The RecJ2 Protein in the Thermophilic Archaeon Thermoplasma acidophilum Is a 3′ 5′ Exonuclease and Associates with a DNA Replication Complex [Microbiology]

March 16th, 2017 by Hiromi Ogino, Sonoko Ishino, Daisuke Kohda, Yoshizumi Ishino

RecJ/cell division cycle 45 (Cdc45) proteins are widely conserved in the three domains of life, i.e., in Bacteria, Eukarya and Archaea. Bacterial RecJ is a 5′ 3′ exonuclease and functions in DNA repair pathways, while using its 5′ 3′ exonuclease activity. Eukaryotic Cdc45 has no identified enzymatic activity, but participates in the CMG complex so named because it is composed of Cdc45, minichromosome maintenance protein complex (MCM) proteins 2-7, and GINS complex proteins (Sld5, Psf11 to 3). Eukaryotic Cdc45 and bacterial/archaeal RecJ share similar amino acid sequences and are considered functional counterparts. In Archaea, a RecJ homolog in Thermococcus kodakarensis was shown to associate with GINS and accelerate its nuclease activity and was therefore designated GAN (GINS-associated nuclease); however, to date, no archaeal RecJ MCM GINS complex has been isolated. The thermophilic archaeon Thermoplasma acidophilum has two RecJ like proteins, designated TaRecJ1 and TaRecJ2. TaRecJ1 exhibited DNA-specific 5′ 3′exonuclease activity, while TaRecJ2 had 3′ 5′ exonuclease activity and preferred RNA over DNA. TaRecJ2, but not TaRecJ1, formed a stable complex with TaGINS in a 2:1 molar ratio. Furthermore, the TaRecJ2-TaGINS complex stimulated activity of TaMCM helicase in vitro, and the TaRecJ2-TaMCM-TaGINS complex was also observed in vivo. However, TaRecJ2 did not interact with TaMCM directly and was not required for the helicase activation in vitro. These findings suggest that the function of archaeal RecJ in DNA replication evolved divergently from Cdc45 despite conservation of the CMG-like complex formation between Archaea and Eukarya.
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Adjacent channelrhodopsin-2 residues within transmembranes 2 and 7 regulate cation selectivity and distribution of the two open states [Membrane Biology]

March 16th, 2017 by Ryan Richards, Robert E. Dempski

Channelrhodopsin-2 (ChR2) is a light-activated channel that can conduct cations of multiple valencies down the electrochemical gradient. Under continuous light exposure, ChR2 transitions from a high conducting open state (O1) to a low conducting open state (O2) with differing ion selectivity. The molecular basis for the O1 to O2 transition and how ChR2 modulates selectivity between states is currently unresolved. To this end, we used steered molecular dynamics, electrophysiology, and kinetic modeling to identify residues that contribute to gating and selectivity in discrete open states. Analysis of steered molecular dynamics experiments identified three transmembrane residues (V86, K93 and N258) that form a putative barrier to ion translocation. Kinetic modeling of photocurrents generated from ChR2 proteins with conservative mutations at these positions demonstrated that these residues contribute to cation selectivity (V86 and N258), the transition between the two open states (V86), open channel stability, and the hydrogen-bonding network (K93I and K93N). These results suggest that this approach can be used to identify residues that contribute to the open state transitions and the discrete ion selectivity within these states. With the rise of ChR2 use in optogenetics, it will be critical to identify residues that contribute to O1 or O2 selectivity and gating to minimize undesirable effects.
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Two alternative binding mechanisms connect the protein translocation Sec71/Sec72 complex with heat shock proteins [Protein Structure and Folding]

March 12th, 2017 by Arati Tripathi, Elisabet C Mandon, Reid Gilmore, Tom A Rapoport

