Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL₅) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL₅ ↔ FL₅ mechanism, in which the final helical state, FL₅, forms from IL₅ with a time constant of 50–200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.     

FapA is an Intrinsically Disordered Chaperone for Pseudomonas Functional Amyloid FapC

Bacterial functional amyloids contribute to biofilm development by bacteria and provide protection from the immune system and prevent antibiotic treatment. Strategies to target amyloid formation and interrupt biofilm formation have attracted recent interest due to their antimicrobial potential. Functional amyloid in Pseudomonas (Fap) includes FapC as the major component of the fibril while FapB is a minor component suggested to function as a nucleator of FapC. The system also includes the small periplasmic protein FapA, which has been shown to regulate fibril composition and morphology. The interplay between these three components is central in Fap fibril biogenesis. Here we present a comprehensive biophysical and spectroscopy analysis of FapA, FapB and FapC and provide insight into their molecular interactions. We show that all three proteins are primarily disordered with some regions with structural propensities for α-helix and β-sheet. FapA inhibits FapC fibrillation by targeting the nucleation step, whereas for FapB the elongation step is modulated. Furthermore, FapA alters the morphology of FapC (more than FapB) fibrils. Complex formation is observed between FapA and FapC, but not between FapA and FapB, and likely involves the N-terminus of FapA. We conclude that FapA is an intrinsically disordered chaperone for FapC that guards against fibrillation within the periplasm. This new understanding of a natural protective mechanism of Pseudomonas against amyloid formations can serve as inspiration for strategies blocking biofilm formation in infections.     

Towards Decoding the Sequence-Based Grammar Governing the Functions of Intrinsically Disordered Protein Regions

A substantial portion of the proteome consists of intrinsically disordered regions (IDRs) that do not fold into well-defined 3D structures yet perform numerous biological functions and are associated with a broad range of diseases. It has been a long-standing enigma how different IDRs successfully execute their specific functions. Further putting a spotlight on IDRs are recent discoveries of functionally relevant biomolecular assemblies, which in some cases form through liquid-liquid phase separation. At the molecular level, the formation of biomolecular assemblies is largely driven by weak, multivalent, but selective IDR-IDR interactions. Emerging experimental and computational studies suggest that the primary amino acid sequences of IDRs encode a variety of their interaction behaviors. In this review, we focus on findings and insights that connect sequence-derived features of IDRs to their conformations, propensities to form biomolecular assemblies, selectivity of interaction partners, functions in the context of physiology and disease, and regulation of function. We also discuss directions of future research to facilitate establishing a comprehensive sequence-function paradigm that will eventually allow prediction of selective interactions and specificity of function mediated by IDRs.     

Dynamic Autoinhibition of the HMGB1 Protein via Electrostatic Fuzzy Interactions of Intrinsically Disordered Regions

Highly negatively charged segments containing only aspartate or glutamate residues (“D/E repeats”) are found in many eukaryotic proteins. For example, the C-terminal 30 residues of the HMGB1 protein are entirely D/E repeats. Using nuclear magnetic resonance (NMR), fluorescence, and computational approaches, we investigated how the D/E repeats causes the autoinhibition of HMGB1 against its specific binding to cisplatin-modified DNA. By varying ionic strength in a wide range (40–900 mM), we were able to shift the conformational equilibrium between the autoinhibited and uninhibited states toward either of them to the full extent. This allowed us to determine the macroscopic and microscopic equilibrium constants for the HMGB1 autoinhibition at various ionic strengths. At a macroscopic level, a model involving the autoinhibited and uninhibited states can explain the salt concentration-dependent binding affinity data. Our data at a microscopic level show that the D/E repeats and other parts of HMGB1 undergo electrostatic fuzzy interactions, each of which is weaker than expected from the macroscopic autoinhibitory effect. This discrepancy suggests that the multivalent nature of the fuzzy interactions enables strong autoinhibition at a macroscopic level despite the relatively weak intramolecular interaction at each site. Both experimental and computational data suggest that the D/E repeats interact preferentially with other intrinsically disordered regions (IDRs) of HMGB1. We also found that mutations mimicking post-translational modifications relevant to nuclear export of HMGB1 can moderately modulate DNA-binding affinity, possibly by impacting the autoinhibition. This study illuminates a functional role of the fuzzy interactions of D/E repeats.     

