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bioRxiv2019

Lithium-ATP: structure characterization and lithium binding

Lithium, in the form of a salt, is a mood stabilizer and a leading drug for the treatment of bipolar disorder. It has a very narrow therapeutic range and a variety of side effects. Lithium can replace magnesium and other cations in enzymes and small molecules, among them ATP, thereby affecting and inhibiting many biochemical pathways. The form of binding of lithium ions to ATP is not known.\n\nHere we extract the binding environment of lithium in solid ATP using a multi-nuclear multi-dimensional solid-state NMR approach.\n\nWe determine that the coordination sphere of lithium includes, at a distance of 3.0({+/-}0.4) [A], three phosphates; the two phosphates closest to the ribose ring from one ATP molecule, and the middle phosphate from another ATP molecule. A water molecule most probably completes the fourth coordination. Despite the use of excess lithium in the preparations, sodium ions still remain bound to the sample, at distances of 4.3-5.5 [A] from Li, and coordinate the first phosphate and two terminal phosphates.\n\nIn conclusion, solid-state NMR enables to unravel the exact coordination of lithium in ATP showing binding to three phosphates from two molecules, none of which are the terminal gamma phosphate. The methods we use are applicable to study lithium bound to a variety of ATP-bound enzymes, or to other cellular targets of lithium, consequently suggesting a molecular basis for its mode of action.

biochemistry↗

bioRxiv2019

Molecular basis of EMRE-dependence of the human mitochondrial calcium uniporter

The mitochondrial uniporter is calcium-activated calcium channel complex critical for cellular signaling and bioenergetics. MCU, the pore-forming subunit of the uniporter, contains two transmembrane domains and is found in all major eukaryotic taxa. In amoeba and fungi, MCU homologs are sufficient to form a functional calcium channel, whereas human MCU exhibits a strict requirement for the metazoan-specific, single-pass transmembrane protein EMRE for conductance. Here, we exploit this evolutionary divergence to decipher the molecular basis of the human MCUs dependence on EMRE. By systematically generating chimeric proteins that consist of EMRE-independent D. discoideum MCU (DdMCU) and H. sapiens MCU (HsMCU), we converged on a stretch of 10 amino acids in DdMCU that can be transplanted to HsMCU to render it EMRE-dependent. We call this region in human MCU the EMRE-dependence domain (EDD). Crosslinking experiments show that HsEMRE directly interacts with MCU at both of its transmembrane domains as well as the EDD. Based on previously published structures of fungal MCU homologs, the EDD segment is located distal to the calcium pores selectivity filter and appears flexible. We propose that EMRE stabilizes EDD of MCU, permitting both channel opening and calcium conductance

biochemistry↗

bioRxiv2019

A role for the Saccharomyces cerevisiae ABCF protein New1 during translation termination

Translation on the ribosome is controlled by numerous accessory proteins and translation factors. In the yeast Saccharomyces cerevisiae, translation elongation requires an essential elongation factor, the ABCF ATPase eEF3. A closely related ABCF ATPase, New1, is encoded by a non-essential gene with a cold sensitivity and ribosome assembly defect knock-out phenotype. Since the exact molecular function of New1 is unknown, it is unclear if the ribosome assembly defect is direct, i.e. New1 is a bona fide ribosome assembly factor, or indirect, for instance due to a defect in protein synthesis. To investigate this, we employed a combination of yeast genetics, cryo-electron microscopy (cryo-EM) and ribosome profiling (Ribo-Seq) to interrogate the molecular function of New1. Overexpression of New1 rescues the inviability of a yeast strain lacking the otherwise strictly essential translation factor eEF3. The structure of the ATPase-deficient (EQ2) New1 mutant locked on the 80S ribosome reveals that New1 binds analogously to the ribosome as eEF3. Finally, Ribo-Seq analysis revealed that loss of New1 leads to ribosome queuing upstream of 3-terminal lysine and arginine codons, including those genes encoding proteins of the cytoplasmic translational machinery. Our results suggest that New1 is a translation factor that fine-tunes the efficiency of translation termination.

