Mechanistic Basis for a Connection between the Catalytic Step and Slow Opening Dynamics of Adenylate Kinase.

Escherichia coli adenylate kinase (AdK) is a small, monomeric enzyme that synchronizes the catalytic step with the enzyme's conformational dynamics to optimize a phosphoryl transfer reaction and the subsequent release of the product. Guided by experimental measurements of low catalytic activity in seven single-point mutation AdK variants (K13Q, R36A, R88A, R123A, R156K, R167A, and D158A), we utilized classical mechanical simulations to probe mutant dynamics linked to product release, and quantum mechanical and molecular mechanical calculations to compute a free energy barrier for the catalytic event. The goal was to establish a mechanistic connection between the two activities. Our calculations of the free energy barriers in AdK variants were in line with those from experiments, and conformational dynamics consistently demonstrated an enhanced tendency toward enzyme opening. This indicates that the catalytic residues in the wild-type AdK serve a dual role in this enzyme's function─one to lower the energy barrier for the phosphoryl transfer reaction and another to delay enzyme opening, maintaining it in a catalytically active, closed conformation for long enough to enable the subsequent chemical step. Our study also discovers that while each catalytic residue individually contributes to facilitating the catalysis, R36, R123, R156, R167, and D158 are organized in a tightly coordinated interaction network and collectively modulate AdK's conformational transitions. Unlike the existing notion of product release being rate-limiting, our results suggest a mechanistic interconnection between the chemical step and the enzyme's conformational dynamics acting as the bottleneck of the catalytic process. Our results also suggest that the enzyme's active site has evolved to optimize the chemical reaction step while slowing down the overall opening dynamics of the enzyme.

The bacterial curli system possesses a potent and selective inhibitor of amyloid formation.

Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation and host colonization. An efficient secretion system and chaperone network ensures that the major curli fiber subunit, CsgA, does not form intracellular amyloid aggregates. We discovered that the periplasmic protein CsgC was a highly effective inhibitor of CsgA amyloid formation. In the absence of CsgC, CsgA formed toxic intracellular aggregates. In vitro, CsgC inhibited CsgA amyloid formation at substoichiometric concentrations and maintained CsgA in a non-ß-sheet-rich conformation. Interestingly, CsgC inhibited amyloid assembly of human α-synuclein, but not Aß42, in vitro. We identified a common D-Q-Φ-X0,1-G-K-N-ζ-E motif in CsgC client proteins that is not found in Aß42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids.

Domain-specific insight into the recognition of BH3-death motifs by the pro-survival Bcl-2 protein.

Programmed mammalian cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of dysfunctional cells. In its intrinsic (mitochondrial) pathway, opposing members of the Bcl-2 (B cell lymphoma 2) protein family meet at the mitochondrial outer membrane (MOM) to control its integrity. Any imbalance can cause disorders, with upregulation of the cell-guarding antiapoptotic Bcl-2 protein itself being common in many, often incurable, cancers. Normally, the Bcl-2 protein itself is embedded in the MOM where it sequesters cell-killing apoptotic proteins such as Bax (Bcl-2-associated X protein) that would otherwise perforate the MOM and subsequently cause cell death. However, the molecular basis of Bcl-2's ability to recognize those apoptotic proteins via their common BH3 death motifs remains elusive due to the lack of structural insight. By employing nuclear magnetic resonance on fully functional human Bcl-2 protein in membrane-mimicking micelles, we identified glycine residues across all functional domains of the Bcl-2 protein and could monitor their residue-specific individual response upon the presence of a Bax-derived 36aa long BH3 domain. The observed chemical shift perturbations allowed us to determine the response and individual affinity of each glycine residue and provide an overall picture of the individual roles by which Bcl-2's functional domains engage in recognizing and inhibiting apoptotic proteins via their prominent BH3 motifs. This way, we provide a unique residue- and domain-specific insight into the molecular functioning of Bcl-2 at the membrane level, an insight also opening up for interfering with this cell-protecting mechanism in cancer therapy.

