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Human HtrA2 is part of the HtrA family of ATP-independent serine proteases that are conserved in both prokaryotes and eukaryotes and localizes to the intermembrane space of the mitochondria. Several recent reports have suggested that HtrA2 is important for maintaining proper mitochondrial homeostasis and may play a role in Alzheimer's disease (AD), which is characterized by the presence of aggregates of the amyloid-beta peptide 1-42 (Abeta1-42). In this study, we analyzed the ability of HtrA2 to delay the aggregation of the model substrate citrate synthase (CS) and of the toxic Abeta1-42 peptide. We found that HtrA2 had a moderate ability to delay the aggregation of CS in vitro, and this activity was significantly enhanced when the PDZ domain was removed suggesting an inhibitory role for this domain on the activity. Additionally, using electron microscopy and nuclear magnetic resonance analyses, we observed that HtrA2 significantly delayed the aggregation of the Abeta1-42 peptide. Interestingly, the protease activity of HtrA2 and its PDZ domain were not essential for the delay of Abeta1-42 peptide aggregation. These results indicate that besides its protease activity, HtrA2 also performs a chaperone function and suggest a role for HtrA2 in the metabolism of intracellular Abeta and in AD. [ABSTRACT FROM AUTHOR]

The fields of allostery and amyloid-related pathologies, such as Parkinson's disease (PD), have been extensively explored individually, but less is known about how amyloids control allostery. Recent advancements have revealed that amyloids can drive allosteric effects in both intrinsically disordered proteins, such as alpha-synuclein (αS), and multi-domain signaling proteins, such as protein kinase A (PKA). Amyloid-driven allostery plays a central role in explaining the mechanisms of gain-of-pathological-function mutations in αS (e.g. E46K, which causes early PD onset) and loss-of-physiological-function mutations in PKA (e.g. A211D, which predisposes to tumors). This review highlights allosteric effects of disease-related mutations and how they can cause exposure of amyloidogenic regions, leading to amyloids that are either toxic or cause aberrant signaling. We also discuss multiple potential modulators of these allosteric effects, such as MgATP and kinase substrates, opening future opportunities to improve current pharmacological interventions against αS and PKA-related pathologies. Overall, we show that amyloid-driven allosteric models are useful to explain the mechanisms underlying disease-related mutations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Human serum albumin (HSA), the most abundant protein in plasma and cerebrospinal fluid, not only serves as a crucial carrier of various exogenous and endogenous ligands but also modulates the aggregation of amyloidogenic proteins, including alpha synuclein (αSyn), which is associated with Parkinson's disease and other α-synucleinopathies. HSA decreases αSyn toxicity through the direct binding to monomeric and oligomeric αSyn species. However, it is possible that HSA also sequesters metal ions that otherwise promote aggregation. Cu(ii) ions, for example, enhance αSyn fibrillization in vitro , while also leading to neurotoxicity by generating reactive oxygen species (ROS). However, it is currently unclear if and how HSA affects Cu(ii)-binding to αSyn. Using an integrated set of NMR experiments, we show that HSA is able to chelate Cu(ii) ions from αSyn more efficiently than standard chelators such as EDTA, revealing an unexpected cooperativity between the HSA metal-binding sites. Notably, fatty acid binding to HSA perturbs this cooperativity, thus interfering with the sequestration of Cu(ii) ions from αSyn. We also observed that glycation of HSA diminished Cu(ii)-binding affinity, while largely preserving the degree of cooperativity between the HSA metal-binding sites. Additionally, our results show that Cu(ii)-binding to HSA stabilizes the interactions of HSA with αSyn primarily at two different regions, i.e. the N-terminus, Tyr 39 and the majority of the C-terminus. Our study not only unveils the effect of fatty acid binding and age-related posttranslational modifications, such as glycation, on the neuroprotective mechanisms of HSA, but also highlights the potential of αSyn as a viable NMR-based sensor to investigate HSA-metal interactions., Competing Interests: There is no conflict of interest to declare., (This journal is © The Royal Society of Chemistry.)

Alpha synuclein (αS) aggregates are the main component of Lewy bodies (LBs) associated with Parkinson's disease (PD). A longstanding question about αS and PD pertains to the autosomal dominant E46K αS mutant, which leads to the early onset of PD and LB dementias. The E46K mutation not only promotes αS aggregation but also stabilizes αS monomers in "closed" conformers, which are compact and aggregation-incompetent. Hence, the mechanism of action of the E46K mutation is currently unclear. Here, we show that αS monomers harboring the E46K mutation exhibit more extensive interactions with fibrils compared to those of WT. Such monomer-fibril interactions are sufficient to allosterically drive transitions of αS monomers from closed to open conformations, enabling αS aggregation. We also show that E46K promotes head-to-tail monomer-monomer interactions in early self-association events. This multipronged mechanism provides a new framework to explain how the E46K mutation and possibly other αS variants trigger early-onset PD., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