biochemistry↗

bioRxiv2019

The membrane insertion of soluble CLIC1 into active chloride channels is triggered by specific divalent cations

The CLIC family of proteins display the unique feature of altering their structure from a soluble form to a membrane-bound chloride channel. CLIC1, a member of this family, can be found in the cytoplasm or in nuclear, ER and plasma membranes, with membrane overexpression linked to tumour proliferation. The molecular switch promoting CLIC1 membrane insertion has been related to environmental factors, but still remains unclear. Here, we use solution NMR studies to confirm that both the soluble and membrane bound forms are in the same oxidation state. Our data from fluorescence assays and chloride efflux assays indicate that Ca2+ and Zn2+ trigger association to the membrane into active chloride channels. We use fluorescence microscopy to confirm that an increase of the intracellular Ca2+ leads to re-localisation of CLIC1 to both plasma and internal membranes. Finally, we show that soluble CLIC1 adopts an equilibrium of oligomeric species, and Ca2+/Zn2+ mediated membrane insertion promotes the formation of a tetrameric assembly. Thus, our results identify Ca2+ and Zn2+ binding as the molecular switch promoting CLIC1 membrane insertion.\n\nSIGNIFICANCE STATEMENTCLIC1, a member of the CLIC family of proteins, is expressed as a soluble protein in cells but can insert in the membrane forming a chloride channel. This chloride channel form is upregulated in different types of cancers including glioblastoma and promote tumour invasiveness and metastasis. The factors promoting CLIC1 membrane insertion nor the mechanism of this process are yet understood. Here, we use a combination of solution NMR, biophysics and fluorescence microscopy to identify Ca2+ and Zn2+ binding as the switch to promote CLIC1 insertion into the membrane to form active chloride channels. We also provide a simple mechanism how such transition to the membrane occurs. Such understanding will enable subsequent studies on the structure of the chloride channel form and its inhibition.

biochemistry↗

bioRxiv2019

Structural basis for tetherin antagonism as a barrier to zoonotic lentiviral transmission

Tetherin is a host defense that physically prevents escape of virions from the plasma membrane. Human tetherin lacks the motif DIWK antagonized by SIV, the antecedent of HIV. Here, we reconstituted the AP-2 clathrin adaptor complex with a simian tetherin and SIV Nef and determined its structure by cryo-EM. Nef refolds the first -helix of the {beta}2 subunit of AP-2 to a {beta} hairpin, creating a binding site for the DIWK sequence. The tetherin binding site in Nef is distinct from those of MHC-I, CD3, and CD4, but overlaps the site for SERINC5 restricting viral infectivity. The structure explains the dependence of SIVs on the host tetherin DIWK sequence and the consequent barrier to human transmission.

biochemistry↗

bioRxiv2019

Role of Water Mediated Interactions in Calcium-Coupled Allostery of Calmodulin Domains

Allosteric communication between distant parts of proteins controls many cellular functions, in which metal ions are widely utilized as effectors to trigger the allosteric cascade. Due to the involvement of strong coordination interactions, the energy landscape dictating the metal ion binding is intrinsically rugged. How metal ions achieve fast binding by overcoming the landscape ruggedness and thereby efficiently mediate protein allostery is elusive. By performing molecular dynamics simulations for the Ca2+ binding mediated allostery of the calmodulin (CaM) domains, each containing two Ca2+ binding helix-loop-helix motifs (EF-hands), we revealed the key role of water-bridged interactions in Ca2+ binding and protein allostery. The bridging water molecules between Ca2+ and binding residue reduces the ruggedness of ligand exchange landscape by acting as a lubricant, facilitating the Ca2+ coupled protein allostery. Calcium-induced rotation of the helices in the EF-hands, with the hydrophobic core serving as the pivot, leads to exposure of hydrophobic sites for target binding. Intriguingly, despite being structurally similar, the response of the two symmetrically arranged EF-hands upon Ca2+ binding is asymmetric. Breakage of symmetry is needed for efficient allosteric communication between the EF-hands. The key roles that water molecules play in driving allosteric transitions are likely to be general in other metal ion mediated protein allostery.