Temporal Analysis of Protein Ubiquitylation and Phosphorylation During Parkin-Dependent Mitophagy

Mitophagy, the selective degradation of mitochondria by autophagy, affects defective mitochondria following damage or stress. At the onset of mitophagy, parkin ubiquitylates proteins on the mitochondrial outer membrane. While the role of parkin at the onset of mitophagy is well understood, less is known about its activity during later stages in the process. Here, we used HeLa cells expressing catalytically active or inactive parkin to perform temporal analysis of the proteome, ubiquitylome, and phosphoproteome during 18 h after induction of mitophagy by mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine. Abundance profiles of proteins downregulated in parkin-dependent manner revealed a stepwise and “outside–in” directed degradation of mitochondrial subcompartments. While ubiquitylation of mitochondrial outer membrane proteins was enriched among early parkin-dependent targets, numerous mitochondrial inner membrane, matrix, and cytosolic proteins were also found ubiquitylated at later stages of mitophagy. Phosphoproteome analysis revealed a possible crosstalk between phosphorylation and ubiquitylation during mitophagy on key parkin targets, such as voltage-dependent anion channel 2.     

Towards Molecular Understanding of the pH Dependence Characterizing NhaA of Which Structural Fold is Shared by Other Transporters

Na⁺/H⁺ antiporters comprise a super-family (CPA) of membrane proteins that are found in all kingdoms of life and are essential in cellular homeostasis of pH, Na⁺ and volume. Their activity is strictly dependent on pH, a property that underpins their role in pH homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystal structure provided insight into the architecture of this molecular machine. However, the mechanism of the strict pH dependence of NhaA is missing. Here, as a follow up of a recent evolutionary analysis that identified a ‘CPA motif’, we rationally designed three E. coli NhaA mutants: D133S, I134T, and the double mutant D133S-I134T. Exploring growth phenotype, transport activity and Li⁺-binding of the mutants, we revealed that Asp133 does not participate directly in proton binding, nor does it directly dictate the pH-dependent transport of NhaA. Strikingly, the variant I134T lost some of the pH control, and the D133S-Il134T double mutant retained Li⁺ binding in a pH independent fashion. Concurrent to loss of pH control, these mutants bound Li⁺ more strongly than the WT. Both positions are in close vicinity to the ion-binding site of the antiporter, attributing the results to electrostatic interaction between these residues and Asp164 of the ion-binding site. This is consistent with pH sensing resulting from direct coupling between cation binding and deprotonation in Asp164, which applies also to other CPA antiporters that are involved in human diseases.     

Targeting the DNA damage response (DDR) by natural compounds

Natural compounds (NC) are an important source of anticancer drugs. The genomic DNA of tumor cells is a major target of conventional anticancer therapeutics (cAT). DNA damage elicits a complex stress response programme termed DNA damage response (DDR), with the PI3-like kinase ATM and ATR being the key regulators. Since the DDR coordinates mechanisms of DNA repair and apoptosis, hence regulating the balance between death and survival, it is an attractive target of novel anticancer strategies. The aim of the study was to identify natural compounds derived from endophytic fungi, lichens, marine sponges or plants that interfere with mechanisms of the DDR. To this end, the cytotoxic and DDR modulating potency of 296 natural compounds, used alone or in combination with the cAT cisplatin (Cis) and doxorubicin (Doxo) was investigated by fluorescence-based analysis of the ATM/ATR-catalyzed S139 phosphorylation of histone 2AX (γH2AX), a surrogate marker of DNA damage-triggered DDR. After initial screening, a total of ten natural compounds were identified that were toxic in pancreatic carcinoma cells and activated the DDR on their own and/or promoted the DDR if used in combination with cAT. Their mode of action was shown to be independent of drug transport mechanisms. Based on their chemical structures, DDR modulatory activity and published data we suggest the marine NC 5-epi-nakijiquinone Q and 5-epi-ilimaquinone as well as the fungal compound secalonic acid F as most promising NC-based drug candidates for future synthesis of DDR-modulating chemical derivatives and their preclinical in vitro and in vivo testing.     