Dissociation between the critical role of ClpB of Francisella tularensis for the heat shock response and the DnaK interaction and its important role for efficient type VI secretion and bacterial virulence.

Francisella tularensis, a highly infectious, intracellular bacterium possesses an atypical type VI secretion system (T6SS), which is essential for its virulence. The chaperone ClpB, a member of the Hsp100/Clp family, is involved in Francisella T6SS disassembly and type VI secretion (T6S) is impaired in its absence. We asked if the role of ClpB for T6S was related to its prototypical role for the disaggregation activity. The latter is dependent on its interaction with the DnaK/Hsp70 chaperone system. Key residues of the ClpB-DnaK interaction were identified by molecular dynamic simulation and verified by targeted mutagenesis. Using such targeted mutants, it was found that the F. novicida ClpB-DnaK interaction was dispensable for T6S, intracellular replication, and virulence in a mouse model, although essential for handling of heat shock. Moreover, by mutagenesis of key amino acids of the Walker A, Walker B, and Arginine finger motifs of each of the two Nucleotide-Binding Domains, their critical roles for heat shock, T6S, intracellular replication, and virulence were identified. In contrast, the N-terminus was dispensable for heat shock, but required for T6S, intracellular replication, and virulence. Complementation of the ΔclpB mutant with a chimeric F. novicida ClpB expressing the N-terminal of Escherichia coli, led to reconstitution of the wild-type phenotype. Collectively, the data demonstrate that the ClpB-DnaK interaction does not contribute to T6S, whereas the N-terminal and NBD domains displayed critical roles for T6S and virulence.

Tandem Ring Opening/Intramolecular [2 + 2] Cycloaddition Reaction for the Synthesis of Cyclobutane Fused Thiazolino-2-Pyridones.

Reaction of thiazoline fused 2-pyridones with alkyl halides in the presence of cesium carbonate opens the thiazoline ring via S-alkylation and generates N-alkenyl functionalized 2-pyridones. In the reaction with propargyl bromide, the thiazoline ring opens and subsequently closes via a [2 + 2] cycloaddition between an in situ generated allene and the α,ß-unsaturated methyl ester. This method enabled the synthesis of a variety of cyclobutane fused thiazolino-2-pyridones, of which a few analogues inhibit amyloid ß1-40 fibril formation. Furthermore, other analogues were able to bind mature α-synuclein and amyloid ß1-40 fibrils. Several thiazoline fused 2-pyridones with biological activity tolerate this transformation, which in addition provides an exocyclic alkene as a potential handle for tuning bioactivity.

K2S2O8-mediated coupling of 6-amino-7-aminomethyl-thiazolino-pyridones with aldehydes to construct amyloid affecting pyrimidine-fused thiazolino-2-pyridones.

We herein present the synthesis of diversely functionalized pyrimidine fused thiazolino-2-pyridones via K2S2O8-mediated oxidative coupling of 6-amino-7-(aminomethyl)-thiazolino-2-pyridones with aldehydes. The developed protocol is mild, has wide substrate scope, and does not require transition metal catalyst or base. Some of the synthesized compounds have an ability to inhibit the formation of Amyloid-ß fibrils associated with Alzheimer's disease, while others bind to mature amyloid-ß and α-synuclein fibrils.

Synthetic NAC 71-82 Peptides Designed to Produce Fibrils with Different Protofilament Interface Contacts.

Alpha-synucleinopathies are featured by fibrillar inclusions in brain cells. Although α-synuclein fibrils display structural diversity, the origin of this diversity is not fully understood. We used molecular dynamics simulations to design synthetic peptides, based on the NAC 71-82 amino acid fragment of α-synuclein, that govern protofilament contacts and generation of twisted fibrillar polymorphs. Four peptides with structures based on either single or double fragments and capped or non-capped ends were selected for further analysis. We determined the fibrillar yield and the structures from these peptides found in the solution after fibrillisation using protein concentration determination assay and circular dichroism spectroscopy. In addition, we characterised secondary structures formed by individual fibrillar complexes using laser-tweezers Raman spectroscopy. Results suggest less mature fibrils, based on the lower relative ß-sheet content for double- than single-fragment peptide fibrils. We confirmed this structural difference by TEM analysis which revealed, in addition to short protofibrils, more elongated, twisted and rod-like fibril structures in non-capped and capped double-fragment peptide systems, respectively. Finally, time-correlated single-photon counting demonstrated a difference in the Thioflavin T fluorescence lifetime profiles upon fibril binding. It could be proposed that this difference originated from morphological differences in the fibril samples. Altogether, these results highlight the potential of using peptide models for the generation of fibrils that share morphological features relevant for disease, e.g., twisted and rod-like polymorphs.