biochemistry↗

bioRxiv2019

Activation loop dynamics are controlled by conformation-selective inhibitors of ERK2

Modulating the dynamics of protein kinases expands the inhibitory mechanisms for small molecules. NMR measurements of the MAP kinase, ERK2, have shown that activation by dual-phosphorylation induces global motions involving exchange between two states, \"L\" and \"R\". We show that ERK inhibitors Vertex-11e and SCH772984 exploit the small energetic difference between L and R to shift the equilibrium in opposing directions, while inhibitor GDC-0994 and ATP analogue AMP-PNP retain L{rightleftharpoons}R exchange. An X-ray structure of active 2P-ERK2 complexed with AMP-PNP reveals a shift in the Gly-rich loop along with domain closure to position the nucleotide in a more catalytically productive conformation relative to inactive 0P-ERK2:ATP. X-ray structures of 2P-ERK2 complexed with Vertex-11e or GDC-0994 recapitulate this closure, which is blocked in a complex with a SCH772984 analogue. Thus, the L[->]R shift in 2P-ERK2 is associated with movements needed to form a competent active site. Solution measurements by hydrogen-exchange mass spectrometry (HX-MS) reveal distinct binding modes for Vertex-11e, GDC-0994 and AMP-PNP to active vs inactive ERK2, where the extent of HX protection matches their degree of R-state formation. In addition, Vertex-11e and SCH772984 show opposite effects on HX near the activation loop, suggesting that L{rightleftharpoons}R exchange involves coupling between the activation loop and the active site. Consequently, these inhibitors differentially affect MAP kinase phosphatase activity towards 2P-ERK2. We conclude that global motions in ERK2 promote productive nucleotide binding, and couple with the activation loop to allow control of dephosphorylation by conformation-selective inhibitors.\n\nSIGNIFICANCE STATEMENTProtein kinases in the RAF/MKK/ERK signaling pathway are dysregulated in cancer and are important targets for inhibitor development. Catalytic activation of the MAP kinase, ERK2, induces global motions involving exchange between two conformational states. Using nuclear magnetic resonance (NMR) and hydrogen-exchange mass spectrometry, we show that inhibitors exploit these motions to trap ERK2 in distinct states. Our findings reveal motions of the activation loop coupled to the active site. Inhibitor binding can control these activation loop dynamics to alter its rate of dephosphorylation by MAP kinase phosphatase.

biochemistry↗

bioRxiv2019

TDP-43 α-helical structure tunes liquid-liquid phase separation and function

Liquid-liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. Present in several MLOs, TDP-43 undergoes LLPS and is linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some disease variants of TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes in a dimeric TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix-helix interaction, which are removed in part by variants including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a mini-gene assay. Therefore, TDP-43 helical region serves as a short but uniquely tunable module that shows promise as for controlling assembly and function in cellular and synthetic biology applications of LLPS.

biochemistry↗

bioRxiv2019

A rationally designed and highly versatile epitope tag for nanobody-based purification, detection and manipulation of proteins

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a novel, rationally designed epitope tag that serves an exceptionally broad spectrum of applications in life sciences while outperforming established tags like the HA, FLAG or myc tags. The ALFA-tag forms a small and stable -helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We developed a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in-vivo detection of proteins. By solving the crystal structure of the complex we were able to design a nanobody mutant (NbALFAPE) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.

biochemistry↗

bioRxiv2019

How ATP is used by P-glycoprotein to export multiple hydrophobic compounds

P-glycoprotein (P-gp; also known as MDR1 or ABCB1) is an ATP-driven multidrug transporter that extrudes various hydrophobic toxic compounds to the extracellular space. P-gp consists of two transmembrane domains (TMDs) that form the substrate translocation pathway and two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. P-gp takes at least two states during transport; the inward-facing (pre-drug transport) conformation, in which the two NBDs are separated and the two TMDs are open to the intracellular side, and the outward-facing (post-drug transport) conformation, in which the NBDs are dimerized and the TMDs are slightly open to the extracellular side. ATP binding and hydrolysis cause conformational changes between the inward-facing and the outward-facing conformations to translocate substrates across the membrane. However, it remains unclear how ATP is used during these conformational changes in living cells. In this study, we investigated the role of ATP binding and hydrolysis during the conformational changes of human P-gp in living cells by using fluorescence resonance energy transfer (FRET). We show that ATP binding causes the conformational change to the outward-facing state and that ATP hydrolysis and subsequent release of {gamma}-phosphate from both NBDs allow the outward-facing state to return to the original inward-facing state.

