Structural Basis of Tirasemtiv Activation of Fast Skeletal Muscle.

Troponin regulates the calcium-mediated activation of skeletal muscle. Muscle weakness in diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy occurs from diminished neuromuscular output. The first direct fast skeletal troponin activator, tirasemtiv, amplifies the response of muscle to neuromuscular input. Tirasemtiv binds selectively and strongly to fast skeletal troponin, slowing the rate of calcium release and sensitizing muscle to calcium. We report the solution NMR structure of tirasemtiv bound to a fast skeletal troponin C-troponin I chimera. The structure reveals that tirasemtiv binds in a hydrophobic pocket between the regulatory domain of troponin C and the switch region of troponin I, which overlaps with that of Anapoe in the X-ray structure of skeletal troponin. Multiple interactions stabilize the troponin C-troponin I interface, increase the affinity of troponin C for the switch region of fast skeletal troponin I, and drive the equilibrium toward the active state.

Calcium binds and rigidifies the dysferlin C2A domain in a tightly coupled manner.

The membrane protein dysferlin (DYSF) is important for calcium-activated plasma membrane repair, especially in muscle fibre cells. Nearly 600 mutations in the DYSF gene have been identified that are causative for rare genetic forms of muscular dystrophy. The dysferlin protein consists of seven C2 domains (C2A-C2G, 13%-33% identity) used to recruit calcium ions and traffic accessory proteins and vesicles to injured membrane sites needed to reseal a wound. Amongst these, the C2A is the most prominent facilitating the calcium-sensitive interaction with membrane surfaces. In this work, we determined the calcium-free and calcium-bound structures of the dysferlin C2A domain using NMR spectroscopy and X-ray crystallography. We show that binding two calcium ions to this domain reduces the flexibility of the Ca2+-binding loops in the structure. Furthermore, calcium titration and mutagenesis experiments reveal the tight coupling of these calcium-binding sites whereby the elimination of one site abolishes calcium binding to its partner site. We propose that the electrostatic potential distributed by the flexible, negatively charged calcium-binding loops in the dysferlin C2A domain control first contact with calcium that promotes subsequent binding. Based on these results, we hypothesize that dysferlin uses a 'calcium-catching' mechanism to respond to calcium influx during membrane repair.

The ER chaperone calnexin controls mitochondrial positioning and respiration.

Chaperones in the endoplasmic reticulum (ER) control the flux of Ca2+ ions into mitochondria, thereby increasing or decreasing the energetic output of the oxidative phosphorylation pathway. An example is the abundant ER lectin calnexin, which interacts with sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). We found that calnexin stimulated the ATPase activity of SERCA by maintaining its redox state. This function enabled calnexin to control how much ER Ca2+ was available for mitochondria, a key determinant for mitochondrial bioenergetics. Calnexin-deficient cells compensated for the loss of this function by partially shifting energy generation to the glycolytic pathway. These cells also showed closer apposition between the ER and mitochondria. Calnexin therefore controls the cellular energy balance between oxidative phosphorylation and glycolysis.

Metabolomic study of disease progression in scrapie prion infected mice; validation of a novel method for brain metabolite extraction.

INTRODUCTION: Prion disease is a form of neurodegenerative disease caused by the misfolding and aggregation of cellular prion protein (PrPC). The neurotoxicity of the misfolded form of prion protein, PrPSc still remains understudied. Here we try to investigate this issue using a metabolomics approach. OBJECTIVES: The intention was to identify and quantify the small-in-size and water-soluble metabolites extracted from mice brains infected with the Rocky Mountain Laboratory isolate of mouse-adapted scrapie prions (RML) and track changes in these metabolites during disease evolution. METHODS: A total of 73 mice were inoculated with RML prions or normal brain homogenate control; brains were harvested at 30, 60, 90, 120 and 150 days post-inoculation (dpi). We devised a high-efficiency metabolite extraction method and used nuclear magnetic resonance spectroscopy to identify and quantify 50 metabolites in the brain extracts. Data were analyzed using multivariate approach. RESULTS: Brain metabolome profiles of RML infected animals displayed continuous changes throughout the course of disease. Among the analyzed metabolites, the most noteworthy changes included increases in myo-inositol and glutamine as well as decreases in 4-aminobutyrate, acetate, aspartate and taurine. CONCLUSION: We report a novel metabolite extraction method for lipid-rich tissue. As all the major metabolites are identifiable and quantifiable by magnetic resonance spectroscopy, this study suggests that tracking of neurochemical profiles could be effective in monitoring the progression of neurodegenerative diseases and useful for assessing the efficacy of candidate therapeutics.