Insight into Functional Membrane Proteins by Solution NMR: The Human Bcl-2 Protein-A Promising Cancer Drug Target.

Evasion from programmed cell death (apoptosis) is the main hallmark of cancer and a major cause of resistance to therapy. Many tumors simply ensure survival by over-expressing the cell-protecting (anti-apoptotic) Bcl-2 membrane protein involved in apoptotic regulation. However, the molecular mechanism by which Bcl-2 protein in its mitochondrial outer membrane location protects cells remains elusive due to the absence of structural insight; and current strategies to therapeutically interfere with these Bcl-2 sensitive cancers are limited. Here, we present an NMR-based approach to enable structural insight into Bcl-2 function; an approach also ideal as a fragment-based drug discovery platform for further identification and development of promising molecular Bcl-2 inhibitors. By using solution NMR spectroscopy on fully functional intact human Bcl-2 protein in a membrane-mimicking micellar environment, and constructs with specific functions remaining, we present a strategy for structure determination and specific drug screening of functional subunits of the Bcl-2 protein as targets. Using 19F NMR and a specific fragment library (Bionet) with fluorinated compounds we can successfully identify various binders and validate our strategy in the hunt for novel Bcl-2 selective cancer drug strategies to treat currently incurable Bcl-2 sensitive tumors.

A novel recombinant expression and purification approach for the full-length anti-apoptotic membrane protein Bcl-2.

Programmed cell death (apoptosis) is an essential mechanism in life that tightly regulates embryogenesis and removal of harmful cells. Besides an extrinsic pathway, an intrinsic (mitochondrial) apoptotic pathway exists where mitochondria are actively involved in cellular clearance in response to internal stress signals. Pro-apoptotic (death) and anti-apoptotic (survival) members of the B cell CLL/lymphoma-2 (Bcl-2) protein family meet at the mitochondrion's surface where they accurately regulate apoptosis. Overexpression of the anti-apoptotic Bcl-2 protein is a hallmark for many types of cancers and in particular for many treatment resistant tumors. Bcl-2 is a membrane protein residing in the mitochondrial outer membrane. Due to its typical membrane protein features including very limited solubility, it is difficult to express and to purify. Therefore, most biophysical and structural studies have used truncated, soluble versions. However, to understand its membrane-coupled function and structure, access to sufficient amount of full-length human Bcl-2 protein is a necessity. Here, we present a novel, E. coli based approach for expression and purification of preparative amounts of the full-length human isoform 2 of Bcl-2 (Bcl-2(2)), solubilized in detergent micelles, which allows for easy exchange of the detergent.

Intramolecular Povarov Reactions for the Synthesis of Chromenopyridine Fused 2-Pyridone Polyheterocycles Binding to α-Synuclein and Amyloid-ß Fibrils.

A BF3·OEt2 catalyzed intramolecular Povarov reaction was used to synthesize 15 chromenopyridine fused thiazolino-2-pyridone peptidomimetics. The reaction works with several O-alkylated salicylaldehydes and amino functionalized thiazolino-2-pyridones, to generate polyheterocycles with diverse substitution. The synthesized compounds were screened for their ability to bind α-synuclein and amyloid ß fibrils in vitro. Analogues substituted with a nitro group bind to mature amyloid fibrils, and the activity moreover depends on the positioning of this functional group.

Inhibition of curli assembly and Escherichia coli biofilm formation by the human systemic amyloid precursor transthyretin.