The ability of Adenosine Triphosphate (ATP) to modulate protein solubility establishes a critical link between ATP homeostasis and proteinopathies, such as Parkinson's (PD). The most significant risk factor for PD is aging, and ATP levels decline dramatically with age. However, the mechanism by which ATP interacts with alpha-synuclein (αS), whose aggregation is characteristic of PD, is currently not fully understood, as is ATP's effect on αS aggregation. Here, we use nuclear magnetic resonance spectroscopy as well as fluorescence, dynamic light scattering and microscopy to show that ATP affects multiple species in the αS self-association cascade. The triphosphate moiety of ATP disrupts long-range electrostatic intramolecular contacts in αS monomers to enhance initial aggregation, while also inhibiting the formation of late-stage β-sheet fibrils by disrupting monomer-fibril interactions. These effects are modulated by magnesium ions and early onset PD-related αS mutations, suggesting that loss of the ATP hydrotropic function on αS fibrillization may play a role in PD etiology., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Protein kinase G (PKG) is an essential regulator of eukaryotic cyclic guanosine monophosphate (cGMP)-dependent intracellular signaling, controlling pathways that are often distinct from those regulated by cyclic adenosine monophosphate (cAMP). Specifically, the C-terminal cyclic-nucleotide-binding domain (CNB-B) of PKG has emerged as a critical module to control allostery and cGMP-selectivity in PKG. While key contributions to the cGMP-versus-cAMP selectivity of CNB-B were previously assessed, only limited knowledge is currently available on how cyclic nucleotide binding rewires the network of hydrogen bonds in CNB-B, and how such rewiring contributes to allostery and cGMP selectivity. To address this gap, we extend the comparative analysis of apo, cAMP- and cGMP-bound CNB-B to H/D fractionation factors (FFs), which are well-suited for assessing backbone hydrogen-bond strengths within proteins. Apo-vs-bound comparisons inform of perturbations arising from both binding and allostery, while cGMP-bound vs cAMP-bound comparisons inform of perturbations that are purely allosteric. The comparative FF analyses of the bound states revealed mixed patterns of hydrogen-bond strengthening and weakening, pointing to inherent frustration, whereby not all hydrogen bonds can be simultaneously stabilized. Interestingly, contrary to expectations, these patterns include a weakening of hydrogen bonds not only within critical recognition and allosteric elements of CNB-B, but also within elements known to undergo rigid-body movement upon cyclic nucleotide binding. These results suggest that frustration may contribute to the reversibility of allosteric conformational shifts by avoiding over-rigidification that may otherwise trap CNB-B in its active state. Considering that PKG CNB-B serves as a prototype for allosteric conformational switches, similar concepts may be applicable to allosteric domains in general.

The cAMP- and cGMP-dependent protein kinases (PKA and PKG) are canonically activated by the corresponding cyclic nucleotides. However, both systems are also sensitive to a wide range of non-canonical allosteric effectors, such as reactive oxygen species, which induce the formation of regulatory inter- and intra-molecular disulfide bridges, and disease-related mutations (DRMs). Here, we present a combined analysis of representative non-canonical allosteric effectors for PKA and PKG, and we identify common molecular mechanisms underlying non-canonical allostery in these kinases, from shifts in dynamical regulatory equilibria to modulation of inter-protomer interactions. In addition, mutations may also drive oligomerization beyond dimerization, and possibly phase transitions, causing loss of kinase inhibitory function and amplifying the allosteric effects of DRMs. Hence non-canonical allosteric stimuli often result in constitutive kinase activation underlying either physiological control of downstream signaling pathways or pathological outcomes, from aortic aneurisms to cancer predisposition. Overall, PKA and PKG emerge as "pan-sensors" going well beyond canonical cyclic nucleotide activation, revealing their versatile roles as central signaling hubs., Competing Interests: Competing interests The authors have declared that no competing interests exist., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cyclic guanosine monophosphate (cGMP) signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs, complementing drugs that target nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory (AI) pseudo-substrate sequences to PKG Iα and Iβ that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is not well understood. Here, we present a crystal structure of PKG Iβ in which the AI sequence and the cyclic nucleotide-binding (CNB) domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG Iβ AI sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I CNB domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wildtype cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG Iβ auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and cooperative activation of PKGs., Competing Interests: RS, JK, LQ, PH, MA, BV, GK, BS, KM, GM, DC, FH, CK No competing interests declared

Exchange proteins directly activated by cAMP (EPAC) are guanine nucleotide exchange factors for the small GTPases, Rap1 and Rap2. They regulate several physiological functions and mitigation of their activity has been suggested as a possible treatment for multiple diseases such as cardiomyopathy, diabetes, chronic pain, and cancer. Several EPAC-specific modulators have been developed, however studies that quantify their structure-activity relationships are still lacking. Here we propose a quantitative structure-activity relationship (QSAR) model for a series of EPAC-specific compounds. The model demonstrated high reproducibility and predictivity and the predictive ability of the model was tested against a series of compounds that were unknown to the model. The compound with the highest predicted affinity was validated experimentally through fluorescence-based competition assays and NMR experiments revealed its mode of binding and mechanism of action as a partial agonist. The proposed QSAR model can, therefore, serve as an effective screening tool to identify promising EPAC-selective drug leads with enhanced potency., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