Heteroarylamide smoothened inhibitors: Discovery of N-[2,4-dimethyl-5-(1-methylimidazol-4-yl)phenyl]-4-(2-pyridylmethoxy)benzamide (AZD8542) and N-[5-(1H-imidazol-2-yl)-2,4-dimethyl-phenyl]-4-(2- pyridylmethoxy)benzamide (AZD7254)

Aberrant hedgehog (Hh) pathway signaling is implicated in multiple cancer types and targeting the Smoothened (SMO) receptor, a key protein of the Hh pathway, has proven effective in treating metastasized basal cell carcinoma. Our lead optimization effort focused on a series of heteroarylamides. We observed that a methyl substitution ortho to the heteroaryl groups on an aniline core significantly improved the potency of this series of compounds. These findings predated the availability of SMO crystal structure in 2013. Here we retrospectively applied quantum mechanics calculations to demonstrate the o-Me substitution favors the bioactive conformation by inducing a dihedral twist between the heteroaryl rings and the core aniline. The o-Me also makes favorable hydrophobic interactions with key residue side chains in the binding pocket. From this effort, two compounds (AZD8542 and AZD7254) showed excellent pharmacokinetics across multiple preclinical species and demonstrated in vivo activity in abrogating the Hh paracrine pathway as well as anti- tumor effects.     

Widespread Arginine Phosphorylation in Staphylococcus aureus

Arginine phosphorylation was only recently discovered to play a significant and relevant role in the Gram-positive bacterium Bacillus subtilis. In addition, arginine phosphorylation was also detected in Staphylococcus aureus, suggesting a widespread role in bacteria. However, the large-scale analysis of protein phosphorylation, and especially those that involve a phosphoramidate bond, comes along with several challenges. The substoichiometric nature of protein phosphorylation requires proper enrichment strategies prior to LC-MS/MS analysis, and the acid instability of phosphoramidates was long thought to impede those enrichments. Furthermore, good spectral quality is required, which can be impeded by the presence of neutral losses of phosphoric acid upon higher energy collision–induced dissociation. Here we show that pArg is stable enough for commonly used Fe³⁺-IMAC enrichment followed by LC-MS/MS and that HCD is still the gold standard for the analysis of phosphopeptides. By profiling a serine/threonine kinase (Stk1) and phosphatase (Stp1) mutant from a methicillin-resistant S. aureus mutant library, we identified 1062 pArg sites and thus the most comprehensive arginine phosphoproteome to date. Using synthetic arginine phosphorylated peptides, we validated the presence and localization of arginine phosphorylation in S. aureus. Finally, we could show that the knockdown of Stp1 significantly increases the overall amount of arginine phosphorylation in S. aureus. However, our analysis also shows that Stp1 is not a direct protein-arginine phosphatase but only indirectly influences the arginine phosphoproteome.     

The chromosome-scale assembly of endive (Cichorium endivia) genome provides insights into the sesquiterpenoid biosynthesis

Endive (Cichorium endivia L.) is a leafy vegetable in the Asteraceae family. Sesquiterpene lactones (STLs) in endive leaves bring a bitter taste that varies between varieties. Despite their importance in breeding varieties with unique flavours, sesquiterpenoid biosynthesis pathways in endive are poorly understood. We assembled a chromosome-scale endive genome of 641 Mb with a contig N50 of 5.16 Mb and annotated 46,711 protein-coding genes. Several gene families, especially terpene synthases (TPS) genes, expanded significantly in the C. endivia genome. STLs biosynthesis-related genes and TPS genes in more bitter varieties have shown a higher level of expression, which could be attributed to genomic variations. Our results penetrate the origin and diversity of bitter taste and facilitate the molecular breeding of endive varieties with unique bitter tastes. The high-quality endive assembly would provide a reference genome for studying the evolution and diversity of Asteraceae.     