Characterizing the inhibition of α-synuclein oligomerization by a pharmacological chaperone that prevents prion formation by the protein PrP.

Aggregation of the disordered protein α-synuclein into amyloid fibrils is a central feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease. Small, pre-fibrillar oligomers of misfolded α-synuclein are thought to be the key toxic entities, and α-synuclein misfolding can propagate in a prion-like way. We explored whether a compound with anti-prion activity that can bind to unfolded parts of the protein PrP, the cyclic tetrapyrrole Fe-TMPyP, was also active against α-synuclein aggregation. Observing the initial stages of aggregation via fluorescence cross-correlation spectroscopy, we found that Fe-TMPyP inhibited small oligomer formation in a dose-dependent manner. Fe-TMPyP also inhibited the formation of mature amyloid fibrils in vitro, as detected by thioflavin T fluorescence. Isothermal titration calorimetry indicated Fe-TMPyP bound to monomeric α-synuclein with a stoichiometry of 2, and two-dimensional heteronuclear single quantum coherence NMR spectra revealed significant interactions between Fe-TMPyP and the C-terminus of the protein. These results suggest commonalities among aggregation mechanisms for α-synuclein and the prion protein may exist that can be exploited as therapeutic targets.

Role of Polyubiquitin Chain Flexibility in the Catalytic Mechanism of Cullin-RING Ubiquitin Ligases.

Cullin-RING ubiquitin ligases are a diverse family of ubiquitin ligases that catalyze the synthesis of K48-linked polyubiquitin (polyUb) chains on a variety of substrates, ultimately leading to their degradation by the proteasome. The cullin-RING enzyme scaffold processively attaches a Ub molecule to the distal end of a growing chain up to lengths of eight Ub monomers. However, the molecular mechanism governing how chains of increasing size are built using a scaffold of largely fixed dimensions is not clear. We developed coarse-grained molecular dynamics simulations to describe the dependence of kcat for cullin-RING ligases on the length and flexibility of the K48-linked polyUb chain attached to the substrate protein, key factors that determine the rate of subsequent Ub attachment to the chain, and therefore, the ensuing biological outcomes of ubiquitination. The results suggest that a number of regulatory mechanisms may lead to variations in the rate of chain elongation for different cullin-RING ligases. Specifically, modulation of the distance between the target lysine and the phosphodegron sequence of the substrate, the distance between the substrate lysine and the active site cysteine of the Ub conjugation enzyme (E2) bound to the cullin-RING scaffold, and flexibility of the bound E2 can lead to significant differences in the processing of K48-linked chains on substrates, potentially leading to differences in biological outcomes.

Insights into the Mechanism of Action of the Two-Peptide Lantibiotic Lacticin 3147.

Lacticin 3147 is a two peptide lantibiotc (LtnA1 and LtnA2) that displays nanomolar activity against many Gram-positive bacteria. Lacticin 3147 may exert its antimicrobial effect by several mechanisms. Isothermal titration calorimetry experiments show that only LtnA1 binds to the peptidoglycan precursor lipid II, which could inhibit peptidoglycan biosynthesis. An experimentally supported model of the resulting complex suggests that the key binding partners are the C-terminus of LtnA1 and pyrophosphate of lipid II. A combination of in vivo and in vitro assays indicates that LtnA1 and LtnA2 can induce rapid membrane lysis without the need for lipid II binding. However, the presence of lipid II substantially increases the activity of lacticin 3147. Furthermore, studies with synthetic LtnA2 analogues containing either desmethyl- or oxa-lanthionine rings confirm that the precise geometry of these rings is essential for this synergistic activity.

Dataset of urinary metabolites measured by 1H NMR analysis of normal human urine.

The data in this article are related to the research entitled, "Assessment of 1H NMR-based metabolomics analysis for normalization of urinary metals against creatinine" (M. Cassiède, S. Nair, M. Dueck, J. Mino, R. McKay, P. Mercier, B. Quémerais, P. Lacy, 2016) [1]. This article describes the analysis of urinary metabolites in normal, healthy individuals by 1H NMR-based metabolomics. NMR spectra of urine samples typically contain hundreds of peaks that must be carefully screened for reproducibility and detectability. An important requirement in the screening of appropriate urinary metabolites is to ensure that they are reproducibly detected. In our study, we applied the peak profiles of 151 known urinary metabolites to 10 normal human urine samples and found that 50 metabolites were reproducibly measured between 600 and 700 MHz magnets in the same samples. The data set has been made publicly available to enable critical or extended analysis.

