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bioRxiv2017

Molecular determinants and bottlenecks in the unbinding dynamics of biotin-streptavidin

Biotin-streptavidin is a very popular system used to gain insight into protein-ligand interactions. In its tetrameric form, it is well-known for its extremely long residence times, being one of the strongest known non-covalent interactions in nature, and is heavily used across the biotechnological industry. In this work we gain understanding into the molecular determinants and bottlenecks in the unbinding of the dimeric biotinstreptavidin system in its wild type and with N23A mutation. Using new enhanced sampling methods with full atomistic resolution, we reproduce the variation caused by N23A mutation in experimentally reported residence time. We also answer a longstanding question regarding cause/effect in the coupled events of bond stretching and bond hydration during unbinding and establish that in this system, it is the bond stretching and not hydration which forms the bottleneck in the early parts of the unbinding. We believe these calculations represent a step forward in the use of atomistic simulations to study pharmacodynamics. An improved understanding of biotin-streptavidin unbinding dynamics should also have direct benefits in biotechnological and nanobiotechnological applications.

biochemistry↗

bioRxiv2017

Breaking the Deadlock of Molecular Chaperones

Protein folding in the cell requires ATP-driven chaperone machines. It is poorly understood, however, how these machines fold proteins. Here we propose that the conserved Hsp70 and Hsp90 chaperones support formation of the folding nucleus by providing a gradient of decreasing hydrophobicity. Early on the folding pathway Hsp70 uses its highly hydrophobic binding pocket to recover a stalled, unproductive folding intermediate. The aggressive nature of Hsp70 action, however, blocks productive folding by grabbing hydrophobic, core-forming segments. This precludes on-pathway nucleation at high, physiological Hsp70 levels. Transfer to the less hydrophobic Hsp90 enables the intermediate to resume forming its folding nucleus. Subsequently, the protein enters a spontaneous folding trajectory towards its native state, independent of the ATPase activities of both Hsp70 and Hsp90. Our findings provide a general mechanistic concept for chaperoned protein folding.

biochemistry↗

bioRxiv2017

Searching for Correlated Conformational Dynamics: Analysis of the NMR Relaxation Dispersions with Akaike's Information Theory and Hierarchical Clustering

ABSTRACTIn this manuscript, I am proposing an approach for identification of correlated exchange in proteins via analysis of the NMR relaxation dispersion data. For a set of spins experiencing exchange, every relaxation dispersion datasets is fit individually and then--globally while paired with every other dataset. The corrected Akaike s Information Criteria (AICc) for individual and global fits are used to evaluate the likelihood of two spins to report on the same dynamic event. Application of hierarchical cluster analysis reveals correlated spin groups using the difference in AICcs as a measure of similarity within the pairs. This approach to detection of correlated dynamics is independent of accuracy of best-fit parameters rendering it less sensitive to experimental noise. High throughput and the absence of the operator bias might make it applicable to a relatively lower quality NMR relaxation dispersion data from large and poorly soluble systems.

biochemistry↗

bioRxiv2017

Chemical control of a CRISPR-Cas9 acetyltransferase

Lysine acetyltransferases (KATs) play a critical role in the regulation of transcription and other genomic functions. However, a persistent challenge is the development of assays capable of defining KAT activity directly in living cells. Towards this goal, here we report the application of a previously reported dCas9-p300 fusion as a transcriptional reporter of KAT activity. First we benchmark the activity of dCas9-p300 relative to other dCas9-based transcriptional activators, and demonstrate its compatibility with second generation short guide RNA architectures. Next, we repurpose this technology to rapidly identify small molecule inhibitors of acetylation-dependent gene expression. These studies validate a recently reported p300 inhibitor chemotype, and reveal a role for p300s bromodomain in dCas9-p300-mediated transcriptional activation. Comparison with other CRISPR-Cas9 transcriptional activators highlights the inherent ligand tuneable nature of dCas9-p300 fusions, suggesting new opportunities for orthogonal gene expression control. Overall, our studies highlight dCas9-p300 as a powerful tool for studying gene expression mechanisms in which acetylation plays a causal role, and provide a foundation for future applications requiring spatiotemporal control over acetylation at specific genomic loci.

biochemistry↗

bioRxiv2017

RNA editing of CAPS1 regulates synaptic vesicle organization, release and retrieval.

