Probing the Conformation of the ISWI ATPase Domain With Genetically Encoded Photoreactive Crosslinkers and Mass Spectrometry

Forné, Ignasi; Ludwigsen, Johanna; Imhof, Axel; Becker, Peter B.; Mueller-Planitz, Felix

doi:10.1074/mcp.m111.012088

Cited by 46 publications

(58 citation statements)

References 39 publications

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“…UV light activates Bpa to cross-link nearby alkyl carbons with a preference for aliphatic residues (10). Bpa substitutions provide highly specific cross-linking information that can resolve unique binding partners in nearby features (11). We tested each purified, radiolabeled variant for cross-linking to RNAP holoenzyme in vitro, identifying 18 variants that cross-linked to one of the two large subunits in RNAP (SI Appendix, Fig.…”

Section: Significancementioning

confidence: 99%

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

Parshin

Shiver

Lee

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Sensing and responding to nutritional status is a major challenge for microbial life. In Escherichia coli, the global response to amino acid starvation is orchestrated by guanosine-3′,5′-bisdiphosphate and the transcription factor DksA. DksA alters transcription by binding to RNA polymerase and allosterically modulating its activity. Using genetic analysis, photo-cross-linking, and structural modeling, we show that DksA binds and acts upon RNA polymerase through prominent features of both the nucleotide-access secondary channel and the active-site region. This work is, to our knowledge, the first demonstration of a molecular function for Sequence Insertion 1 in the β subunit of RNA polymerase and significantly advances our understanding of how DksA binds to RNA polymerase and alters transcription.transcription regulation | stringent response | protein cross-linking | molecular modeling | lineage-specific insertions S ensing and responding to nutritional status is one of the major challenges of microbial life. In Escherichia coli, the global regulatory response to amino acid starvation is orchestrated by the second messenger guanosine-3′,5′-bisdiphosphate (ppGpp), which is a widely conserved master regulator (1). Accumulation of ppGpp during amino acid scarcity triggers the stringent response, which down-regulates expression of rRNA and tRNA while increasing expression of amino acid biosynthetic enzymes. In E. coli, ppGpp works synergistically with the transcription factor DksA to initiate the stringent response (2, 3). Both ppGpp and DksA are critical for survival of stress and virulence in many pathogenic proteobacteria (4).DksA is a relatively small protein with a prominent N-terminal coiled-coil domain and a globular C-terminal domain consisting of a Zn 2+ -binding region and a C-terminal α-helix (3). It belongs to a class of regulators that bind directly to RNA polymerase (RNAP) without contacting DNA (5). DksA modulates RNAP activity by preventing formation of or destabilizing the intermediate complex (RPi) on the pathway to the open complex (RPo), which is competent for initiation. For promoters with intrinsically unstable open complexes, such as rRNA promoters, DksA binding leads to decreased transcription (2).DksA is a critical determinant of the stringent response and a model system for an important class of transcription regulators, making it essential to understand how DksA interacts with RNAP at the molecular level. High-resolution structural information of the DksA/RNAP interaction is currently unavailable. Current models agree that the coiled-coil domain of DksA inserts into the secondary channel of RNAP, the channel used by NTPs to access the active site; that the secondary channel rim helices of β' subunit are critical for DksA binding; and that residues at the tip of the coiled-coil of DksA are important for its activity. However, the precise placement of DksA is unknown. With the number of critical features that can be accessed through the secondary channel, even small changes in the model can ...

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Section: Significancementioning

confidence: 99%

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

Parshin

Shiver

Lee

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Data Analysis-Mass spectrometry data from the crosslinked PTEN samples were analyzed using Crossfinder version 1.0 (20,21) with the default parameters. Prior to analysis with Crossfinder, MS/MS data were converted to mzXML files using ProteoWizard.…”

Section: Methodsmentioning

confidence: 99%

“…Irradiation of BB-4p-PTEN was followed by alkaline phosphatase treatment (to remove the mass spectrometry electrospray ionization suppressive effects of the phosphates) and trypsinization and avidin treatment to enrich for biotin-containing peptides. These peptides were then subjected to LC-MS/MS, and then the data were analyzed using Crossfinder (20,21). This led to the identification of a cross-linked peptide that contained a C-terminal peptide ( 379 CSDTTDSD-PENEP(B)DEDK bio 396 ) attached to an N-terminal peptide ( 42 LEGVYRNNIDDVVR 55 ) (Fig.…”

Section: Individual Tail Phosphorylation Effects On Ptenmentioning

confidence: 99%

Molecular Features of Phosphatase and Tensin Homolog (PTEN) Regulation by C-terminal Phosphorylation