Structure of phosphorylated UBL domain and insights into PINK1-orchestrated parkin activation.

Mutations in PARK2 and PARK6 genes are responsible for the majority of hereditary Parkinson's disease cases. These genes encode the E3 ubiquitin ligase parkin and the protein kinase PTEN-induced kinase 1 (PINK1), respectively. Together, parkin and PINK1 regulate the mitophagy pathway, which recycles damaged mitochondria following oxidative stress. Native parkin is inactive and exists in an autoinhibited state mediated by its ubiquitin-like (UBL) domain. PINK1 phosphorylation of serine 65 in parkin's UBL and serine 65 of ubiquitin fully activate ubiquitin ligase activity; however, a structural rationale for these observations is not clear. Here, we report the structure of the phosphorylated UBL domain from parkin. We find that destabilization of the UBL results from rearrangements to hydrophobic core packing that modify its structure. Altered surface electrostatics from the phosphoserine group disrupt its intramolecular association, resulting in poorer autoinhibition in phosphorylated parkin. Further, we show that phosphorylation of both the UBL domain and ubiquitin are required to activate parkin by releasing the UBL domain, forming an extended structure needed to facilitate E2-ubiquitin binding. Together, the results underscore the importance of parkin activation by the PINK1 phosphorylation signal and provide a structural picture of the unraveling of parkin's ubiquitin ligase potential.

Assessment of 1H NMR-based metabolomics analysis for normalization of urinary metals against creatinine.

BACKGROUND: Proton nuclear magnetic resonance (1H NMR, or NMR) spectroscopy and inductively coupled plasma-mass spectrometry (ICP-MS) are commonly used for metabolomics and metal analysis in urine samples. However, creatinine quantification by NMR for the purpose of normalization of urinary metals has not been validated. We assessed the validity of using NMR analysis for creatinine quantification in human urine samples in order to allow normalization of urinary metal concentrations. METHODS: NMR and ICP-MS techniques were used to measure metabolite and metal concentrations in urine samples from 10 healthy subjects. For metabolite analysis, two magnetic field strengths (600 and 700MHz) were utilized. In addition, creatinine concentrations were determined by using the Jaffe method. RESULTS: Creatinine levels were strongly correlated (R2=0.99) between NMR and Jaffe methods. The NMR spectra were deconvoluted with a target database containing 151 metabolites that are present in urine. A total of 50 metabolites showed good correlation (R2=0.7-1.0) at 600 and 700MHz. Metal concentrations determined after NMR-measured creatinine normalization were comparable to previous reports. CONCLUSIONS: NMR analysis provided robust urinary creatinine quantification, and was sufficient for normalization of urinary metal concentrations. We found that NMR-measured creatinine-normalized urinary metal concentrations in our control subjects were similar to general population levels in Canada and the United Kingdom.

Antimicrobial lipopeptide tridecaptin A1 selectively binds to Gram-negative lipid II.

Tridecaptin A1 (TriA1) is a nonribosomal lipopeptide with selective antimicrobial activity against Gram-negative bacteria. Here we show that TriA1 exerts its bactericidal effect by binding to the bacterial cell-wall precursor lipid II on the inner membrane, disrupting the proton motive force. Biochemical and biophysical assays show that binding to the Gram-negative variant of lipid II is required for membrane disruption and that only the proton gradient is dispersed. The NMR solution structure of TriA1 in dodecylphosphocholine micelles with lipid II has been determined, and molecular modeling was used to provide a structural model of the TriA1-lipid II complex. These results suggest that TriA1 kills Gram-negative bacteria by a mechanism of action using a lipid-II-binding motif.

(1)H-NMR urinary metabolomic profiling for diagnosis of gastric cancer.

BACKGROUND: Metabolomics has shown promise in gastric cancer (GC) detection. This research sought to identify whether GC has a unique urinary metabolomic profile compared with benign gastric disease (BN) and healthy (HE) patients. METHODS: Urine from 43 GC, 40 BN, and 40 matched HE patients was analysed using (1)H nuclear magnetic resonance ((1)H-NMR) spectroscopy, generating 77 reproducible metabolites (QC-RSD <25%). Univariate and multivariate (MVA) statistics were employed. A parsimonious biomarker profile of GC vs HE was investigated using LASSO regularised logistic regression (LASSO-LR). Model performance was assessed using Receiver Operating Characteristic (ROC) curves. RESULTS: GC displayed a clear discriminatory biomarker profile; the BN profile overlapped with GC and HE. LASSO-LR identified three discriminatory metabolites: 2-hydroxyisobutyrate, 3-indoxylsulfate, and alanine, which produced a discriminatory model with an area under the ROC of 0.95. CONCLUSIONS: GC patients have a distinct urinary metabolite profile. This study shows clinical potential for metabolic profiling for early GC diagnosis.

Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis.

The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N-terminal ubiquitin-like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin-binding interface, provide a model for the phosphoubiquitin-parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin-binding site used by the E2~Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2~Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin.

Structure of the HHARI catalytic domain shows glimpses of a HECT E3 ligase.

The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn²âº-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.

Ataxin-3 is a multivalent ligand for the parkin Ubl domain.

The ubiquitin signaling pathway consists of hundreds of enzymes that are tightly regulated for the maintenance of cell homeostasis. Parkin is an E3 ubiquitin ligase responsible for conjugating ubiquitin onto a substrate protein, which itself can be ubiquitinated. Ataxin-3 performs the opposing function as a deubiquitinating enzyme that can remove ubiquitin from parkin. In this work, we have identified the mechanism of interaction between the ubiquitin-like (Ubl) domain from parkin and three C-terminal ubiquitin-interacting motifs (UIMs) in ataxin-3. (1)H-(15)N heteronuclear single-quantum coherence titration experiments revealed that there are weak direct interactions between all three individual UIM regions of ataxin-3 and the Ubl domain. Each UIM utilizes the exposed ß-grasp surface of the Ubl domain centered around the I44 patch that did not vary in the residues involved or the surface size as a function of the number of ataxin-3 UIMs involved. Further, the apparent dissociation constant for ataxin-3 decreased as a function of the number of UIM regions used in experiments. A global multisite fit of the nuclear magnetic resonance titration data, based on three identical binding ligands, resulted in a KD of 669 ± 62 µM for each site. Our observations support a multivalent ligand binding mechanism employed by the parkin Ubl domain to recruit multiple UIM regions in ataxin-3 and provide insight into how these two proteins function together in ubiquitination-deubiquitination pathways.

A molecular explanation for the recessive nature of parkin-linked Parkinson's disease.

Mutations in the park2 gene, encoding the RING-inBetweenRING-RING E3 ubiquitin ligase parkin, cause 50% of autosomal recessive juvenile Parkinsonism cases. More than 70 known pathogenic mutations occur throughout parkin, many of which cluster in the inhibitory amino-terminal ubiquitin-like domain, and the carboxy-terminal RING2 domain that is indispensable for ubiquitin transfer. A structural rationale showing how autosomal recessive juvenile Parkinsonism mutations alter parkin function is still lacking. Here we show that the structure of parkin RING2 is distinct from canonical RING E3 ligases and lacks key elements required for E2-conjugating enzyme recruitment. Several pathogenic mutations in RING2 alter the environment of a single surface-exposed catalytic cysteine to inhibit ubiquitination. Native parkin adopts a globular inhibited conformation in solution facilitated by the association of the ubiquitin-like domain with the RING-inBetweenRING-RING C-terminus. Autosomal recessive juvenile Parkinsonism mutations disrupt this conformation. Finally, parkin autoubiquitinates only in cis, providing a molecular explanation for the recessive nature of autosomal recessive juvenile Parkinsonism.

Towards automatic metabolomic profiling of high-resolution one-dimensional proton NMR spectra.

Nuclear magnetic resonance (NMR) and Mass Spectroscopy (MS) are the two most common spectroscopic analytical techniques employed in metabolomics. The large spectral datasets generated by NMR and MS are often analyzed using data reduction techniques like Principal Component Analysis (PCA). Although rapid, these methods are susceptible to solvent and matrix effects, high rates of false positives, lack of reproducibility and limited data transferability from one platform to the next. Given these limitations, a growing trend in both NMR and MS-based metabolomics is towards targeted profiling or "quantitative" metabolomics, wherein compounds are identified and quantified via spectral fitting prior to any statistical analysis. Despite the obvious advantages of this method, targeted profiling is hindered by the time required to perform manual or computer-assisted spectral fitting. In an effort to increase data analysis throughput for NMR-based metabolomics, we have developed an automatic method for identifying and quantifying metabolites in one-dimensional (1D) proton NMR spectra. This new algorithm is capable of using carefully constructed reference spectra and optimizing thousands of variables to reconstruct experimental NMR spectra of biofluids using rules and concepts derived from physical chemistry and NMR theory. The automated profiling program has been tested against spectra of synthetic mixtures as well as biological spectra of urine, serum and cerebral spinal fluid (CSF). Our results indicate that the algorithm can correctly identify compounds with high fidelity in each biofluid sample (except for urine). Furthermore, the metabolite concentrations exhibit a very high correlation with both simulated and manually-detected values.