Calcium-dependent activator protein for secretion 1 (CAPS1) facilitates the docking and priming of synaptic and dense core vesicles. A conserved hairpin structure in the CAPS1 pre-mRNA allows an post-transcriptional adenosine-to-inosine RNA editing event to alter a genomically-encoded glutamate to a glycine codon. Functional comparisons of CAPS1 protein isoforms in primary hippocampal neurons show that elevation of edited CAPS1 isoforms facilitates presynaptic vesicle clustering and turnover. Conversely, non-edited CAPS1 isoforms slow evoked release, increase spontaneous fusion, and loosen the clustering of synaptic vesicles. Therefore, CAPS1 editing promotes organization of the vesicle pool in a way that is beneficial for evoked release, while non-edited isoforms promote more lax vesicle organization that widens distribution, attenuates evoked release and eases the control of spontaneous fusion. Overall, RNA editing of CAPS1 is a mechanism to fine tune neurotransmitter release.\n\nIMPACT STATEMENTPost-transcriptional RNA editing of CAPS1 is a mechanism to regulate neurotransmitter release from synaptic vesicles.

biochemistry↗

bioRxiv2017

Detection of domain motion in NADPH-cytochrome P450 oxidoreductase through polarization anisotropy measurements

Conformational transitions between closed and open states in the NADPH-cytochrome P450 oxidoreductase (POR) play a critical role in its electron-transport function. In this study, we determined rotational diffusion coefficients of the EDANS fluorophore attached to the cytosolic POR construct lacking the N-terminal transmembrane region. We identified two dynamic modes, slow and fast, which are interpreted as the rotational diffusion of POR as a whole and the local domain motion, respectively. Timescale of the local rotational diffusion component suggests that it may correspond to the transient opening of the fully oxidized POR structure.

biochemistry↗

bioRxiv2017

Effect of ligands on stability of H-Ras GTPase

The G domain of a small monomeric GTPase Ras contains a nucleotide-binding pocket and a magnesium-binding site essential for the Ras function in cellular signaling. The G domain also has another (allosteric) ion-binding site on the rear surface of the G domain, which function is still unknown. In this paper, we detailed the effect of calcium and magnesium ions on stability of Ras bound to GDP, GTP, and GTP-mimic GppNHp. We revealed that the remote allosteric ion-binding site contributes very significantly to stability of Ras in the GDP-bound conformation, but nearly not at all--when Ras is bound to a GTP mimic. These findings highlight that further studies of the remote ion-binding site are warranted to reveal its role in the Ras function.

biochemistry↗

bioRxiv2017

Identification of Iridoid Synthases from Nepeta species: Iridoid cyclization does not determine nepetalactone stereochemistry

Graphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC=\"FIGDIR/small/179572_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (91K):\norg.highwire.dtl.DTLVardef@cc600dorg.highwire.dtl.DTLVardef@1b6240dorg.highwire.dtl.DTLVardef@f8e1beorg.highwire.dtl.DTLVardef@1acb9dd_HPS_FORMAT_FIGEXP M_FIG Iridoid synthase from Nepeta cateria (catnip) and Nepeta mussinii, have been cloned and characterized.\n\nC_FIG AbstractNepetalactones are iridoid monoterpenes with a broad range of biological activities produced by plants in the Nepeta genus. However, none of the genes for nepetalactone biosynthesis have been discovered. Here we report the transcriptomes of two Nepeta species, each with distinctive profiles of nepetalactone stereoisomers. As a starting point for investigation of nepetalactone biosynthesis in Nepeta, these transcriptomes were used to identify candidate genes for iridoid synthase homologs, an enzyme that has been shown to form the core iridoid skeleton in several iridoid producing plant species. Iridoid synthase homologs identified from the transcriptomes were cloned, heterologously expressed, and then assayed with the 8-oxogeranial substrate. These experiments revealed that catalytically active iridoid synthase enzymes are present in Nepeta, though there are unusual mutations in key active site residues. Nevertheless, these enzymes exhibit similar catalytic activity and product profile compared to previously reported iridoid synthases from other plants. Notably, four nepetalactone stereoisomers with differing stereochemistry at the 4 and 7 positions - which are generated during the iridoid synthase reaction - are observed at different ratios in various Nepeta species. This work strongly suggests that the variable stereochemistry at these 4 and 7 positions of nepetalactone diastereomers is established further downstream in the iridoid pathway in Nepeta. Overall, this work provides a gateway into the biosynthesis of nepetalactones in Nepeta.\n\nHighlightsO_LISpecies within the Nepeta genus (such as catnip) produce nepetalactone iridoids\nC_LIO_LIThe enzymes that produce the iridoid scaffold of nepetalactone were identified from two species of Nepeta\nC_LIO_LIThe iridoid synthase enzymes are not responsible for the stereochemical variation in these iridoids\nC_LI