Allosteric regulation is essential to control biological function. In addition, allosteric sites offer a promising venue for selective drug targeting. However, accurate mapping of allosteric sites remains challenging since allostery relies on often subtle, yet functionally relevant, structural and dynamical changes. A viable approach proposed to overcome such challenge is chemical shift covariance analysis (CHESCA). Although CHESCA offers an exhaustive map of allosteric networks, it is critical to define the core allosteric sites to be prioritized in subsequent functional studies or in the design of allosteric drugs. Here, we propose two new CHESCA-based methodologies, called temperature CHESCA (T-CHESCA) and CLASS-CHESCA, aimed at narrowing down allosteric maps to the core allosteric residues. Both T- and CLASS-CHESCAs rely on the invariance of core inter-residue correlations to changes in the chemical shifts of the active and inactive conformations interconverting in fast exchange. In T-CHESCA the chemical shifts of such states are modulated through temperature changes, while in CLASS-CHESCA through variations in the spin-active nuclei involved in pairwise correlations. T- and CLASS-CHESCAs, as well as complete-linkage CHESCA, were applied to the cAMP-binding domain of the exchange protein directly activated by cAMP (EPAC), which serves as a prototypical allosteric switch. Residues consistently identified by the three CHESCA methods were found in previously identified EPAC allosteric core sites. Hence, T-, CLASS-, and CL-CHESCA provide a toolset to establish allosteric site hierarchy and triage allosteric sites to be further analyzed by mutations and functional assays. Furthermore, the core allosteric networks selectively revealed through T- and CLASS-CHESCA are expected to facilitate the mechanistic understanding of disease-related mutations and the design of selective allosteric modulators., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Allosteric pluripotency arises when the functional response of an allosteric receptor to an allosteric stimulus depends on additional allosteric modulators. Here, we discuss allosteric pluripotency as observed in the prototypical Protein Kinase A (PKA) as well as in other signaling systems, from typical multidomain signaling proteins to bacterial enzymes. We identify key drivers of pluripotent allostery and illustrate how hypothesizing allosteric pluripotency may solve apparent discrepancies currently present in the literature regarding the dual nature of known allosteric modulators. We also outline the implications of allosteric pluripotency for cellular signaling and allosteric drug design, and analyze the challenges and opportunities opened by the pluripotent nature of allostery., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Novel therapeutic strategies are needed to control the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) pandemic. Here, we present a protocol to anchor the SARS-CoV-2 spike (S-)protein in the cytoplasmic membranes of erythrocyte liposomes. A surfactant was used to stabilize the S-protein's structure in the aqueous environment before insertion and to facilitate reconstitution of the S-proteins in the erythrocyte membranes. The insertion process was studied using coarse grained Molecular Dynamics (MD) simulations. Liposome formation and S-protein anchoring was studied by dynamic light scattering (DLS), ELV-protein co-sedimentation assays, fluorescent microcopy and cryo-TEM. The Erythro-VLPs (erythrocyte based virus like particles) have a well defined size of ∼200 nm and an average protein density on the outer membrane of up to ∼300 proteins/μm2. The correct insertion and functional conformation of the S-proteins was verified by dose-dependent binding to ACE-2 (angiotensin converting enzyme 2) in biolayer interferometry (BLI) assays. Seroconversion was observed in a pilot mouse trial after 14 days when administered intravenously, based on enzyme-linked immunosorbent assays (ELISA). This red blood cell based platform can open novel possibilities for therapeutics for the coronavirus disease (COVID-19) including variants, and other viruses in the future., Competing Interests: The authors have declared that no competing interests exist.