The biosynthesis of many eukaryotic proteins requires accurate targeting to and translocation across the endoplasmic reticulum (ER) membrane. Post-translational protein translocation in yeast requires both the Sec61 translocation channel, and a complex of four additional proteins: Sec63, Sec62, Sec71, and Sec72. The structure and function of these proteins are largely unknown. This pathway also requires the cytosolic Hsp70 protein Ssa1, but whether Ssa1 associates with the translocation machinery to target protein substrates to the membrane is unclear. Here, we use a combined structural and biochemical approach to explore the role of Sec71/Sec72 subcomplex in post-translational protein translocation. To this end, we report a crystal structure of the Sec71/Sec72 complex, which revealed that Sec72 contains a tetratricopeptide repeat (TPR) domain that is anchored to the ER membrane by Sec71. We also determined the crystal structure of this TPR domain with a C-terminal peptide derived from Ssa1, which suggests how Sec72 interacts with full-length Ssa1. Surprisingly, Ssb1, a cytoplasmic Hsp70 that binds ribosome- associated nascent polypeptide chains also binds to the TPR domain of Sec72, even though it lacks the TPR-binding C-terminal residues of Ssa1. We demonstrate that Ssb1 binds through its ATPase domain to the TPR domain, an interaction that leads to inhibition of nucleotide exchange. Taken together, our results suggest that translocation substrates can be recruited to the Sec71/72 complex either post-translationally through Ssa1 or co-translationally through Ssb1.
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A New General Method for Simultaneous Fitting of Temperature- and Concentration-Dependence of Reaction Rates Yields Kinetic and Thermodynamic Parameters for HIV Reverse Transcriptase Specificity [DNA and Chromosomes]

March 2nd, 2017 by An Li, Jessica L. Ziehr, Kenneth A. Johnson

Recent studies have demonstrated the dominant role of induced-fit in enzyme specificity of HIV reverse transcriptase and many other enzymes. However, relevant thermodynamic parameters are lacking and equilibrium thermodynamic methods are of no avail because the key parameters can only determined by kinetic measurement. By modifying KinTek Explorer software, we present a new general method for globally fitting data collected over a range of substrate concentrations and temperatures and apply it to HIV reverse transcriptase. Fluorescence stopped-flow methods were used to record the kinetics of enzyme conformational changes that monitor nucleotide binding and incorporation. The nucleotide concentration dependence was measured at temperatures ranging from 5 to 37C and the raw data were fit globally to derive a single set of rate constants at 37C and a set of activation enthalpy terms to account for the kinetics at all other temperatures. This comprehensive analysis afforded thermodynamic parameters for nucleotide binding (Kd, ΔG, ΔH, ΔS at 37C), and kinetic parameters for enzyme conformational changes and chemistry (rate constants and activation enthalpy). Comparisons between wild-type enzyme and a mutant resistant to nucleoside analogs used to treat HIV infections reveal that the ground state binding is weaker and the activation enthalpy for the conformational change step is significantly larger for the mutant. Further studies to explore the structural underpinnings of the observed thermodynamics and kinetics of the conformational change step may help to design better analogs to treat HIV infections and other diseases. Our new method is generally applicable to enzyme and chemical kinetics.
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Structural Basis for the Lesion-scanning Mechanism of the Bacterial MutY DNA Glycosylase [Enzymology]

January 27th, 2017 by Lan Wang, Srinivas Chakravarthy, Gregory L Verdine

The highly mutagenic A:oxoG (8-oxoguanine) base-pair is generated mainly by misreplication of the C:oxoG base-pair, the oxidation product of the C:G base-pair. A:oxoG base-pair is particularly insidious because neither base in it carries faithful information to direct the repair of the other. The bacterial MutY (MUTYH in humans) adenine DNA glycosylase is able to initiate the repair of A:oxoG by selectively cleaving the A base from the A:oxoG base-pair. The difference between faithful repair and wreaking mutagenic havoc on the genome lies in the accurate discrimination between two structurally similar base-pairs: A:oxoG and A:T. Here we present two crystal structures of the MutY N-terminal domain in complex with either undamaged DNA or DNA containing an intrahelical lesion. These structures have captured for the first time, a DNA glycosylase scanning the genome for a damaged base in the very first stage of lesion-recognition and the base-extrusion pathway. The mode of interaction observed here has suggested a common lesion-scanning mechanism across the entire helix-hairpin-helix superfamily to which MutY belongs. In addition, small-angle X-ray scattering (SAXS) studies together with accompanying biochemical assays have suggested a possible role played by the C-terminal oxoG-recognition domain of MutY in lesion-scanning.