During biofilm formation, Escherichia coli and other Enterobacteriaceae produce an extracellular matrix consisting of curli amyloid fibers and cellulose. The precursor of curli fibers is the amyloidogenic protein CsgA. The human systemic amyloid precursor protein transthyretin (TTR) is known to inhibit amyloid-ß (Aß) aggregation in vitro and suppress the Alzheimer's-like phenotypes in a transgenic mouse model of Aß deposition. We hypothesized that TTR might have broad antiamyloid activity because the biophysical properties of amyloids are largely conserved across species and kingdoms. Here, we report that both human WT tetrameric TTR (WT-TTR) and its engineered nontetramer-forming monomer (M-TTR, F87M/L110M) inhibit CsgA amyloid formation in vitro, with M-TTR being the more efficient inhibitor. Preincubation of WT-TTR with small molecules that occupy the T4 binding site eliminated the inhibitory capacity of the tetramer; however, they did not significantly compromise the ability of M-TTR to inhibit CsgA amyloidogenesis. TTR also inhibited amyloid-dependent biofilm formation in two different bacterial species with no apparent bactericidal or bacteriostatic effects. These discoveries suggest that TTR is an effective antibiofilm agent that could potentiate antibiotic efficacy in infections associated with significant biofilm formation.

A Capped Peptide of the Aggregation Prone NAC 71-82 Amino Acid Stretch of α-Synuclein Folds into Soluble ß-Sheet Oligomers at Low and Elevated Peptide Concentrations.

Although Lewy bodies and Lewy neurites are hallmarks of Parkinson's disease (PD) and dementia with Lewy bodies (DLB), misfolded α-synuclein oligomers are nowadays believed to be key for the development of these diseases. Attempts to target soluble misfolded species of the full-length protein have been limited so far, probably due to the fast aggregation kinetics and burial of aggregation prone segments in final cross-ß-sheet fibrils. A previous characterisation study of fibrils prepared from a capped peptide of the non-amyloid ß-component (NAC) 71-82 amino acid stretch of α-synuclein demonstrated an increased aggregation propensity resulting in a cross-ß-structure that is also found in prion proteins. From this, it was suggested that capped NAC 71-82 peptide oligomers would provide interesting motifs with a capacity to regulate disease development. Here, we demonstrated, from a series of circular dichroism spectroscopic measurements and molecular dynamics simulations, the molecular-environment-sensitive behaviour of the capped NAC 71-82 peptide in a solution phase and the formation of ß-sheet oligomeric structures in the supernatant of a fibrillisation mixture. These results highlighted the use of the capped NAC 71-82 peptide as a motif in the preparation of oligomeric ß-sheet structures that potentially could be used in therapeutic strategies in the fight against progressive neurodegenerative disorders, such as PD and DLB.

Viral mimetic priming enhances α-synuclein-induced degeneration: Implications for Parkinson's disease.

Evidence is accumulating to suggest that viral infections and consequent viral-mediated neuroinflammation may contribute to the etiology of idiopathic Parkinson's disease. Moreover, viruses have been shown to influence α-synuclein oligomerization as well as the autophagic clearance of abnormal intra-cellular proteins aggregations, both of which are key neuropathological events in Parkinson's disease pathogenesis. To further investigate the interaction between viral-mediated neuroinflammation and α-synuclein aggregation in the context of Parkinson's disease, this study sought to determine the impact of viral neuroinflammatory priming on α-synuclein aggregate-induced neuroinflammation and neurotoxicity in the rat nigrostriatal pathway. To do so, male Sprague-Dawley rats were intra-nigrally injected with a synthetic mimetic of viral dsRNA (poly I:C) followed two weeks later by a peptidomimetic small molecule which accelerates α-synuclein fibril formation (FN075). The impact of the viral priming on α-synuclein aggregation-induced neuroinflammation, neurodegeneration and motor dysfunction was assessed. We found that prior administration of the viral mimetic poly I:C significantly exacerbated or precipitated the α-synuclein aggregate induced neuropathological and behavioral effects. Specifically, sequential exposure to the two challenges caused a significant increase in nigral microgliosis (p < 0.001) and astrocytosis (p < 0.01); precipitated a significant degeneration of the nigrostriatal cell bodies (p < 0.05); and precipitated a significant impairment in forelimb kinesis (p < 0.01) and sensorimotor integration (p < 0.01). The enhanced sensitivity of the nigrostriatal neurons to pathological α-synuclein aggregation after viral neuroinflammatory priming further suggests that viral infections may contribute to the etiology and pathogenesis of Parkinson's disease.