biochemistry↗

bioRxiv2019

How thermophilic Gram-positive organisms perform extracellular electron transfer: characterization of the cell surface terminal reductase OcwA

Extracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or added-value compounds. Thermincola potens JR is a promising Gram-positive bacterium to be used in these systems because it is thermophilic. In this paper we describe the structural and functional properties of the nonaheme cytochrome OcwA, which is the terminal reductase of this organism. The structure of OcwA, determined at 2.2[A] resolution shows that the overall-fold and organization of the hemes are not related to other metal reductases and instead are similar to that of multiheme cytochromes involved in the biogeochemical cycles of nitrogen and sulfur. We show that, in addition to solid electron acceptors, OcwA can also reduce soluble electron shuttles and oxyanions. These data reveal that OcwA can take the role of a respiratory swiss-army knife allowing this organism to grow in environments with rapidly changing availability of terminal electron acceptors without the need for transcriptional regulation and protein synthesis.\n\nImportanceThermophilic Gram-positive organisms were recently shown to be a promising class of organisms to be used in bioelectrochemical systems for the production of electrical energy. These organisms present a thick peptidoglycan layer that was thought to preclude them to perform extracellular electron transfer (i.e. exchange catabolic electrons with solid electron acceptors outside of the cell). In this manuscript we describe the structure and functional mechanisms of the multiheme cytochrome OcwA, the terminal reductase of the Gram-positive bacterium Thermincola potens JR found at the cell surface of this organism. The results presented here show that this protein is unrelated with terminal reductases found at the cell surface of other electroactive organisms. Instead, OcwA is similar to terminal reductases of soluble electron acceptors. Our data reveals that terminal oxidoreductases of soluble and insoluble substrates are evolutionarily related, providing novel insights into the evolutionary pathway of multiheme cytochromes.

biochemistry↗

bioRxiv2019

Chromatin structure-dependent histone incorporation revealed by a genome-wide deposition assay

In eukaryotes, histone variant distribution within the genome is the key epigenetic feature. To understand how each histone variant is targeted to the genome, we developed a new method, in which epitope-tagged histone complexes are introduced into permeabilized cells and incorporated into their chromatin. We found that the incorporation of histones H2A and H2A.Z mainly occurred at less condensed chromatin (open), suggesting that the condensed chromatin (closed) is a barrier for histone incorporation. To overcome this barrier, H2A, but not H2A.Z, uses a replication-coupled deposition mechanism. This led to the recapitulation of the pre-existing chromatin structure: the genome-wide even distribution of H2A and the exclusion of H2A.Z from the closed chromatin. Intriguingly, an H2A.Z mutant with mutations in the developmentally essential region was incorporated into closed chromatin. Our study revealed that the combination of chromatin structure and DNA replication dictates the differential histone deposition for maintaining the epigenetic chromatin states.

