Biomedical subjects

James M Berger

Publications and source records attributed to James M Berger.

At least 19 recordsLinked to original sources

PubMed2006

Structure of the yeast histone H3-ASF1 interaction: implications for chaperone mechanism, species-specific interactions, and epigenetics.

BACKGROUND: The histone H3/H4 chaperone Asf1 (anti-silencing function 1) is required for the establishment and maintenance of proper chromatin structure, as well as for genome stability in eukaryotes. Asf1 participates in both DNA replication-coupled (RC) and replication-independent (RI) histone deposition reactions in vitro and interacts with complexes responsible for both pathways in vivo. Asf1 is known to directly bind histone H3, however, high-resolution structural information about the geometry of this interaction was previously unknown. RESULTS: Here we report the structure of a histone/histone chaperone interaction. We have solved the 2.2 A crystal structure of the conserved N-terminal immunoglobulin fold domain of yeast Asf1 (residues 2-155) bound to the C-terminal helix of yeast histone H3 (residues 121-134). The structure defines a histone-binding patch on Asf1 consisting of both conserved and yeast-specific residues; mutation of these residues abrogates H3/H4 binding affinity. The geometry of the interaction indicates that Asf1 binds to histones H3/H4 in a manner that likely blocks sterically the H3/H3 interface of the nucleosomal four-helix bundle. CONCLUSION: These data clarify how Asf1 regulates histone stoichiometry to modulate epigenetic inheritance. The structure further suggests a physical model in which Asf1 contributes to interpretation of a "histone H3 barcode" for sorting H3 isoforms into different deposition pathways.

Animals↗

PubMed2006

Structural insights into RNA-dependent ring closure and ATPase activation by the Rho termination factor.

Hexameric helicases and translocases are required for numerous essential nucleic-acid transactions. To better understand the mechanisms by which these enzymes recognize target substrates and use nucleotide hydrolysis to power molecular movement, we have determined the structure of the Rho transcription termination factor, a hexameric RNA/DNA helicase, with single-stranded RNA bound to the motor domains of the protein. The structure reveals a closed-ring "trimer of dimers" conformation for the hexamer that contains an unanticipated arrangement of conserved loops required for nucleic-acid translocation. RNA extends across a shallow intersubunit channel formed by conserved amino acids required for RNA-stimulated ATP hydrolysis and translocation and directly contacts a conserved lysine, just upstream of the catalytic GKT triad, in the phosphate-binding (P loop) motif of the ATP-binding pocket. The structure explains the molecular effects of numerous mutations and provides new insights into the links between substrate recognition, ATP turnover, and coordinated strand movement.

Adenosine Triphosphatases↗

PubMed2006

Regulation of bacterial priming and daughter strand synthesis through helicase-primase interactions.

The replisome is a multi-component molecular machine responsible for rapidly and accurately copying the genome of an organism. A central member of the bacterial replisome is DnaB, the replicative helicase, which separates the parental duplex to provide templates for newly synthesized daughter strands. A unique RNA polymerase, the DnaG primase, associates with DnaB to repeatedly initiate thousands of Okazaki fragments per replication cycle on the lagging strand. A number of studies have shown that the stability and frequency of the interaction between DnaG and DnaB determines Okazaki fragment length. More recent work indicates that each DnaB hexamer associates with multiple DnaG molecules and that these primases can coordinate with one another to regulate their activities at a replication fork. Together, disparate lines of evidence are beginning to suggest that Okazaki fragment initiation may be controlled in part by crosstalk between multiple primases bound to the helicase.

DNA↗

PubMed2006

Structural basis for topoisomerase VI inhibition by the anti-Hsp90 drug radicicol.

Members of the GHL ATPase superfamily, including type II topoisomerases, Hsp90-class chaperones, and MutL, all share a common GHKL-type ATP-binding fold and act as nucleotide-controlled 'molecular clamps'. These enzymes' ATP-binding sites have proven to be rich drug targets, and certain inhibitors of type II topoisomerases and Hsp90 bind to this region and competitively inhibit these enzymes. Recently, it was found that radicicol, a drug known to block Hsp90 function, also inhibits the archaeal type IIB topoisomerase topo VI. Here, we use X-ray crystallography to show that despite low sequence identity ( approximately 10-12%) between topo VI and Hsp90, radicicol binds to the ATPase sites of these two enzymes in an equivalent manner. We further demonstrate that radicicol inhibits both the dimerization of the topo VI ATPase domains and ATP hydrolysis, two critical steps in the enzyme's strand passage reaction. This work contributes to a growing set of structures detailing the interactions between GHL-family proteins and various drugs, and reveals radicicol as a versatile scaffold for targeting distantly related GHL enzymes.