biochemistry↗

bioRxiv2017

Tadpole-like conformations of huntingtin exon 1 with expanded polyglutamine engenders novel interactions in cells

Soluble huntingtin exon 1 (Httex1) with expanded polyglutamine (polyQ) engenders neurotoxicity in Huntingtons disease. To uncover the physical basis of this toxicity, we performed structural studies of soluble Httex1 for wild type and mutant polyQ lengths. Nuclear magnetic resonance experiments show evidence for conformational rigidity across the polyQ region. In contrast, hydrogen-deuterium exchange shows absence of backbone amide protection, suggesting negligible persistence of hydrogen bonds. The seemingly conflicting results are explained by all-atom simulations, which show that Httex1 adopts tadpole-like structures with a globular head encompassing the N-terminal amphipathic and polyQ regions and the tail encompassing the C-terminal proline-rich region. The surface area of the globular domain increases monotonically with polyQ length. This stimulates sharp increases in gain-of-function interactions in cells for expanded polyQ, and one of these interactions is with the stress-granule protein Fus. Our results highlight plausible connections between Httex1 structure and routes to neurotoxicity.

biochemistry↗

bioRxiv2017

Structural analyses reveal that the ATG2A-WIPI4 complex functions as a membrane tether for autophagosome biogenesis

Autophagy is an enigmatic cellular process in which double-membrane compartments, called autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the phosphatidylinositol-3-phosphate (PI3P)-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER-phagophore association and/or tethers vesicles to the ER-phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.

biochemistry↗

bioRxiv2017

Asymmetric MukB ATPases are regulated independently by the N- and C-terminal domains of MukF kleisin

The Escherichia coli SMC complex, MukBEF, acts in chromosome segregation. MukBEF shares the distinctive architecture of other SMC complexes, with one prominent difference; unlike other kleisins, MukF forms dimers through its N-terminal domain. We show that a 4-helix bundle adjacent to the MukF dimerization domain interacts functionally with the MukB coiled-coiled neck adjacent to the ATPase head, forming an asymmetric tripartite complex, as in other SMC complexes. Since MukF dimerization is preserved during this interaction, MukF directs the formation of dimer of dimers MukBEF complexes, observed previously in vivo. The MukF N- and C-terminal domains stimulate ATPase independently and additively, consistent with them each targeting only one of the two MukB ATPase active sites in the asymmetric complex. We demonstrate that MukF interaction with the MukB neck turns over during cycles of ATP binding and hydrolysis in vivo and that impairment of this interaction leads to MukBEF release from chromosomes.

biochemistry↗

bioRxiv2017

Structural insights into the architecture of human Importin4_histone H3/H4_Asf1a complex and its histone H3 tail binding

Importin4 transports histone H3/H4 in complex with Asf1a to the nucleus for chromatin assembly. Importin4 recognizes the nuclear localization sequence located at the N-terminal tail of histones. Here, we analyzed the structures and interactions of human Importin4, histones and Asf1a by cross-linking mass spectrometry, X-ray crystallography, negative-stain electron microscopy, small-angle X-ray scattering and integrative modeling. The XL-MS data showed that the C-terminal region of Importin4 interacts extensively with the histone H3 tail. We determined the crystal structure of the C-terminal region of Importin4 bound to the histone H3 peptide, thus revealing that the acidic path in Importin4 accommodates the histone H3 tail and that histone H3 Lys14 is the primary residue interacting with Importin4. Furthermore, the molecular architecture of the Importin4_histone H3/H4_Asf1a complex was produced through an integrative modeling approach. Overall, this work provides structural insights into how Importin4 recognizes histones and their chaperone complex.