Prioritizing conservation areas and vulnerability analyses of the genus Pinus L. (Pinaceae) in Mexico

Mexico hosts the highest species richness of pines (Pinus, Pinaceae) worldwide; however, the priority areas for their conservation in the country are unknown. In this study, the ecological niche of the 50 native pine species was modeled. Then, through a multi-criteria analysis, the priority areas for the conservation of the genus Pinus were identified according to the spatial patterns of richness, geographic rareness, irreplaceability, the level of vulnerability of their habitat and the status of legal protection. The results revealed that the regions with high species richness differed from those with high endemism. Also, most pine species have undergone processes of habitat degradation, having been the endemic species the most affected. The priority areas covered regions with high species richness, high endemism, and highly degraded forests, located at mountainous portions of the Baja California Peninsula, the Sierra Madre Occidental, the Sierra Madre Oriental, the Trans-Mexican Volcanic Belt, and the Sierra Madre del Sur. A low proportion of priority areas overlapped with protected areas or terrestrial regions considered priorities for biological conservation. These results suggest that conservation efforts for this genus should be focused beyond regions with high species richness and current protected areas. Besides, the priority areas identified in this study can be the basis to create biological corridors and new protected areas, which could contribute significantly to the conservation of this genus in Mexico.     

Molecular dynamics and simulation analysis against superoxide dismutase (SOD) target of Micrococcus luteus with secondary metabolites from Bacillus licheniformis recognized by genome mining approach

Micrococcus luteus, also known as M. luteus, is a bacterium that inhabits mucous membranes, human skin, and various environmental sources. It is commonly linked to infections, especially among individuals who have compromised immune systems. M. luteus is capable of synthesizing the enzyme superoxide dismutase (SOD) as a component of its protective response to reactive oxygen species (ROS). This enzyme serves as a promising target for drug development in various diseases. The current study utilized a subtractive genomics approach to identify potential therapeutic targets from M. luteus. Additionally, genome mining was employed to identify and characterize the biosynthetic gene clusters (BGCs) responsible for the production of secondary metabolites in Bacillus licheniformis (B. licheniformis), a bacterium known for its production of therapeutically relevant secondary metabolites. Subtractive genomics resulted in identification of important extracellular protein SOD as a drug target that plays a crucial role in shielding cells from damage caused by ROS. Genome mining resulted in identification of five potential ligands (secondary metabolites) from B. licheniformis such as, Bacillibactin (BAC), Paenibactin (PAE), Fengycin (FEN), Surfactin (SUR) and Lichenysin (LIC). Molecular docking was used to predict and analyze the binding interactions between these five ligands and target protein SOD. The resulting protein–ligand complexes were further analyzed for their motions and interactions of atoms and molecules over 250 ns using molecular dynamics (MD) simulation analysis. The analysis of MD simulations suggests, Bacillibactin as the probable candidate to arrest the activities of SOD. All the five compounds reported in this study were found to act by directly/indirectly interacting with ROS molecules, such as superoxide radicals (O₂–) and hydrogen peroxide (H₂O₂), and transforming them into less reactive species. This antioxidant activity contributes to its protective effects against oxidative stress-induced damage in cells making them likely candidate for various applications, including in the development of antioxidant-based therapies, nutraceuticals, and functional foods.     

P300 Interacted With N-Myc and Regulated Its Protein Stability via Altering Its Post-Translational Modifications in Neuroblastoma

MYCN amplification is an independent risk factor for poor prognosis in neuroblastoma (NB), but its protein product cannot be directly targeted because of protein structure. Thus, this study aimed to explore novel ways to indirectly target N-Myc by regulating its post-translational modifications (PTMs) and therefore protein stability. N-Myc coimmunoprecipitation combined with HPLC–MS/MS identified 16 PTM residues and 114 potential N-Myc-interacting proteins. Notably, both acetylation and ubiquitination were identified on lysine 199 of N-Myc. We then discovered that p300, which can interact with N-Myc, modulated the protein stability of N-Myc in MYCN-amplified NB cell lines and simultaneously regulated the acetylation level and ubiquitination level on lysine-199 of N-Myc protein in vitro. Furthermore, p300 correlated with poor prognosis in NB patients. Taken together, p300 can be considered as a potential therapeutic target to treat MYCN-amplified NB patients, and other identified PTMs and interacting proteins also provide potential targets for further study.     