biochemistry↗

bioRxiv2019

Simple rules govern the diversity of bacterial nicotianamine-like metallophores

In metal-scarce environments, some pathogenic bacteria produce opine-type metallophores mainly to face the hosts nutritional immunity. This is the case of staphylopine, pseudopaline and yersinopine, identified in Staphylococcus aureus, Pseudomonas aeruginosa and Yersinia pestis respectively. These metallophores are synthesized by two (CntLM) or three enzymes (CntKLM), CntM catalyzing the last step of biosynthesis using diverse substrates (pyruvate or -ketoglutarate), pathway intermediates (xNA or yNA) and cofactors (NADH or NADPH), depending on the species. Here, we explored substrate specificity of CntM by combining bioinformatics and structural analysis with chemical synthesis and enzymatic studies. We found that NAD(P)H selectivity was mainly due to the amino acid at position 33 (S. aureus numbering) which ensures a preferential binding to NADPH when it is an arginine. Moreover, whereas CntM from P. aeruginosa preferentially uses yNA over xNA, the staphylococcal enzyme is not stereospecific. Most importantly, selectivity towards -ketoacids is largely governed by a single residue at position 150 of CntM (S. aureus numbering): an aspartate at this position ensures selectivity towards pyruvate whereas an alanine leads to the consumption of both pyruvate and -ketoglutarate. Modifying this residue in P. aeruginosa led to a complete reversal of selectivity. Thus, opine-type metallophore diversity is mainly mediated by the absence/presence of a cntK gene encoding a histidine racemase, and the presence of an aspartate/alanine at position 150 of CntM. These two simple rules predict the production of a fourth metallophore by Paenibacillus mucilaginosus, which was confirmed in vitro and called bacillopaline.

biochemistry↗

bioRxiv2019

UHPLC/MS based large-scale targeted metabolomics method for multiple-biological matrix assay

This new method has the capacity to dynamically analyse the metabolome of interest in diverse biological matrixes by offering coverage of rat urine, plasma, liver, brain, intestine, stomach, heart, spleen, lung, faeces, fresh plant tissues, cells and microbes. In addition, this new method enables specific and efficient analysis of microdontia metabolomes, non-microdontia and whole cell metabolomes, as well as can engage in absolute determination of 84 key clinical-wide metabolites in different biological matrixes, to enable the complementary support of clinical diagnosis and classification of diseases. To demonstrate the applicable capacity of this new method, multiple-matrixes differential metabolomes were firstly characterized using this new method to coordinate metabolic modifications underlie hepatitis induced by carbon tetrachloride (CCL4) in rats, such finding provides novel insight into the pathogenesis and therapeutics of hepatitis in clinic. Altogether, we are fully confident that this new metabolomics method will be widely welcomed by scientists in different niches to solve their key questions accordingly.

biochemistry↗

bioRxiv2019

A New Minimally Invasive Technique for Repairing Achilles Tendon Rupture: A Biomechanical Study

IntroductionThe incidence of Achilles tendon rupture shows a gradually increasing trend, which is mainly managed by minimally invasive treatment due to its advantages, such as low wound infection rate. At present, the firmness of the commonly applied minimally invasive suture method for Achilles tendon remains controversial. Our research group has developed a novel suture method for Achilles tendon, which has achieved favorable clinical outcomes. Therefore, this experiment aimed to explore the optimal approach to repair Achilles tendon rupture through comparing the biomechanical strength of the commonly used Achilles tendon suture methods currently.\n\nMaterials and methods6 fresh frozen human cadaveric Achilles tendon specimens were sutured by three kinds of technique, and were tested through the cyclical loading after repair.\n\nResultsResults of cyclical loading showed that, the repair using the new technique was stronger after 10 cycles, 1000 cycles, and rupture. Moreover, the new technique had displayed superior anti-deformation strength to that of the Ma-Griffith technique.\n\nConclusionsOur experimental results demonstrate that, the new technique proposed by our research group can attain comparable biomechanical properties to those of the Krachow technique. However, the sample size in this study is small, and further clinical trials are warranted.

biochemistry↗

bioRxiv2019

Categorical Assignment of Pulmonary Embolism is a Simple and More Accurate Indicator of Right Ventricular Dysfunction and Short Team Mortality