Bax to the future - A novel, high-yielding approach for purification and expression of full-length Bax protein for structural studies.

Mitochondria-mediated apoptosis (programmed cell death) involves a sophisticated signaling and regulatory network that is regulated by the Bcl-2 protein family. Members of this family have either pro- or anti-apoptotic functions. An important pro-apoptotic member of this family is the cytosolic Bax. This protein is crucial for the onset of apoptosis by perforating the mitochondrial outer membrane (MOM). This process can be seen as point of no return, since disintegration of the MOM leads to the release of apotogenic factors such as cytochrome c into the cytosol triggering the activation of caspases and subsequent apoptotic steps. Bax is able to interact with the MOM with both its termini, making it inherently difficult to express in E. coli. In this study, we present a novel approach to express and purify full-length Bax with significantly increased yields, when compared to the commonly applied strategy. Using a double fusion approach with an N-terminal GST-tag and a C-terminal Intein-CBD-tag, we were able to render both Bax termini inactive and prevent disruptive interactions from occurring during gene expression. By deploying an Intein-CBD-tag at the C-terminus we were further able to avoid the introduction of any artificial residues, hence ensuring the native like activity of the membrane-penetrating C-terminus of Bax. Further, by engineering a His6-tag to the C-terminus of the CBD-tag we greatly improved the robustness of the purification procedure. We report yields for pure, full-length Bax protein that are increased by an order of magnitude, when compared to commonly used Bax expression protocols.

Pyridine-Fused 2-Pyridones via Povarov and A3 Reactions: Rapid Generation of Highly Functionalized Tricyclic Heterocycles Capable of Amyloid Fibril Binding.

We here describe the use of three-component reactions to synthesize tricyclic pyridine ring-fused 2-pyridones. The developed protocols have a wide substrate scope and allow for the installation of diverse chemical functionalities on the tricyclic central fragment. Several of these pyridine-fused rigid polyheterocycles are shown to bind to Aß and α-synuclein fibrils, which are associated with neurodegenerative diseases.

Copper binding triggers compaction in N-terminal tail of human copper pump ATP7B.

Protein conformational changes are fundamental to biological reactions. For copper ion transport, the multi-domain protein ATP7B in the Golgi network receives copper from the cytoplasmic copper chaperone Atox1 and, with energy from ATP hydrolysis, moves the metal to the lumen for loading of copper-dependent enzymes. Although anticipated, conformational changes involved in ATP7B's functional cycle remain elusive. Using spectroscopic methods we here demonstrate that the four most N-terminal metal-binding domains in ATP7B, upon stoichiometric copper addition, adopt a more compact arrangement which has a higher thermal stability than in the absence of copper. In contrast to previous reports, no stable complex was found in solution between the metal-binding domains and the nucleotide-binding domain of ATP7B. Metal-dependent movement of the first four metal-binding domains in ATP7B may be a trigger that initiates the overall catalytic cycle.

Correlated inter-domain motions in adenylate kinase.

Correlated inter-domain motions in proteins can mediate fundamental biochemical processes such as signal transduction and allostery. Here we characterize at structural level the inter-domain coupling in a multidomain enzyme, Adenylate Kinase (AK), using computational methods that exploit the shape information encoded in residual dipolar couplings (RDCs) measured under steric alignment by nuclear magnetic resonance (NMR). We find experimental evidence for a multi-state equilibrium distribution along the opening/closing pathway of Adenylate Kinase, previously proposed from computational work, in which inter-domain interactions disfavour states where only the AMP binding domain is closed. In summary, we provide a robust experimental technique for study of allosteric regulation in AK and other enzymes.

Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro.

After Ctr1-mediated copper ion (Cu) entry into the human cytoplasm, chaperones Atox1 and CCS deliver Cu to P1B-type ATPases and to superoxide dismutase, respectively, via direct protein-protein interactions. Although the two Cu chaperones are presumed to work along independent pathways, we here assessed cross-reactivity between Atox1 and the first domain of CCS (CCS1) using biochemical and biophysical methods in vitro. By NMR we show that CCS1 is monomeric although it elutes differently from Atox1 in size exclusion chromatography (SEC). This property allows separation of Atox1 and CCS1 by SEC and, combined with the 254/280 nm ratio as an indicator of Cu loading, we demonstrate that Cu can be transferred from one protein to the other. Cu exchange also occurs with full-length CCS and, as expected, the interaction involves the metal binding sites since mutation of Cu-binding cysteine in Atox1 eliminates Cu transfer from CCS1. Cross-reactivity between CCS and Atox1 may aid in regulation of Cu distribution in the cytoplasm.

Macromolecular crowding effects on two homologs of ribosomal protein s16: protein-dependent structural changes and local interactions.

Proteins function in cellular environments that are crowded with biomolecules, and in this reduced available space, their biophysical properties may differ from those observed in dilute solutions in vitro. Here, we investigated the effects of a synthetic macromolecular crowding agent, dextran 20, on the folded states of hyperthermophilic (S16Thermo) and mesophilic (S16Meso) homologs of the ribosomal protein S16. As expected for an excluded-volume effect, the resistance of the mesophilic protein to heat-induced unfolding increased in the presence of dextran 20, and chemical denaturation experiments at different fixed temperatures showed the macromolecular crowding effect to be temperature-independent. Förster resonance energy transfer experiments show that intramolecular distances between an intrinsic Trp residue and BODIPY-labeled S16Meso depend on the level of the crowding agent. The BODIPY group was attached at three specific positions in S16Meso, allowing measurements of three intraprotein distances. All S16Meso variants exhibited a decrease in the average Trp-BODIPY distance at up to 100 mg/mL dextran 20, whereas the changes in distance became anisotropic (one distance increased, two distances decreased) at higher dextran concentrations. In contrast, the two S16Thermo mutants did not show any changes in Trp-BODIPY distances upon increase of dextran 20 concentrations. It should be noted that the fluorescence quantum yields and lifetimes of BODIPY attached to the two S16 homologs decreased gradually in the presence of dextran 20. To investigate the origin of this decrease, we studied the BODIPY quantum yield in three protein variants in the presence of a tyrosine-labeled dextran. The experiments revealed distinct tyrosine quenching behaviors of BODIPY in the three variants, suggesting a dynamic local interaction between dextran and one particular S16 variant.

Folding of an unfolded protein by macromolecular crowding in vitro.

Protein folding in vivo takes place in a highly crowded environment. The resulting excluded volume forces are thought to stabilize folded forms of proteins. In agreement, many in vitro studies have shown that the presence of macromolecular crowding agents increases the stability of folded proteins but often by only a few kJ per mol. Although it should not matter at what position in the transition between folded and unfolded forms the effect of crowding is employed, there have been no studies assessing whether excluded volume forces alone can correctly fold polypeptides that are mostly unfolded. However, some studies have indicated that the effect of crowding becomes larger the more destabilized the protein is (but still being folded), suggesting that the crowding effect may be exaggerated for unfolded proteins. To address this question directly, we turned to a destabilized mutant of protein L that is mostly unfolded in water but can be folded upon addition of salt. We find that the effect of 200 mg/mL Dextran 20 on the folding equilibrium constant for unfolded protein L (ΔΔGU ≈ 2 kJ mol(-1)) matches the crowding effects found on the folded wild type protein and the mutant when prefolded by salt. This result indicates that the excluded volume effect is independent of starting protein stability and that crowding can shift the reaction toward the folded form when the polypeptide is in the transition region between folded and unfolded states.