Early-Onset Parkinson Mutation Remodels Monomer-Fibril Interactions to Allosterically Amplify Synuclein's Amyloid Cascade.

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.

QSAR models reveal new EPAC-selective allosteric modulators.

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.

An auto-inhibited state of protein kinase G and implications for selective activation.

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 Iß 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 Iß 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 Iß 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 Iß 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.

Allosteric pluripotency: challenges and opportunities.

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.

Non-Canonical Allostery in Cyclic Nucleotide Dependent Kinases.

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.

State-selective frustration as a key driver of allosteric pluripotency.

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 (ΔG R,Gap) relative to the effective free energy of R1a binding to the catalytic subunit of PKA (ΔG R:C) dictates whether the antagonism-to-agonism switch occurs. However, the key drivers of ΔG R,Gap are not fully understood. Here, by analyzing an R1a mutant that selectively silences allosteric pluripotency, we show that a major determinant of ΔG 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 ΔG R,Gap to values comparable to ΔG 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.

Backbone resonance assignment of the cAMP-binding domains of the protein kinase A regulatory subunit Iα.

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 1H, 13C, and 15 N backbone assignments of cAMP-bound RIα (119-379) CBDs (BMRB No. 50920).

Noncanonical protein kinase A activation by oligomerization of regulatory subunits as revealed by inherited Carney complex mutations.

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.

Atomic Resolution Map of Hierarchical Self-Assembly for an Amyloidogenic Protein Probed through Thermal 15N-R2 Correlation Matrices.

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 15N - R2 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,15N - R2 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 15N - R2 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 15N - R2 with a positive ΔR2/ΔT slope. These two hallmarks are systematically probed through a thermal R2 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.

Mechanism of hERG inhibition by gating-modifier toxin, APETx1, deduced by functional characterization.

BACKGROUND: Human ether-à-go-go-related gene potassium channel 1 (hERG) is a voltage-gated potassium channel, the voltage-sensing domain (VSD) of which is targeted by a gating-modifier toxin, APETx1. APETx1 is a 42-residue peptide toxin of sea anemone Anthopleura elegantissima and inhibits hERG by stabilizing the resting state. A previous study that conducted cysteine-scanning analysis of hERG identified two residues in the S3-S4 region of the VSD that play important roles in hERG inhibition by APETx1. However, mutational analysis of APETx1 could not be conducted as only natural resources have been available until now. Therefore, it remains unclear where and how APETx1 interacts with the VSD in the resting state. RESULTS: We established a method for preparing recombinant APETx1 and determined the NMR structure of the recombinant APETx1, which is structurally equivalent to the natural product. Electrophysiological analyses using wild type and mutants of APETx1 and hERG revealed that their hydrophobic residues, F15, Y32, F33, and L34, in APETx1, and F508 and I521 in hERG, in addition to a previously reported acidic hERG residue, E518, play key roles in the inhibition of hERG by APETx1. Our hypothetical docking models of the APETx1-VSD complex satisfied the results of mutational analysis. CONCLUSIONS: The present study identified the key residues of APETx1 and hERG that are involved in hERG inhibition by APETx1. These results would help advance understanding of the inhibitory mechanism of APETx1, which could provide a structural basis for designing novel ligands targeting the VSDs of KV channels.

CHESPA/CHESCA-SPARKY: automated NMR data analysis plugins for SPARKY to map protein allostery.

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.

Allosteric inhibition explained through conformational ensembles sampling distinct "mixed" states.

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.

Allosteric Mechanisms of Nonadditive Substituent Contributions to Protein-Ligand Binding.

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.

An NMR based phosphodiesterase assay.

We propose a phosphodiesterase assay based on 1D 1H 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 KM and kcat parameters and to identify phosphodiesterase inhibitors.

Molecular Mechanism for the Suppression of Alpha Synuclein Membrane Toxicity by an Unconventional Extracellular Chaperone.

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 (αSn) toxicity through a three-pronged mechanism. First, endogenous HSA targets αSn 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.

Mechanism of allosteric inhibition in the Plasmodium falciparum cGMP-dependent protein kinase.