Human Serum Albumin (HSA), the most abundant protein in plasma, serves a diverse repertoire of biological functions including regulation of oncotic pressure and redox potential, transport of serum solutes, but also chaperoning of misfolded proteins. Here we review how HSA interacts with a wide spectrum of client proteins including intrinsically disordered proteins (IDPs) such as Aβ, the islet amyloid peptide (IAPP), alpha synuclein and stressed globular proteins such as insulin. The comparative analysis of the HSA chaperone - client interactions reveals that the amyloid-inhibitory function of HSA arises from at least four emerging mechanisms. Two mechanisms (the monomer stabilizer model and the monomer competitor model) involve the direct binding of HSA to either IDP monomers or oligomers, while other mechanisms (metal chelation and membrane protection) rely on the indirect modulation by HSA of other factors that drive IDP aggregation. While HSA is not the only extracellular chaperone, given its abundance, HSA is likely to account for a significant fraction of the chaperoning effects in plasma, thus opening new therapeutic opportunities in the context of the peripheral sink hypothesis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Malaria is a life-threatening infectious disease primarily caused by the Plasmodium falciparum parasite. The increasing resistance to current antimalarial drugs and their side effects has led to an urgent need for novel malaria drug targets, such as the P. falciparum cGMP-dependent protein kinase (pfPKG). However, PKG plays an essential regulatory role also in the human host. Human cGMP-dependent protein kinase (hPKG) and pfPKG are controlled by structurally homologous cGMP-binding domains (CBDs). Here, we show that despite the structural similarities between the essential CBDs in pfPKG and hPKG, their respective allosteric networks differ significantly. Through comparative analyses of chemical shift covariance analyses, molecular dynamics simulations, and backbone internal dynamics measurements, we found that conserved allosteric elements within the essential CBDs are wired differently in pfPKG and hPKG to implement cGMP-dependent kinase activation. Such pfPKG versus hPKG rewiring of allosteric networks was unexpected because of the structural similarity between the two essential CBDs. Yet, such finding provides crucial information on which elements to target for selective inhibition of pfPKG versus hPKG, which may potentially reduce undesired side effects in malaria treatments., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The aberrant self-assembly of intrinsically disordered proteins (IDPs) into soluble oligomers and their interactions with biological membranes underlie the pathogenesis of numerous neurodegenerative diseases, including Alzheimer's disease. Catechins have emerged as useful tools to reduce the toxicity of IDP oligomers by modulating their interactions with membranes. However, the structural determinants of catechin binding to IDP oligomers and membranes remain largely elusive. Here, we assemble a catechin library by combining several naturally occurring chemical modifications and, using a coupled NMR-statistical approach, we map at atomic resolution the interactions of such library with the Alzheimer's-associated amyloid-beta (Aβ) oligomers and model membranes. Our results reveal multiple catechin affinity drivers and show that the combination of affinity-reducing covalent changes may lead to unexpected net gains in affinity. Interestingly, we find that the positive cooperativity is more prevalent for Aβ oligomers than membrane binding, and that the determinants underlying catechin recognition by membranes are markedly different from those dissected for Aβ oligomers. Notably, we find that the unanticipated positive cooperativity arises from the critical regulatory role of the gallate catechin moiety, which recruits previously disengaged substituents into the binding interface and leads to an overall greater compaction of the receptor-bound conformation. Overall, the previously elusive structural attributes mapped here provide an unprecedented foundation to establish structure-activity relationships of catechins., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Protein kinase G (PKG) is a major receptor of cGMP, and controls signaling pathways distinct from those regulated by cAMP. However, the contributions of the two substituents that differentiate cGMP from cAMP (i.e. 6-oxo and 2-NH 2 ) to the cGMP-versus-cAMP selectivity of PKG remain unclear. Here, using NMR to map how binding affinity and dynamics of the protein and ligand vary along a ligand double-substitution cycle, we show that the contributions of the two substituents to binding affinity are surprisingly non-additive. Such non-additivity stems primarily from mutual protein-ligand conformational selection, whereby not only does the ligand select for a preferred protein conformation upon binding, but also, the protein selects for a preferred ligand conformation. The 6-oxo substituent mainly controls the conformational equilibrium of the bound protein, while the 2-NH 2 substituent primarily controls the conformational equilibrium of the unbound ligand (i.e. syn versus anti). Therefore, understanding the conformational dynamics of both the protein and ligand is essential to explain the cGMP-versus-cAMP selectivity of PKG., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Protein kinase A (PKA) is the main receptor for the universal cAMP second messenger. PKA is a tetramer with two catalytic (C) and two regulatory (R) subunits, each including two tandem cAMP-binding domains, i.e. CBD-A and -B. Activation of the complex occurs with cAMP binding first to CBD-B, followed by a second molecule of cAMP binding to CBD-A, which causes the release of the active C-subunit. Unlike previous constructs for eukaryotic cAMP-binding domains (CBDs), the 29.5 kDa construct analyzed here [i.e. RIα (119-379)] spans the CBDs in full and provides insight into inter-domain communication. In this note we report the 1 H, 13 C, and 15  N backbone assignments of cAMP-bound RIα (119-379) CBDs (BMRB No. 50920)., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Allosteric pluripotency arises when an allosteric effector switches from agonist to antagonist depending on the experimental conditions. For example, the Rp-cAMPS ligand of Protein Kinase A (PKA) switches from agonist to antagonist as the MgATP concentration increases and/or the kinase substrate affinity or concentration decreases. Understanding allosteric pluripotency is essential to design effective allosteric therapeutics with minimal side effects. Allosteric pluripotency of PKA arises from divergent allosteric responses of two homologous tandem cAMP-binding domains, resulting in a free energy landscape for the Rp-cAMPS-bound PKA regulatory subunit R1a in which the ground state is kinase inhibition-incompetent and the kinase inhibition-competent state is excited. The magnitude of the free energy difference between the ground non-inhibitory and excited inhibitory states (Δ G R,Gap ) relative to the effective free energy of R1a binding to the catalytic subunit of PKA (Δ G R:C ) dictates whether the antagonism-to-agonism switch occurs. However, the key drivers of Δ G R,Gap are not fully understood. Here, by analyzing an R1a mutant that selectively silences allosteric pluripotency, we show that a major determinant of Δ G R,Gap unexpectedly arises from state-selective frustration in the ground inhibition-incompetent state of Rp-cAMPS-bound R1a. Such frustration is caused by steric clashes between the phosphate-binding cassette and the helices preceding the lid, which interact with the phosphate and base of Rp-cAMPS, respectively. These clashes are absent in the excited inhibitory state, thus reducing the Δ G R,Gap to values comparable to Δ G R:C , as needed for allosteric pluripotency to occur. The resulting model of allosteric pluripotency is anticipated to assist the design of effective allosteric modulators., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