Chen

Dempsey

Thomas

et al. 2016

Journal of Biological Chemistry

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PTEN is a tumor suppressor that functions to negatively regulate the PI3K/AKT pathway as the lipid phosphatase for phosphatidylinositol 3,4,5-triphosphate. Phosphorylation of a cluster of Ser/Thr residues (amino acids 380 -385) on the C-terminal tail serves to alter the conformational state of PTEN from an open active state to a closed inhibited state, resulting in a reduction of plasma membrane localization and inhibition of enzyme activity. The relative contribution of each phosphorylation site to PTEN autoinhibition and the structural basis for the conformational closure is still unclear. To further the structural understanding of PTEN regulation by C-terminal tail phosphorylation, we used protein semisynthesis to insert stoichiometric and site-specific phospho-Ser/Thr(s) in the C-terminal tail of PTEN. Additionally, we employed photo-cross-linking to map the intramolecular PTEN interactions of the phospho-tail. Systematic evaluation of the PTEN C-tail phospho-cluster showed autoinhibition, and conformational closure was influenced by the aggregate effect of multiple phospho-sites rather than dominated by a single phosphorylation site. Moreover, photo-crosslinking suggested a direct interaction between the PTEN C-tail and a segment in the N-terminal region of the catalytic domain. Mutagenesis experiments provided additional insights into how the PTEN phospho-tail interacts with both the C2 and catalytic domains. PTEN3 is a phosphatidylinositol 3,4,5-triphosphate (PIP3) lipid phosphatase that is frequently inactivated in cancer by mutation, epigenetic silencing, or post-translational modifications (PTMs) (1-8). Loss of PTEN function allows unabated production of PIP3 from PIP2 by PI3Ks and stimulates the AKT protein kinase signaling pathway and cancer cell proliferation (1, 9, 10). Understanding the mechanisms of PTEN regulation is therefore critical to the development of therapeutics that can treat a wide variety of cancers.PTEN is a ϳ45-kDa protein composed of an N-terminal PTP-type phosphatase domain, a midsection phospholipid membrane interacting C2 domain, and a 50-residue regulatory tail (1). A crystal structure of the core PTEN catalytic-C2 region has revealed intimate interactions between the catalytic and C2 domains but omitted the C-terminal tail, which is considered to be flexible and largely unstructured (11). One of the most intensively studied set of PTMs in PTEN is a cluster of four C-terminal phosphorylations at Ser 380 , Thr 382 , Thr 383 , and Ser 385 (12-15). These phosphorylation events have been suggested to be catalyzed by protein kinases casein kinase 2 and/or glycogen synthase kinase 3␤ and demonstrated to drive a conformational change that inhibits membrane interactions and reduces catalytic activity (12-15).Several reports that shed light on the molecular basis for this phospho-dependent regulatory event suggest that the PTEN tail phosphorylation induces conformational closure involving intramolecular interactions between the tail and the CBR3 loop of the C2 domain of PTEN (14 -16). H...

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“…Probing the conformation of the ISWI ATPase domain with genetically encoded photoreactive cross-linkers and mass spectrometry allowed us to take advantage of the UV-reactive p-benzoyl-p-phenylalanine. It could be shown that the ATPase lobes strongly rotate against each other, a movement postulated earlier to be necessary to achieve a catalytically competent state (Forne et al 2012).…”

Section: Biohybrid Systems: Modifying Proteins Containing Unnatural Amentioning

confidence: 99%

Protein Tectons in Synthetic Biology

Schiller

2014

Synthetic Biology

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The expansion of cellular functions via novel modular building blocks, namely unnatural amino acids, their site-selective genetically encoded cotranslational incorporation into proteins, requires the redesign and expansion of the translational network with additional components, the orthogonal tRNA and tRNA synthetase. At the next level protein tectons (tecton = architectural building block) constitute complex genetically encoded "material libraries" inside the cell. These protein tectons are architectural building blocks allowing for complex supramolecular self-assembly inside the cell, forming cellular compartments or constituting 3D matrix mimicry of the extracellular matrix outside the cell. In addition they form the basis for biohybrid materials in protein/enzyme engineering, nanotechnology and regenerative medicine. The defined modification of protein tectons utilizing chemical biology allows for the selective bioconjugation e.g. of unnatural amino acids, via bioorthogonal chemical reactions introducing novel chemical entities expanding the repertoire of "posttranslational" protein modifications in vitro and in vivo for various applications.

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Probing the Conformation of the ISWI ATPase Domain With Genetically Encoded Photoreactive Crosslinkers and Mass Spectrometry

Cited by 46 publications

References 39 publications

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

DksA regulates RNA polymerase in Escherichia coli through a network of interactions in the secondary channel that includes Sequence Insertion 1

Molecular Features of Phosphatase and Tensin Homolog (PTEN) Regulation by C-terminal Phosphorylation

Protein Tectons in Synthetic Biology

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