biochemistry↗

bioRxiv2017

Development of High-Throughput Screening Assays for Inhibitors of ETS Transcription Factors

ETS transcription factors from the ERG and ETV1/4/5 subfamilies are overexpressed in the majority of prostate cancer patients and contribute to disease progression. Here, we develop two in vitro assays for the interaction of ETS transcription factors with DNA that are amenable for high throughput screening. Using ETS1 as a model, these assays were applied to screen 110 compounds derived from a high-throughput virtual screen. We find that the use of lower affinity DNA-binding sequences, similar to those which ERG and ETV1 bind to in prostate cells, allowed for higher inhibition from many of these test compounds. Further pilot experiments demonstrated that the in vitro assays are robust for ERG, ETV1, and ETV5, three of the ETS transcription factors that are overexpressed in prostate cancer.

biochemistry↗

bioRxiv2017

Inhibition of Intracellular Lipolysis Promotes Cancer Cell Adaptation to Hypoxia

Tumor tissues are chronically exposed to hypoxia owing to aberrant vascularity. Lipid droplet (LD) accumulation is a hallmark of hypoxic cancer cells, yet how LDs form and function during hypoxia remains poorly understood. Herein, we report that in various cancer cells upon oxygen deprivation, HIF-1 activation down-modulates LD catabolism mediated by adipose triglyceride lipase (ATGL), the key enzyme for intracellular lipolysis. Proteomics and functional analyses identified hypoxia-inducible gene 2 (HIG2), a HIF-1 target, as a new inhibitor of ATGL. Knockout of HIG2 enhanced LD breakdown and fatty acid (FA) oxidation, leading to increased ROS production and apoptosis in hypoxic cancer cells as well as impaired growth of tumor xenografts. All of these effects were reversed by co-ablation of ATGL. Thus, by inhibiting ATGL, HIG2 acts downstream of HIF-1 to sequester FAs in LDs away from the mitochondrial pathways for oxidation and ROS generation, thereby sustaining cancer cell survival in hypoxia.

biochemistry↗

bioRxiv2017

On the Structure and Mechanism of Two-Pore Channels

In eukaryotes, two-pore channels (TPC1-3) comprise a family of ion channels that regulate the conductance of Na+ and Ca2+ ions across cellular membranes. TPC1-3 form endolysosomal channels, but TPC3 can also function in the plasma membrane. TPC1/3 are voltage-gated channels, but TPC2 opens in response to binding endolysosome-specific lipid phosphatidylinositol-3,5-diphosphate (PI(3,5)P2). Filoviruses, such as Ebola, exploit TPC-mediated ion release as a means of escape from the endolysosome during infection. Antagonists that block TPC1/2 channel conductance abrogate filoviral infections. TPC1/2 form complexes with the mechanistic target of rapamycin complex 1 (mTORC1) at the endolysosomal surface that couple cellular metabolic state and cytosolic nutrient concentrations to the control of membrane potential and pH. We determined the X-ray structure of TPC1 from Arabidopsis thaliana (AtTPC1) to 2.87[A] resolution-one of the two first reports of a TPC channel structure. Here we summarize these findings and the implications that the structure may have for understanding endolysosomal control mechanisms and their role in human health.\n\nAbbreviations

biochemistry↗

bioRxiv2017

GIGYF2 mediates post-transcriptional mRNA repression through recruitment of the CCR4/NOT complex