Super-resolution visualization of caveolae deformation in response to osmotic stress [Signal Transduction]

January 17th, 2017 by Lu Yang, Suzanne Scarlata

Caveolae are protein dense plasma membrane domains structurally composed of caveolin -1 or -3 along with other proteins. Our previous studies have shown that caveolae enhance calcium signals generated through the Gαq/phospholipase Cβ signaling pathway, and that subjecting cells to hypo-osmotic stress reverses this enhancement. In this study, we have used super-resolution fluorescence microscopy supplemented by fluorescence correlation studies to determine the structural factors that underlie this behavior. We find similar and significant populations of Gαq and one of its receptors, bradykinin type 2 receptor (β2R), as well as Gαi and its coupled 2-adrenergic receptor (βAR), localize to caveolae domains. While mild osmotic stress deforms caveolae altering interactions between caveolae and these proteins, it does not affect the general structure and the localization of caveolae components remain largely unchanged. Additionally, in contrast to calcium signals mediated through Gαq-B2R, osmotic stress does not affect cAMP signals mediated through Gαi and βAR. Structurally, we find that mild osmotic stress corresponding roughly to a pressure of 3.82 N/m2 increases the domain diameter by ~30% and increases the fluorescence intensity in the center of the domain mouth suggesting a flattening of the invagination. Approximate calculations show that caveolae in muscle tissue have the strength to handle the stress of muscle movement.

Autoinhibition of the Nuclease ARTEMIS is Mediated by a Physical Interaction between Its Catalytic and C-terminal Domains [Immunology]

January 12th, 2017 by Doris Niewolik, Ingrid Peter, Carmen Butscher, Klaus Schwarz

The nuclease ARTEMIS is essential for the development of B and T lymphocytes. It is required for opening DNA hairpins generated during antigen receptor gene assembly from variable (V), diversity (D) and joining (J) subgenic elements (V(D)J recombination). As a member of the non-homologous end joining pathway it is also involved in repairing a subset of pathological DNA double-strand breaks. Loss of ARTEMIS function therefore results in radiosensitive severe combined immunodeficiency (RS-SCID). The hairpin-opening activity is dependent on the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which can bind to and phosphorylate ARTEMIS. The ARTEMIS C-terminus is dispensable for cellular V(D)J recombination and in vitro nuclease assays with C-terminally truncated ARTEMIS show DNA PKcs-independent hairpin-opening activity. Therefore it has been postulated that ARTEMIS is regulated via autoinhibition by its C-terminus. To obtain evidence for the autoinhibition model, we performed co-immunoprecipitation experiments with combinations of ARTEMIS mutants. We show that an N-terminal fragment comprising the catalytic domain can interact both with itself and with a C-terminal fragment. Amino acid exchanges N456A+S457A+E458Q in the C-terminus of full-length ARTEMIS resulted in unmasking of the N-terminus and in increased ARTEMIS activity in cellular V(D)J recombination assays. Mutations in ARTEMIS-deficient patients impaired the interaction with the C-terminus and also affected protein stability. The interaction between the N- and C-terminal domains was not DNA-PKcs dependent and phosphomimetic mutations in the C-terminal domain did not result in unmasking of the catalytic domain. Our experiments provide strong evidence that a physical interaction between the C-terminal and catalytic domains mediates ARTEMIS autoinhibition.

RIT1 GTPase Regulates Sox2 Transcriptional Activity and Hippocampal Neurogenesis [Signal Transduction]