Adenosine Triphosphatases↗

PubMed2006

Structural basis for ATP-dependent DnaA assembly and replication-origin remodeling.

In bacteria, the initiation of replication is controlled by DnaA, a member of the ATPases associated with various cellular activities (AAA+) protein superfamily. ATP binding allows DnaA to transition from a monomeric state into a large oligomeric complex that remodels replication origins, triggers duplex melting and facilitates replisome assembly. The crystal structure of AMP-PCP-bound DnaA reveals a right-handed superhelix defined by specific protein-ATP interactions. The observed quaternary structure of DnaA, along with topology footprint assays, indicates that a right-handed DNA wrap is formed around the initiation nucleoprotein complex. This model clarifies how DnaA engages and unwinds bacterial origins and suggests that additional, regulatory AAA+ proteins engage DnaA at filament ends. Eukaryotic and archaeal initiators also have the structural elements that promote open-helix formation, indicating that a spiral, open-ring AAA+ assembly forms the core element of initiators in all domains of life.

Adenosine Triphosphate↗

PubMed2006

Nucleotide-dependent conformational changes in the DnaA-like core of the origin recognition complex.

Structural details of initiator proteins for DNA replication have provided clues to the molecular events in this process. EM reconstructions of the Drosophila melanogaster origin recognition complex (ORC) reveal nucleotide-dependent conformational changes in the core of the complex. All five AAA+ domains in ORC contain a conserved structural element that, in DnaA, promotes formation of a right-handed helix, indicating that helical AAA+ substructures may be a feature of all initiators. A DnaA helical pentamer can be docked into ORC, and the location of Orc5 uniquely positions this core. The results suggest that ATP-dependent conformational changes observed in ORC derive from reorientation of the AAA+ domains. By analogy to the DNA-wrapping activity of DnaA, we posit that ORC together with Cdc6 prepares origin DNA for helicase loading through mechanisms related to the established pathway of prokaryotes.

Adenosine Triphosphate↗

PubMed2006

Structural and computational characterization of the SHV-1 beta-lactamase-beta-lactamase inhibitor protein interface.

Beta-lactamase inhibitor protein (BLIP) binds a variety of class A beta-lactamases with affinities ranging from micromolar to picomolar. Whereas the TEM-1 and SHV-1 beta-lactamases are almost structurally identical, BLIP binds TEM-1 approximately 1000-fold tighter than SHV-1. Determining the underlying source of this affinity difference is important for understanding the molecular basis of beta-lactamase inhibition and mechanisms of protein-protein interface specificity and affinity. Here we present the 1.6A resolution crystal structure of SHV-1.BLIP. In addition, a point mutation was identified, SHV D104E, that increases SHV.BLIP binding affinity from micromolar to nanomolar. Comparison of the SHV-1.BLIP structure with the published TEM-1.BLIP structure suggests that the increased volume of Glu-104 stabilizes a key binding loop in the interface. Solution of the 1.8A SHV D104K.BLIP crystal structure identifies a novel conformation in which this binding loop is removed from the interface. Using these structural data, we evaluated the ability of EGAD, a program developed for computational protein design, to calculate changes in the stability of mutant beta-lactamase.BLIP complexes. Changes in binding affinity were calculated within an error of 1.6 kcal/mol of the experimental values for 112 mutations at the TEM-1.BLIP interface and within an error of 2.2 kcal/mol for 24 mutations at the SHV-1.BLIP interface. The reasonable success of EGAD in predicting changes in interface stability is a promising step toward understanding the stability of the beta-lactamase.BLIP complexes and computationally assisted design of tight binding BLIP variants.

Amino Acid Sequence↗

PubMed2006

Activation of the DExD/H-box protein Dbp5 by the nuclear-pore protein Gle1 and its coactivator InsP6 is required for mRNA export.

The DExD/H-box ATPase Dbp5 is essential for nuclear mRNA export, although its precise role in this process remains poorly understood. Here, we identify the nuclear pore protein Gle1 as a cellular activator of Dbp5. Dbp5 alone is unable to stably bind RNA or effectively hydrolyse ATP under physiological conditions, but addition of Gle1 dramatically stimulates these activities. A gle1 point mutant deficient for Dbp5 stimulation in vitro displays an mRNA export defect in vivo, indicating that activation of Dbp5 is an essential function of Gle1. Interestingly, Gle1 binds directly to inositol hexakisphosphate (InsP6) and InsP6 potentiates the Gle1-mediated stimulation of Dbp5. Dominant mutations in DBP5 and GLE1 that rescue mRNA export phenotypes associated with the lack of InsP6 mimic the InsP6 effects in vitro. Our results define specific functions for Gle1 and InsP6 in mRNA export and suggest that local activation of Dbp5 at the nuclear pore is critical for mRNA export.