Initially identified as a factor involved in tyrosine kinase receptor signalling, GRB10-interacting GYF protein 2 (GIGYF2) has later been shown to interact with the 5 cap-binding protein m4EHP as part of a translation repression complex, and to mediate post-transcriptional repression of tethered reporter mRNAs. We recently observed that GIGYF2 also interacts with the miRNA-induced silencing complex and modulates its translation repression activity. Here we have further investigated how GIGYF2 represses mRNA function. In RNA tethering reporter assays we show that GIGYF2 exerts its action through a combination of translational repression and stimulated mRNA decay. Using truncation variants we identify two distinct effector domains within GIGYF2. In this assay GIGYF2-mediated repression is independent of m4EHP but dependent on the deadenylation activity of the CCR4/NOT complex. We further show that GIGYF2 interacts with multiple subunits of the CCR4/NOT complex and interestingly depletion of the CNOT1 scaffold subunit does not affect GIGYF2-mediated repression. Finally, we identify endogenous mRNA targets of GIGYF2 that recapitulate m4EHP - independent repression. Altogether, we propose that GIGYF2 has two distinct mechanisms of repression: one depends on m4EHP binding and affects translation, the other is m4EHP-independent and relies on the deadenylation activity of the CCR4/NOT complex.

biochemistry↗

bioRxiv2017

Fishing for New Bt Receptors in Diamondback Moth

Bt toxins bind to receptors in the brush border membrane of the insect gut and create pores, leading to insect death. Bt-resistant insects demonstrate reduced binding of the Bt toxins to gut membranes. However, our understanding of the gut receptors involved in Bt toxin binding, and which receptors confer resistance to these toxins is incomplete, especially in diamondback moth (Plutella xylostella), a major agricultural pest. Identifying receptors has remained challenging because we lack sufficiently sensitive methods to detect Bt receptor interactions. Here, we report a modified far-immunoblotting technique, which revealed a broad spectrum of binding targets for the Bt toxins Cry1Ac, Cry1Ab, and Cry1Bd in diamondback moth. We confirm the role of the glucosinolate sulfatases GSS1 and GSS2 in Cry1Bd toxicity. GSS1 and GSS2 bind directly to Cry1Bd, and their expression is crucial for Cry1Bd toxicity. These results improve our understanding of the molecular mechanisms of Bt toxicity.\n\nAUTHOR SUMMARYThe Bt toxins, from the soil bacterium Bacillus thuringiensis, have wide applications in agriculture as insecticides applied to plants or expressed in genetically modified crops. Bt toxins bind to receptors in the brush border membrane of the insect gut and create pores leading to insect death. The success of the Bt toxins in controlling insect pests has been hindered by the emergence of resistant insects, which show reduced binding of Bt to their gut membranes. Although ongoing research has identified a few receptors, many remain unknown and the mechanisms by which these receptors cause resistance remain unclear. Here, we used a modified far-immunoblotting technique to identify proteins that bind to the toxins Cry1Ac, Cry1Ab, and Cry1Bd in the diamondback moth. This identified two glucosinolate sulfatases that bind directly to Cry1Bd; also, the toxicity of Cry1Bd requires expression of these glucosinolate sulfatases. Therefore, identification of these candidate receptors improves our understanding of Bt function and resistance.

biochemistry↗

bioRxiv2017

A high-throughput analysis method of microdroplet PCR coupled with fluorescence spectrophotometry

Here we report a novel microdroplet PCR method combined with fluorescence spectrophotometry (MPFS), which allows for qualitative, quantitative and high -throughput detection of multiple DNA targets. In this study, each pair of primers was labeled with a specific fluorophore. Through microdroplet PCR, a target DNA was amplified and labeled with the same fluorophore. After products purification, the DNA products tagged with different fluorophores could be analyzed qualitatively by the fluorescent intensity determination. The relative fluorensence unit was also measured to construct the standard curve and to achieve quantitative analysis. In a reaction, the co -amplified products with different fluorophores could be simultaneously analyzed to achieve high -throughput detection. We used four kinds of GM maize as a model to confirm this theory. The qualitative results revealed high specificity and sensitivity of 0.5% (w / w). The quantitative results revealed that the limit of detection was 103copies and with good repeatability. Moreover, reproducibility assay were further performed using four foodborne pathogenic bacteria. Consequently, the same qualitative, quantitative and high-throughput results were confirmed as the four GM maize.

biochemistry↗