The Plasmodium falciparum cGMP-dependent protein kinase ( Pf PKG) is required for the progression of the Plasmodium 's life cycle and is therefore a promising malaria drug target. Pf PKG includes four cGMP-binding domains (CBD-A to CBD-D). CBD-D plays a crucial role in Pf PKG regulation as it is the primary determinant for the inhibition and cGMP-dependent activation of the catalytic domain. Hence, it is critical to understand how CBD-D is allosterically regulated by cGMP. Although the apo versus holo conformational changes of CBD-D have been reported, information on the intermediates of the activation pathway is currently lacking. Here, we employed molecular dynamics simulations to model four key states along the thermodynamic cycle for the cGMP-dependent activation of the Pf PKG CBD-D domain. The simulations were compared to NMR data, and they revealed that the Pf PKG CBD-D activation pathway samples a compact intermediate in which the N- and C-terminal helices approach the central β-barrel. In addition, by comparing the cGMP-bound active and inactive states, the essential binding interactions that differentiate these states were identified. The identification of structural and dynamical features unique to the cGMP-bound inactive state provides a promising basis to design Pf PKG-selective allosteric inhibitors as a viable treatment for malaria.

Familial mutations of the protein kinase A (PKA) R1α regulatory subunit lead to a generalized predisposition for a wide range of tumors, from pituitary adenomas to pancreatic and liver cancers, commonly referred to as Carney complex (CNC). CNC mutations are known to cause overactivation of PKA, but the molecular mechanisms underlying such kinase overactivity are not fully understood in the context of the canonical cAMP-dependent activation of PKA. Here, we show that oligomerization-induced sequestration of R1α from the catalytic subunit of PKA (C) is a viable mechanism of PKA activation that can explain the CNC phenotype. Our investigations focus on comparative analyses at the level of structure, unfolding, aggregation, and kinase inhibition profiles of wild-type (wt) PKA R1α, the A211D and G287W CNC mutants, as well as the cognate acrodysostosis type 1 (ACRDYS1) mutations A211T and G287E. The latter exhibit a phenotype opposite to CNC with suboptimal PKA activation compared with wt. Overall, our results show that CNC mutations not only perturb the classical cAMP-dependent allosteric activation pathway of PKA, but also amplify significantly more than the cognate ACRDYS1 mutations nonclassical and previously unappreciated activation pathways, such as oligomerization-induced losses of the PKA R1α inhibitory function., Competing Interests: The authors declare no competing interest.

Motivation: Correlated Nuclear Magnetic Resonance (NMR) chemical shift changes identified through the CHEmical Shift Projection Analysis (CHESPA) and CHEmical Shift Covariance Analysis (CHESCA) reveal pathways of allosteric transitions in biological macromolecules. To address the need for an automated platform that implements CHESPA and CHESCA and integrates them with other NMR analysis software packages, we introduce here integrated plugins for NMRFAM-SPARKY that implement the seamless detection and visualization of allosteric networks., Availability and Implementation: CHESCA-SPARKY and CHESPA-SPARKY are available in the latest version of NMRFAM-SPARKY from the National Magnetic Resonance Facility at Madison (http://pine.nmrfam.wisc.edu/download_packages.html), the NMRbox Project (https://nmrbox.org) and to subscribers to the SBGrid (https://sbgrid.org). The assigned spectra involved in this study and tutorial videos using this dataset are available at https://sites.google.com/view/chescachespa-sparky., Supplementary Information: Supplementary data are available at Bioinformatics Online., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Soluble oligomers formed by amyloidogenic intrinsically disordered proteins are some of the most cytotoxic species linked to neurodegeneration. Due to the transient and heterogeneous nature of such oligomeric intermediates, the underlying self-association events often remain elusive. NMR relaxation measurements sensitive to zero-frequency spectral densities (J(0)), such as the 15 N - R 2 rates, are ideally suited to map sites of self-association at atomic resolution without the need of exogenous labels. Such experiments exploit the dynamic exchange between NMR visible monomers and slowly tumbling oligomers. However, 15 N - R 2 rates are also sensitive to intrinsic monomer dynamics, and it is often difficult to discern these contributions from those arising from exchange with oligomers. Another challenge pertains to defining a hierarchy of self-association. Here, using the archetypical amyloidogenic protein alpha synuclein (αS), we show that the temperature-dependence of 15 N - R 2 effectively identifies self-association sites with reduced bias from internal dynamics. The key signature of the residues involved in self-association is a nonlinear temperature-dependence of 15 N - R 2 with a positive ΔR 2 /ΔT slope. These two hallmarks are systematically probed through a thermal R 2 correlation matrix, from which the network of residues involved in self-association as well as the hierarchy of αS self-association sites is extracted through agglomerative clustering. We find that aggregation is initiated by residues within the NAC region that is solvent inaccessible in αS fibrils and eventually extends to the N-terminal segment harboring familial PD mutations. These hierarchical self-association maps help dissect the essential drivers of oligomerization and reveal how amyloid inhibitors affect oligomer formation.