Most malaria deaths are caused by the protozoan parasite Plasmodium falciparum Its life cycle is regulated by a cGMP-dependent protein kinase (PfPKG), 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 PfPKG inhibition are incompletely understood. Here, we show that 8-NBD-cGMP is an effective PfPKG antagonist. Using comparative NMR analyses of a key regulatory domain, PfD, 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 PfPKG 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 PfPKG, without losing affinity for the cGMP analog. Our proposed model may facilitate the rational design of PfPKG-selective inhibitors for improved management of malaria.

Recent Advances in EPAC-Targeted Therapies: A Biophysical Perspective.

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.

The structure of the apo cAMP-binding domain of HCN4 - a stepping stone toward understanding the cAMP-dependent modulation of the hyperpolarization-activated cyclic-nucleotide-gated ion channels.

The hyperpolarization-activated cyclic-nucleotide-gated (HCN) ion channels control nerve impulse transmission and cardiac pacemaker activity. The modulation by cAMP is critical for the regulatory function of HCN in both neurons and cardiomyocytes, but the underlying mechanism is not fully understood. Here, we show how the structure of the apo cAMP-binding domain of the HCN4 isoform has contributed to a model for the cAMP-dependent modulation of the HCN ion-channel. This model recapitulates the structural and dynamical changes that occur along the thermodynamic cycle arising from the coupling of cAMP-binding and HCN self-association equilibria. The proposed model addresses some of the questions previously open about the auto-inhibition of HCN and its cAMP-induced activation, while opening new opportunities for selectively targeting HCN through allosteric ligands. A remaining challenge is the investigation of HCN dimers and their regulatory role. Overcoming this challenge will require the integration of crystallography, cryo electron microscopy, NMR, electrophysiology and simulations.

A Highly Dynamic Loop of the Pseudomonas aeruginosa PA14 Type IV Pilin Is Essential for Pilus Assembly.

Type IVa pili (T4aP) are long, thin surface filaments involved in attachment, motility, biofilm formation, and DNA uptake. They are important virulence factors for many bacteria, including Pseudomonas aeruginosa, an opportunistic pathogen and common cause of hospital-acquired infections. Each helical filament contains thousands of monomers of the major pilin subunit, PilA. Each P. aeruginosa strain expresses one of five phylogenetically distinct major pilins, which vary in sequence and the nature of their associated accessory protein(s). Here, we present the backbone resonance assignment of the C-terminal domain of the group III PilA from strain PA14, a highly virulent, globally distributed clone. Secondary structure probabilities calculated from chemical shifts were in excellent agreement with previous homology modeling using a group V pilin structural template. The analysis revealed that the distal segment of the αß loop had high microsecond-millisecond dynamics compared with other loop regions. Shortening of this segment by internal deletion abrogated pilus assembly in a dominant negative manner, suggesting a potential role in pilin polymerization. Pilin conformations that support optimal interactions of both the conserved hydrophobic N-termini in the pilus core and hydrophilic loops creating the filament surface may be necessary to produce stable filaments.

Free energy landscape remodeling of the cardiac pacemaker channel explains the molecular basis of familial sinus bradycardia.

The hyperpolarization-activated and cyclic nucleotide-modulated ion channel (HCN) drives the pacemaker activity in the heart, and its malfunction can result in heart disorders. One such disorder, familial sinus bradycardia, is caused by the S672R mutation in HCN, whose electrophysiological phenotypes include a negative shift in the channel activation voltage and an accelerated HCN deactivation. The outcomes of these changes are abnormally low resting heart rates. However, the molecular mechanism underlying these electrophysiological changes is currently not fully understood. Crystallographic investigations indicate that the S672R mutation causes limited changes in the structure of the HCN intracellular gating tetramer, but its effects on protein dynamics are unknown. Here, we utilize comparative S672R versus WT NMR analyses to show that the S672R mutation results in extensive perturbations of the dynamics in both apo- and holo-forms of the HCN4 isoform, reflecting how S672R remodels the free energy landscape for the modulation of HCN4 by cAMP, i.e. the primary cyclic nucleotide modulator of HCN channels. We show that the S672R mutation results in a constitutive shift of the dynamic auto-inhibitory equilibrium toward inactive states of HCN4 and broadens the free-energy well of the apo-form, enhancing the millisecond to microsecond dynamics of the holo-form at sites critical for gating cAMP binding. These S672R-induced variations in dynamics provide a molecular basis for the electrophysiological phenotypes of this mutation and demonstrate that the pathogenic effects of the S672R mutation can be rationalized primarily in terms of modulations of protein dynamics.