December 22nd, 2016 by Sajad Mir, Weikang Cai, Douglas A. Andres

Adult neurogenesis, the process of generating mature neurons from neuronal progenitor cells, makes critical contributions to neural circuitry and brain function under both healthy and disease states. Neurogenesis is a highly regulated process, in which diverse environmental and physiological stimuli are relayed to resident neural stem cell populations to control the transcription of genes involved in their self-renewal and differentiation. Understanding the molecular mechanisms governing neurogenesis is necessary for the development of translational strategies to harness this process for neuronal repair. Here we report that the Ras-related GTPase, RIT1, serves to control the sequential proliferation and differentiation of adult hippocampal neural/stem progenitor cells (NPCs), with in vivo expression of active RIT1 driving robust adult neurogenesis. Gene expression profiling analysis demonstrates increased expression of a specific set of transcription factors known to govern adult neurogenesis in response to active RIT1 expression in the hippocampus, including sex-determining region Y-related HMG box 2 (Sox2), a well-established regulator of stem cell self-renewal and neurogenesis. In adult hippocampal neuronal precursor cells (HNPCs), RIT1 controls an Akt-dependent signaling cascade, resulting in the stabilization and transcriptional activation of phosphorylated Sox2. Together, these studies support a role for RIT1 in relaying niche-derived signals to NPCs to control transcription of genes involved in self-renewal and differentiation.

Molecular Identification of D-Ribulokinase in Budding Yeast and Mammals [Enzymology]

December 1st, 2016 by Charandeep Singh, Enrico Glaab, Carole L. Linster

Proteomes of even well characterized organisms still contain a high percentage of proteins with unknown or uncertain molecular and/or biological function. A significant fraction of those proteins are predicted to have catalytic properties. Here we aimed at identifying the function of the Saccharomyces cerevisiae Ydr109c protein and of its human homolog FGGY, both of which belong to the broadly conserved FGGY family of carbohydrate kinases. Functionally identified members of this family phosphorylate 3- to 7-carbon sugars or sugar derivatives, but the endogenous substrate of S. cerevisiae Ydr109c and human FGGY has remained unknown. Untargeted metabolomics analysis of an S. cerevisiae deletion mutant of YDR109C revealed ribulose as one of the metabolites with the most significantly changed intracellular concentration as compared to a wild-type strain. In human HEK293 cells, ribulose could only be detected when ribitol was added to the cultivation medium and under this condition, FGGY silencing led to ribulose accumulation. Biochemical characterization of the recombinant purified Ydr109c and FGGY proteins showed a clear substrate preference of both kinases for D-ribulose over a range of other sugars and sugar derivatives tested, including L-ribulose. Detailed sequence and structural analyses of Ydr109c and FGGY as well as homologs thereof furthermore allowed the definition of a 5-residue D-ribulokinase signature motif (TCSLV). The physiological role of the herein identified eukaryotic D-ribulokinase remains unclear, but we speculate that S. cerevisiae Ydr109c and human FGGY could act as metabolite repair enzymes, serving to re-phosphorylate free D-ribulose generated by promiscuous phosphatases from D-ribulose-5-phosphate. In human cells, FGGY can additionally participate in ribitol metabolism.

A Conserved Tripeptide Sequence at the C-terminus of the Poxvirus DNA Processivity Factor D4 is Essential for Protein Integrity and Function [Microbiology]

November 11th, 2016 by Nuth, M., Guan, H., Ricciardi, R. P.

Vaccinia virus (VACV) is a poxvirus member, and the VACV D4 protein serves both as a uracil-DNA glycosylase (UDG) and as an essential component required for processive DNA synthesis. The VACV A20 protein has no known catalytic function itself, but associates with D4 to form the D4-A20 heterodimer that functions as the poxvirus DNA processivity factor. The heterodimer enables the DNA polymerase to efficiently synthesize extended strands of DNA. Upon characterizing the interaction between D4 and A20, we observed that the C-terminus of D4 is susceptible to perturbation. Further analysis demonstrated that a conserved hexapeptide stretch at the extreme C-terminus of D4 is essential for maintaining protein integrity, as assessed by its requirement for the production of soluble recombinant protein that is functional in processive DNA synthesis. From the known crystal structures of D4, the C-terminal hexapeptide is shown to make intramolecular contact with residues spanning the protein's inner core. Our mutational analysis revealed that a tripeptide motif (215-GFI-217) within the hexapeptide comprises apparent residues necessary for the contact. Prediction of protein disorder identified the hexapeptide and several regions upstream of Gly-215 that comprise residues of the interface surfaces of the D4-A20 heterodimer. Our study suggests that 215-GFI-217 anchors these potentially dynamic upstream regions of the protein in order to maintain protein integrity. Unlike UDGs from diverse sources, where the C-termini are disordered and do not form comparable intramolecular contacts, this feature may be unique to orthopoxviruses.
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