent in the DISC1Δ22aa splice variant, resulting in reduced association with proteins from the dynein motor complex, viz., NDE-like 1 (NDEL1) and lissencephaly 1 (LIS1), which are crucial during mitosis. By employing surface plasmon resonance, we show that the oligomeric DISC1 C-region has an increased affinity and shows cooperativity in binding to LIS1 and NDEL1, in contrast to the noncooperative binding mode exhibited by the monomeric version. Based on the derived structural models, we propose that the association between the binding partners involves two neighboring subunits of DISC1 C-region oligomers. Altogether, our findings highlight the significance of the DISC1 C-region as a crucial factor governing the balance between its physiological role as a multifunctional scaffold protein and aggregation-related aberrations with potential significance for disease.

Aqueous ionic liquids redistribute local enzyme stability via long-range perturbation pathways.

Ionic liquids (IL) and aqueous ionic liquids (aIL) are attractive (co-)solvents for biocatalysis due to their unique properties. On the other hand, the incubation of enzymes in IL or aIL often reduces enzyme activity. Recent studies proposed various aIL-induced effects to explain the reduction, classified as direct effects, e.g., local dehydration or competitive inhibition, and indirect effects, e.g., structural perturbations or disturbed catalytic site integrity. However, the molecular origin of indirect effects has largely remained elusive. Here we show by multi-µs long molecular dynamics simulations, free energy computations, and rigidity analyses that aIL favorably interact with specific residues of Bacillus subtilis Lipase A (BsLipA) and modify the local structural stability of this model enzyme by inducing long-range perturbations of noncovalent interactions. The perturbations percolate over neighboring residues and eventually affect the catalytic site and the buried protein core. Validation against a complete experimental site saturation mutagenesis library of BsLipA (3620 variants) reveals that the residues of the perturbation pathways are distinguished sequence positions where substitutions highly likely yield significantly improved residual activity. Our results demonstrate that identifying these perturbation pathways and specific IL ion-residue interactions there effectively predicts focused variant libraries with improved aIL tolerance.

Glutamine synthetase as a central element in hepatic glutamine and ammonia metabolism: novel aspects.

Glutamine synthetase (GS) in the liver is expressed in a small perivenous, highly specialized hepatocyte population and is essential for the maintenance of low, non-toxic ammonia levels in the organism. However, GS activity can be impaired by tyrosine nitration of the enzyme in response to oxidative/nitrosative stress in a pH-sensitive way. The underlying molecular mechanism as investigated by combined molecular simulations and in vitro experiments indicates that tyrosine nitration can lead to a fully reversible and pH-sensitive regulation of protein function. This approach was also used to understand the functional consequences of several recently described point mutations of human GS with clinical relevance and to suggest an approach to restore impaired GS activity.

Mechanism of Fully Reversible, pH-Sensitive Inhibition of Human Glutamine Synthetase by Tyrosine Nitration.

Glutamine synthetase (GS) catalyzes an ATP-dependent condensation of glutamate and ammonia to form glutamine. This reaction-and therefore GS-are indispensable for the hepatic nitrogen metabolism. Nitration of tyrosine 336 (Y336) inhibits human GS activity. GS nitration and the consequent loss of GS function are associated with a broad range of neurological diseases. The mechanism by which Y336 nitration inhibits GS, however, is not understood. Here, we show by means of unbiased MD simulations, binding, and configurational free energy computations that Y336 nitration hampers ATP binding but only in the deprotonated and negatively charged state of residue 336. By contrast, for the protonated and neutral state, our computations indicate an increased binding affinity for ATP. pKa computations of nitrated Y336 within GS predict a pKa of ∼5.3. Thus, at physiological pH, nitrated Y336 exists almost exclusively in the deprotonated and negatively charged state. In vitro experiments confirm these predictions, in that, the catalytic activity of nitrated GS is decreased at pH 7 and 6 but not at pH 4. These results indicate a novel, fully reversible, pH-sensitive mechanism for the regulation of GS activity by tyrosine nitration.

Binding modes of thioflavin T and Congo red to the fibril structure of amyloid-ß(1-42).

Binding modes for the amyloid-ß(1-42) fibril fluorescent dyes thioflavin T and Congo red were predicted by molecular dynamics simulations and binding free energy calculations. Both probes bind on the fibril surface to primarily hydrophobic grooves, with their long axis oriented almost parallel to the fibril axis. The computed binding affinities are in agreement with experimental values. The binding modes also explain observables from previous structural studies and, thus, provide a starting point for the systematic search and design of novel molecules, which may improve in vitro diagnostics for Alzheimer's disease.

Small-molecule inhibitors of nisin resistance protein NSR from the human pathogen Streptococcus agalactiae.

Lantibiotics are antimicrobial peptides produced by Gram-positive bacteria and active in the nanomolar range. Nisin is the most intensely studied and used lantibiotic, with applications as food preservative and recognized potential for clinical usage. However, different bacteria that are pathogenic for humans and do not produce nisin, including Streptococcus agalactiae, show an innate resistance that has been related to the nisin resistance protein (NSR), a membrane-associated protease. Here, we report the first-in-class small-molecule inhibitors of SaNSR identified by virtual screening based on a previously derived structural model of the nisin/NSR complex. The inhibitors belong to three different chemotypes, of which the halogenated phenyl-urea derivative NPG9 is the most potent one. Co-administration of NPG9 with nisin yields increased potency compared to nisin alone in SaNSR-expressing bacteria. The binding mode of NPG9, predicted with molecular docking and validated by extensive molecular dynamics simulations, confirms a structure-activity relationship derived from the in vivo data. Saturation transfer difference-NMR experiments demonstrate direct binding of NPG9 to SaNSR and agree with the predicted binding mode. Our results demonstrate the potential to overcome SaNSR-related lantibiotic resistance by small molecules.

Partially inserted nascent chain unzips the lateral gate of the Sec translocon.

The Sec translocon provides the lipid bilayer entry for ribosome-bound nascent chains and thus facilitates membrane protein biogenesis. Despite the appreciated role of the native environment in the translocon:ribosome assembly, structural information on the complex in the lipid membrane is scarce. Here, we present a cryo-electron microscopy-based structure of bacterial translocon SecYEG in lipid nanodiscs and elucidate an early intermediate state upon insertion of the FtsQ anchor domain. Insertion of the short nascent chain causes initial displacements within the lateral gate of the translocon, where α-helices 2b, 7, and 8 tilt within the membrane core to "unzip" the gate at the cytoplasmic side. Molecular dynamics simulations demonstrate that the conformational change is reversed in the absence of the ribosome, and suggest that the accessory α-helices of SecE subunit modulate the lateral gate conformation. Site-specific cross-linking validates that the FtsQ nascent chain passes the lateral gate upon insertion. The structure and the biochemical data suggest that the partially inserted nascent chain remains highly flexible until it acquires the transmembrane topology.

Targeting HSP90 dimerization via the C terminus is effective in imatinib-resistant CML and lacks the heat shock response.

Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38-) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.