Is there nascent structure in the intrinsically disordered region of troponin I?

In striated muscle, the binding of calcium to troponin C (TnC) results in the removal of the C-terminal region of the inhibitory protein troponin I (TnI) from actin. While structural studies of the muscle system have been successful in determining the overall organization of most of the components involved in force generation at the atomic level, the structure and dynamics of the C-terminal region of TnI remains controversial. This domain of TnI is highly flexible, and it has been proposed that this intrinsically disordered region (IDR) regulates contraction via a "fly-casting" mechanism. Different structures have been presented for this region using different methodologies: a single α-helix, a "mobile domain" containing a small ß-sheet, an unstructured region, and a two helix segment. To investigate whether this IDR has in fact any nascent structure, we have constructed a skeletal TnC-TnI chimera that contains the N-domain of TnC (1-90), a short linker (GGAGG), and the C-terminal region of TnI (97-182) and have acquired ¹5N NMR relaxation data for this chimera. We compare the experimental relaxation parameters with those calculated from molecular dynamic simulations using four models based upon the structural studies. Our experimental results suggest that the C-terminal region of TnI does not contain any defined secondary structure, supporting the "fly-casting" mechanism. We interpret the presence of a "plateau" in the ¹5N NMR relaxation data as being an intrinsic property of IDRs. We also identified a more rigid adjacent region of TnI that has implications for muscle performance under ischemic conditions.

The effect of the cosolvent trifluoroethanol on a tryptophan side chain orientation in the hydrophobic core of troponin C.

The unique biophysical properties of tryptophan residues have been exploited for decades to monitor protein structure and dynamics using a variety of spectroscopic techniques, such as fluorescence and nuclear magnetic resonance (NMR). We recently designed a tryptophan mutant in the regulatory N-domain of cardiac troponin C (F77W-cNTnC) to study the domain orientation of troponin C in muscle fibers using solid-state NMR. In our previous study, we determined the NMR structure of calcium-saturated mutant F77W-V82A-cNTnC in the presence of 19% 2,2,2-trifluoroethanol (TFE). TFE is a widely used cosolvent in the biophysical characterization of the solution structures of peptides and proteins. It is generally assumed that the structures are unchanged in the presence of cosolvents at relatively low concentrations, and this has been verified for TFE at the level of the overall secondary and tertiary structure for several calcium regulatory proteins. Here, we present the NMR solution structure of the calcium saturated F77W-cNTnC in presence of its biological binding partner troponin I peptide (cTnI(144-163)) and in the absence of TFE. We have also characterized a panel of six F77W-cNTnC structures in the presence and absence TFE, cTnI(144-163), and the extra mutation V82A, and used (19)F NMR to characterize the effect of TFE on the F77(5fW) analog. Our results show that although TFE did not perturb the overall protein structure, TFE did induce a change in the orientation of the indole ring of the buried tryptophan side chain from the anticipated position based upon homology with other proteins, highlighting the potential dangers of the use of cosolvents.

Metabolite profiling of the intraerythrocytic malaria parasite Plasmodium falciparum by (1)H NMR spectroscopy.

NMR spectroscopy was used to identify and quantify compounds in extracts prepared from mature trophozoite-stage Plasmodium falciparum parasites isolated by saponin-permeabilisation of the host erythrocyte. One-dimensional (1)H NMR spectroscopy and four two-dimensional NMR techniques were used to identify more than 50 metabolites. The intracellular concentrations of over 40 metabolites were estimated from the (1)H NMR spectra of extracts prepared by four extraction methods: perchloric acid, methanol/water, methanol/chloroform/water, and methanol alone. The metabolites quantified included: the majority of the biological alpha-amino acids; 4-aminobutyric acid; mono-, di- and tri-carboxylic acids; nucleotides; polyamines; myo-inositol; and phosphocholine and phosphoethanolamine. The parasites also contained a significant concentration (up to 12 mM) of the exogenous buffering agent, HEPES. Although the metabolite profiles obtained with each extraction method were broadly similar, perchloric acid was found to have significant advantages over the other extraction media.