Several risk stratification tools are available to predict short-term mortality in patients with acute pulmonary embolism (PE). Right ventricular (RV) dysfunction, which is common to intermediate and high risk PE, is an independent predictor of mortality and may be a faster and simpler way to assess patient risk in acute care settings. We evaluated 571 patients presenting with acute PE as the primary diagnosis, stratifying them by the Pulmonary Embolism Severity Index (PESI), by the BOVA score, or categorically as low risk (no RV dysfunction by imaging), intermediate risk (RV dysfunction by imaging), or high risk PE (RV dysfunction by imaging with sustained hypotension). Using imaging data to firstly define the presence of RV dysfunction, and plasma cardiac troponin T (cTnT) and NT-proBNP as additional evidence for myocardial strain, we evaluated the PESI and BOVA scoring systems compared to categorical assignment of PE as low risk, submassive, and massive PE. Cardiac biomarkers poorly distinguished between PESI classes and BOVA stages in patients with acute PE. Cardiac TnT and NT-proBNP easily distinguished low risk from submassive PE with an area under the curve (AUC) of 0.84 (95% C.I. 0.73 - 0.95, p< 0.0001), and 0.88 (95% C.I. 0.79-0.97, p< 0.0001), respectively, and low risk from massive PE with an area under the curve (AUC) of 0.89 (95% C.I. 0.78 - 1.00, p< 0.0001), and 0.89 (95% C.I. 0.82-0.95, p< 0.0001), respectively. Predicted short-term mortality by PESI score or BOVA stage was lower than the observed mortality for submassive PE by a two-fold order of magnitude. These data suggest the presence of RV dysfunction in the context of acute PE is sufficient for the purposes of risk stratification, while more complicated risk stratification algorithms may under-estimate short-term mortality risk.

biochemistry↗

bioRxiv2019

A general LC/MS-based RNA sequencing method for direct analysis of multiple-base modifications in RNA mixtures

A complete understanding of the structural and functional potential of RNA requires understanding of chemical modifications and noncanonical bases; this in turn requires advances in current sequencing methods to be able to sequence not only canonical ribonucleotides, but at the same time directly sequence these nonstandard moieties. Here, we present the first direct and modification type-independent RNA sequencing method via integration of a hydrophobic end-labeling strategy with of 2-D mass-retention time LC/MS analysis to allow de novo sequencing of RNA mixtures and enhance sample usage efficiency. Our method can directly read out the complete sequence, while identifying, locating, and quantifying base modifications accurately in both single and mixed RNA samples containing multiple different modifications at single-base resolution. Our method can also quantify stoichiometry/percentage of modified RNA vs. its canonical counterpart RNA, simulating a real biological sample where modifications exist but may not be 100% at a particular site of the RNA. This method is a critical step towards fully sequencing real complex cellular RNA samples of any type and containing any modification types and can also be used in the quality control of modified therapeutic RNAs.

biochemistry↗

bioRxiv2019

The TFIIH components p44/p62 act as a damage sensor during nucleotide excision repair

Nucleotide excision repair (NER) protects the genome following exposure to diverse types of DNA damage, including UV light and chemotherapeutics. Mutations in mammalian NER genes lead to diseases such as xeroderma pigmentosum, trichothiodystrophy, and Cockayne syndrome. In eukaryotes, the major transcription factor TFIIH is the central hub of NER. The core components of TFIIH include the helicases XPB, XPD, and five structural subunits. Two of these structural TFIIH proteins, p44 and p62 remain relatively unstudied; p44 is known to regulate the helicase activity of XPD during NER whereas p62s role is thought to be structural. However, a recent cryo-EM structure shows that p44, p62, and XPD make extensive contacts within TFIIH, with part of p62 occupying XPDs DNA binding site. This observation implies a more extensive role in DNA repair beyond the structural integrity of TFIIH. Here, we show that p44 stimulates XPDs ATPase but upon encountering DNA damage, further stimulation is only observed when p62 is part of the ternary complex; suggesting a role for the p44/p62 heterodimer in TFIIHs mechanism of damage detection. Using single molecule imaging, we demonstrate that p44/p62 independently interacts with DNA; it is seen to diffuse, however, in the presence of UV-induced DNA lesions the complex stalls. Combined with the analysis of a recent cryo-EM structure we suggest that p44/p62 acts as a novel DNA-binding entity within TFIIH that is capable of recognizing DNA damage. This revises our understanding of TFIIH and prompts more extensive investigation into the core subunits for an active role during both DNA repair and transcription.

biochemistry↗