Parkinson's disease (PD) is the second most common neurodegenerative disease. Currently, PD has no treatment. The neuronal protein α-synuclein (αS) plays an important role in PD. However, the molecular mechanisms governing its physiological and pathological roles are not fully understood. It is becoming widely acknowledged that the biological roles of αS involve interactions with biological membranes. In these biological processes there is a fine-tuned interplay between lipids affecting the properties of αS and αS affecting lipid metabolism, αS binding to membranes, and membrane damage. In this review, the intricate interactions between αS and membranes will be reviewed and a discussion of the relationship between αS and neuronal membrane structural plasticity in health and disease will be made. It is proposed that in healthy neurons the conformational flexibilities of αS and the neuronal membranes are coupled to assist the physiological roles of αS. However, in circumstances where their conformational flexibilities are decreased or uncoupled, there is a shift toward cell toxicity. Strategies to modulate toxic αS-membrane interactions are potential approaches for the development of new therapies for PD. Future work using specific αS molecular species as well as membranes with specific physicochemical properties should widen our understanding of the intricate biological roles of αS which, in turn, would propel the development of new strategies for the treatment of PD., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Given the breadth and depth of the scientific contributions of Sir Christopher Dobson, with over 870 publications to date, it is inconceivable to convey in a single review the impact of his work and its legacy. This review therefore primarily focuses on his contributions to the development of strategies for preventing aberrant protein misfolding. The first section of this review highlights his seminal work on the elucidation of the microscopic nucleation processes underlying protein aggregation. Next, we discuss the specific inhibition of these steps by candidate drugs and biologics, with a particular emphasis on the role of molecular chaperones. In the final section, we review how protein aggregation principles can be exploited for the rational design of novel and more potent aggregation inhibitors. These milestones serve as excellent examples of the profound impact of Dobson's seminal work on fundamental science and its translation into drug discovery., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Allosteric modulation provides an effective avenue for selective and potent enzyme inhibition. Here, we summarize and critically discuss recent advances on the mechanisms of allosteric partial agonists for three representative signalling enzymes activated by cyclic nucleotides: the cAMP-dependent protein kinase (PKA), the cGMP-dependent protein kinase (PKG), and the exchange protein activated by cAMP (EPAC). The comparative analysis of partial agonism in PKA, PKG and EPAC reveals a common emerging theme, i.e. the sampling of distinct "mixed" conformational states, either within a single domain or between distinct domains. Here, we show how such "mixed" states play a crucial role in explaining the observed functional response, i.e. partial agonism and allosteric pluripotency, as well as in maximizing inhibition while minimizing potency losses. In addition, by combining Nuclear Magnetic Resonance (NMR), Molecular Dynamics (MD) simulations and Ensemble Allosteric Modeling (EAM), we also show how to map the free-energy landscape of conformational ensembles containing "mixed" states. By discussing selected case studies, we illustrate how MD simulations and EAM complement NMR to quantitatively relate protein dynamics to function. The resulting NMR- and MD-based EAMs are anticipated to inform not only the design of new generations of highly selective allosteric inhibitors, but also the choice of multidrug combinations., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Authors.)

Quantifying chemical substituent contributions to ligand-binding free energies is challenging due to nonadditive effects. Protein allostery is a frequent cause of nonadditivity, but the underlying allosteric mechanisms often remain elusive. Here, we propose a general NMR-based approach to elucidate such mechanisms and we apply it to the HCN4 ion channel, whose cAMP-binding domain is an archetypal conformational switch. Using NMR, we show that nonadditivity arises not only from concerted conformational transitions, but also from conformer-specific effects, such as steric frustration. Our results explain how affinity-reducing functional groups may lead to affinity gains if combined. Surprisingly, our approach also reveals that nonadditivity depends markedly on the receptor conformation. It is negligible for the inhibited state but highly significant for the active state, opening new opportunities to tune potency and agonism of allosteric effectors., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is linked to the abnormal accumulation of amyloid β peptide (Aβ) aggregates in the brain. Silybin B, a natural compound extracted from milk thistle ( Silybum marianum ), has been shown to significantly inhibit Aβ aggregation in vitro and to exert neuroprotective properties in vivo . However, further explorations of silybin B's clinical potential are currently limited by three main factors: (a) poor solubility, (b) instability in blood serum, and (c) only partial knowledge of silybin's mechanism of action. Here, we address these three limitations. We demonstrate that conjugation of a trehalose moiety to silybin significantly increases both water solubility and stability in blood serum without significantly compromising its antiaggregation properties. Furthermore, using a combination of biophysical techniques with different spatial resolution, that is, TEM, ThT fluorescence, CD, and NMR spectroscopy, we profile the interactions of the trehalose conjugate with both Aβ monomers and oligomers and evidence that silybin may shield the "toxic" surfaces formed by the N-terminal and central hydrophobic regions of Aβ. Finally, comparative analysis with silybin A, a less active diastereoisomer of silybin B, revealed how even subtle differences in chemical structure may entail different effects on amyloid inhibition. The resulting insight on the mechanism of action of silybins as aggregation inhibitors is anticipated to facilitate the future investigation of silybin's therapeutic potential.