The Structure of Saccharomyces cerevisiae Arginyltransferase 1 (ATE1)

Eukaryotic post-translational arginylation, mediated by the family of enzymes known as the arginyltransferases (ATE1s), is an important post-translational modification that can alter protein function and even dictate cellular protein half-life. Multiple major biological pathways are linked to the fidelity of this process, including neural and cardiovascular developments, cell division, and even the stress response. Despite this significance, the structural, mechanistic, and regulatory mechanisms that govern ATE1 function remain enigmatic. To that end, we have used X-ray crystallography to solve the crystal structure of ATE1 from the model organism Saccharomyces cerevisiae ATE1 (ScATE1) in the apo form. The three-dimensional structure of ScATE1 reveals a bilobed protein containing a GCN5-related N-acetyltransferase (GNAT) fold, and this crystalline behavior is faithfully recapitulated in solution based on size-exclusion chromatography-coupled small angle X-ray scattering (SEC-SAXS) analyses and cryo-EM 2D class averaging. Structural superpositions and electrostatic analyses point to this domain and its domain-domain interface as the location of catalytic activity and tRNA binding, and these comparisons strongly suggest a mechanism for post-translational arginylation. Additionally, our structure reveals that the N-terminal domain, which we have previously shown to bind a regulatory [Fe-S] cluster, is dynamic and disordered in the absence of metal bound in this location, hinting at the regulatory influence of this region. When taken together, these insights bring us closer to answering pressing questions regarding the molecular-level mechanism of eukaryotic post-translational arginylation.     

A Conserved Motif in Intracellular Loop 1 Stabilizes the Outward-Facing Conformation of TmrAB

The ATP binding cassette (ABC) family of transporters moves small molecules (lipids, sugars, peptides, drugs, nutrients) across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) is currently an outstanding question. Here we use sequence coevolution analyses together with biochemical characterization to investigate the role of a highly conserved region in intracellular loop 1 we define as the GRD motif in coordinating domain rearrangements in the heterodimeric peptide exporter from Thermus thermophilus, TmrAB. Mutations in the GRD motif alter ATPase activity as well as transport. Disulfide crosslinking, evolutionary trace, and evolutionary coupling analysis reveal that these effects are likely due to the destabilization of a network in which the GRD motif in TmrA bridges residues of the Q-loop, X-loop, and ABC motif in the NBDs to residues in the TmrAB peptide substrate binding site, thus providing an avenue for conformational coupling. We further find that disruption of this network in TmrA versus TmrB has different functional consequences, hinting at an intrinsic asymmetry in heterodimeric ABC transporters extending beyond that of the NBDs. These results support a mechanism in which the GRD motifs help coordinate a transition to an outward open conformation, and each half of the transporter likely plays a different role in the conformational cycle of TmrAB.     

Structural insights into the Switching Off of the Interaction between the Archaeal Ribosomal Stalk and aEF1A by Nucleotide Exchange Factor aEF1B

The ribosomal stalk protein plays a crucial role in functional interactions with translational GTPase factors. It has been shown that the archaeal stalk aP1 binds to both GDP- and GTP-bound conformations of aEF1A through its C-terminal region in two different modes. To obtain an insight into how the aP1•aEF1A binding mode changes during the process of nucleotide exchange from GDP to GTP on aEF1A, we have analyzed structural changes in aEF1A upon binding of the nucleotide exchange factor aEF1B. The isolated archaeal aEF1B has nucleotide exchange ability in the presence of aa-tRNA but not deacylated tRNA, and increases activity of polyphenylalanine synthesis 4-fold. The aEF1B mutation, R90A, results in loss of its original nucleotide exchange activity but retains a remarkable ability to enhance polyphenylalanine synthesis. These results suggest an additional functional role for aEF1B other than in nucleotide exchange. The crystal structure of the aEF1A•aEF1B complex, resolved at 2.0 Å resolution, shows marked rotational movement of domain 1 of aEF1A compared to the structure of aEF1A•GDP•aP1, and this conformational change results in disruption of the original aP1 binding site between domains 1 and 3 of aEF1A. The loss of aP1 binding to the aEF1A•aEF1B complex was confirmed by native gel analysis. The results suggest that aEF1B plays a role in switching off the interaction between aP1 and aEF1A•GDP, as well as in nucleotide exchange, and promote translation elongation.