Protein misfolding as well as the subsequent self-association and deposition of amyloid aggregates is implicated in the progression of several neurodegenerative disorders including Alzheimer's and Parkinson's diseases. Modulators of amyloidogenic aggregation serve as essential tools to dissect the underlying molecular mechanisms and may offer insight on potential therapeutic solutions. These modulators include green tea catechins, which are potent inhibitors of amyloid aggregation. Although catechins often exhibit poor pharmacokinetic properties and bioavailability, they are still essential tools for identifying the drivers of amyloid aggregation and for developing other aggregation modulators through structural mimicry. As an illustration of such strategies, here we review how catechins have been used to map the toxic surfaces of oligomeric amyloid-like species and develop catechin-based phenolic compounds with enhanced anti-amyloid activity.

We propose a phosphodiesterase assay based on 1D 1 H NMR to monitor the hydrolysis of cyclic nucleotides directly, without requiring tags or the addition of exogenous reagents. The method is suitable to measure phosphodiesterase K M and k cat parameters and to identify phosphodiesterase inhibitors.

Most malaria deaths are caused by the protozoan parasite Plasmodium falciparum Its life cycle is regulated by a cGMP-dependent protein kinase ( Pf PKG), whose inhibition is a promising antimalaria strategy. Allosteric kinase inhibitors, such as cGMP analogs, offer enhanced selectivity relative to competitive kinase inhibitors. However, the mechanisms underlying allosteric Pf PKG inhibition are incompletely understood. Here, we show that 8-NBD-cGMP is an effective Pf PKG antagonist. Using comparative NMR analyses of a key regulatory domain, Pf D, in its apo , cGMP-bound, and cGMP analog-bound states, we elucidated its inhibition mechanism of action. Using NMR chemical shift analyses, molecular dynamics simulations, and site-directed mutagenesis, we show that 8-NBD-cGMP inhibits Pf PKG not simply by reverting a two-state active versus inactive equilibrium, but by sampling also a distinct inactive "mixed" intermediate. Surface plasmon resonance indicates that the ability to stabilize a mixed intermediate provides a means to effectively inhibit Pf PKG, without losing affinity for the cGMP analog. Our proposed model may facilitate the rational design of Pf PKG-selective inhibitors for improved management of malaria., (© 2020 Byun et al.)

Alpha synuclein (αS) oligomers are a key component of Lewy bodies implicated in Parkinson's disease (PD). Although primarily intracellular, extracellular αS exocytosed from neurons also contributes to PD pathogenesis through a prion-like transmission mechanism. Here, we show at progressive degrees of resolution that the most abundantly expressed extracellular protein, human serum albumin (HSA), inhibits αS oligomer (αS n ) toxicity through a three-pronged mechanism. First, endogenous HSA targets αS n with sub-μM affinity via solvent-exposed hydrophobic sites, breaking the catalytic cycle that promotes αS self-association. Second, HSA remodels αS oligomers and high-MW fibrils into chimeric intermediates with reduced toxicity. Third, HSA unexpectedly suppresses membrane interactions with the N-terminal and central αS regions. Overall, our findings suggest that the extracellular proteostasis network may regulate αS cell-to-cell transmission not only by reducing the populations of membrane-binding competent αS oligomers but possibly also by shielding the membrane interface from residual toxic species.

The exchange protein activated by cAMP (EPAC) is a promising drug target for a wide disease range, from neurodegeneration and infections to cancer and cardiovascular conditions. A novel partial agonist of the EPAC isoform 1 (EPAC1), I942, was recently discovered, but its mechanism of action remains poorly understood. Here, we utilize NMR spectroscopy to map the I942-EPAC1 interactions at atomic resolution and propose a mechanism for I942 partial agonism. We found that I942 interacts with the phosphate binding cassette (PBC) and base binding region (BBR) of EPAC1, similar to cyclic adenosine monophosphate (cAMP). These results not only reveal the molecular basis for the I942 vs cAMP mimicry and competition, but also suggest that the partial agonism of I942 arises from its ability to stabilize an inhibition-incompetent activation intermediate distinct from both active and inactive EPAC1 states. The mechanism of action of I942 may facilitate drug design for EPAC-related diseases.

Polo-like kinases (Plks) are key cell cycle regulators. They contain a kinase domain followed by a polo-box domain that recognizes phosphorylated substrates and enhances their phosphorylation. The regulatory subunit of the Dbf4-dependent kinase complex interacts with the polo-box domain of Cdc5 (the sole Plk in Saccharomyces cerevisiae) in a phosphorylation-independent manner. We have solved the crystal structures of the polo-box domain of Cdc5 on its own and in the presence of peptides derived from Dbf4 and a canonical phosphorylated substrate. The structure bound to the Dbf4-peptide reveals an additional density on the surface opposite to the phospho-peptide binding site that allowed us to propose a model for the interaction. We found that the two peptides can bind simultaneously and non-competitively to the polo-box domain in solution. Furthermore, point mutations on the surface opposite to the phosphopeptide binding site of the polo-box domain disrupt the interaction with the Dbf4 peptide in solution and cause an early anaphase arrest phenotype distinct from the mitotic exit defect typically observed in cdc5 mutants. Collectively, our data illustrates the importance of non-canonical interactions mediated by the polo-box domain and provide key mechanistic insights into the combinatorial recognition of substrates by Polo-like kinases.

The universal second messenger cAMP regulates diverse intracellular processes by interacting with ubiquitously expressed proteins, such as Protein Kinase A (PKA) and the Exchange Protein directly Activated by cAMP (EPAC). EPAC is implicated in multiple pathologies, thus several EPAC-specific inhibitors have been identified in recent years. However, the mechanisms and molecular interactions underlying the EPAC inhibition elicited by such compounds are still poorly understood. Additionally, being hydrophobic low molecular weight species, EPAC-specific inhibitors are prone to forming colloidal aggregates, which result in non-specific aggregation-based inhibition (ABI) in aqueous systems. Here, we review from a biophysical perspective the molecular basis of the specific and non-specific interactions of two EPAC antagonists-CE3F4R, a non-competitive inhibitor, and ESI-09, a competitive inhibitor of EPAC. Additionally, we discuss the value of common ABI attenuators (e.g., TX and HSA) to reduce false positives at the expense of introducing false negatives when screening aggregation-prone compounds. We hope this review provides the EPAC community effective criteria to evaluate similar compounds, aiding in the optimization of existing drug leads, and informing the development of the next generation of EPAC-specific inhibitors.

Degradable low-fouling hydrogels are ideal vehicles for drug and cell delivery. For each application, hydrogel degradation rate must be re-optimized for maximum therapeutic benefit. We developed a method to rapidly and predictably tune degradation rates of low-fouling poly(oligo(ethylene glycol)methyl ether methacrylate) (P(EG) x MA) hydrogels by modifying two interdependent variables: (1) base-catalysed crosslink degradation kinetics, dependent on crosslinker electronics (electron withdrawing groups (EWGs)); and, (2) polymer hydration, dependent on the molecular weight ( M W ) of poly(ethylene glycol) (PEG) pendant groups. By controlling PEG M W and EWG strength, P(EG) x MA hydrogels were tuned to degrade over 6 to 52 d. A 6-member P(EG) x MA copolymer library yielded slow and fast degrading low-fouling hydrogels suitable for short- and long-term delivery applications. The degradation mechanism was also applied to RGD-functionalized poly(carboxybetaine methacrylamide) (PCBMAA) hydrogels to achieve slow (∼50 d) and fast (∼13 d) degrading low-fouling, bioactive hydrogels., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

A common source of false positives in drug discovery is ligand self-association into large colloidal assemblies that nonspecifically inhibit target proteins. However, the mechanisms of aggregation-based inhibition (ABI) and ABI-attenuation by additives, such as Triton X-100 (TX) and human serum albumin (HSA), are not fully understood. Here, we investigate the molecular basis of ABI and ABI-attenuation through the lens of NMR and coupled thermodynamic cycles. We unexpectedly discover a new class of aggregating ligands that exhibit negligible interactions with proteins but act as competitive sinks for the free inhibitor, resulting in bell-shaped dose-response curves. TX attenuates ABI by converting inhibitory, protein-binding aggregates into nonbinding coaggregates, whereas HSA minimizes nonspecific ligand interactions by functioning as a reservoir for free inhibitor and preventing self-association. Hence, both TX and HSA are useful tools to minimize false positives arising from nonspecific binding but at the cost of potentially introducing false negatives due to suppression of specific interactions.

Soluble amyloid beta assemblies (Aβ n ) are neurotoxic and play a central role in the early phases of the pathogenesis cascade leading to Alzheimer's disease. However, the current knowledge about the molecular determinants of Aβ n toxicity is at best scant. Here, we comparatively analyze Aβ n prepared in the absence or presence of a catechin library that modulates cellular toxicity. By combining solution NMR with dynamic light scattering, fluorescence spectroscopy, electron microscopy, wide-angle X-ray diffraction and cell viability assays, we identify a cluster of unique molecular signatures that distinguish toxic vs. nontoxic Aβ assemblies. These include the exposure of a hydrophobic surface spanning residues 17-28 and the concurrent shielding of the highly charged N-terminus. We show that the combination of these two dichotomous structural transitions promotes the colocalization and insertion of β-sheet rich Aβ n into the membrane, compromising membrane integrity. These previously elusive toxic surfaces mapped here provide an unprecedented foundation to establish structure-toxicity relationships of Aβ assemblies.