Active Transport, Cell Nucleus↗

PubMed2006

A microfluidic device for kinetic optimization of protein crystallization and in situ structure determination.

The unprecedented economies of scale and unique mass transport properties of microfluidic devices made them viable nano-volume protein crystallization screening platforms. However, realizing the full potential of microfluidic crystallization requires complementary technologies for crystal optimization and harvesting. In this paper, we report a microfluidic device which provides a link between chip-based nanoliter volume crystallization screening and structure analysis through "kinetic optimization" of crystallization reactions and in situ structure determination. Kinetic optimization through systematic variation of reactor geometry and actuation of micromechanical valves is used to screen a large ensemble of kinetic trajectories that are not practical with conventional techniques. Using this device, we demonstrate control over crystal quality, reliable scale-up from nanoliter volume reactions, facile harvesting and cryoprotectant screening, and protein structure determination at atomic resolution from data collected in-chip.

Aldose-Ketose Isomerases↗

PubMed2006

Crystal structure and nonhomologous end-joining function of the ligase component of Mycobacterium DNA ligase D.

DNA ligase D (LigD) is a large polyfunctional enzyme involved in nonhomologous end-joining (NHEJ) in mycobacteria. LigD consists of a C-terminal ATP-dependent ligase domain fused to upstream polymerase and phosphoesterase modules. Here we report the 2.4 angstroms crystal structure of the ligase domain of Mycobacterium LigD, captured as the covalent ligase-AMP intermediate with a divalent metal in the active site. A chloride anion on the protein surface coordinated by the ribose 3'-OH and caged by arginine and lysine side chains is a putative mimetic of the 5'-phosphate at a DNA nick. Structure-guided mutational analysis revealed distinct requirements for the adenylylation and end-sealing reactions catalyzed by LigD. We found that a mutation of Mycobacterium LigD that ablates only ligase activity results in decreased fidelity of NHEJ in vivo and a strong bias of mutagenic events toward deletions instead of insertions at the sealed DNA ends. This phenotype contrasts with the increased fidelity of double-strand break repair in deltaligD cells or in a strain in which only the polymerase function of LigD is defective. We surmise that the signature error-prone quality of bacterial NHEJ in vivo arises from a dynamic balance between the end-remodeling and end-sealing steps.

Amino Acid Sequence↗

PubMed2006

Molecular basis for G protein control of the prokaryotic ATP sulfurylase.

Sulfate assimilation is a critical component of both primary and secondary metabolism. An essential step in this pathway is the activation of sulfate through adenylation by the enzyme ATP sulfurylase (ATPS), forming adenosine 5'-phosphosulfate (APS). Proteobacterial ATPS overcomes this energetically unfavorable reaction by associating with a regulatory G protein, coupling the energy of GTP hydrolysis to APS formation. To discover the molecular basis of this unusual role for a G protein, we biochemically characterized and solved the X-ray crystal structure of a complex between Pseudomonas syringae ATPS (CysD) and its associated regulatory G protein (CysN). The structure of CysN*D shows the two proteins in tight association; however, the nucleotides bound to each subunit are spatially segregated. We provide evidence that conserved switch motifs in the G domain of CysN allosterically mediate interactions between the nucleotide binding sites. This structure suggests a molecular mechanism by which conserved G domain architecture is used to energetically link GTP turnover to the production of an essential metabolite.

Amino Acid Sequence↗

PubMed2006

Evolutionary relationships and structural mechanisms of AAA+ proteins.

Complex cellular events commonly depend on the activity of molecular "machines" that efficiently couple enzymatic and regulatory functions within a multiprotein assembly. An essential and expanding subset of these assemblies comprises proteins of the ATPases associated with diverse cellular activities (AAA+) family. The defining feature of AAA+ proteins is a structurally conserved ATP-binding module that oligomerizes into active arrays. ATP binding and hydrolysis events at the interface of neighboring subunits drive conformational changes within the AAA+ assembly that direct translocation or remodeling of target substrates. In this review, we describe the critical features of the AAA+ domain, summarize our current knowledge of how this versatile element is incorporated into larger assemblies, and discuss specific adaptations of the AAA+ fold that allow complex molecular manipulations to be carried out for a highly diverse set of macromolecular targets.

Adenosine Triphosphatases↗

PubMed2005

Crosstalk between primase subunits can act to regulate primer synthesis in trans.

The coordination of primase function within the replisome is an essential but poorly understood feature of lagging strand synthesis. By using crystallography and small-angle X-ray scattering (SAXS), we show that functional elements of bacterial primase transition between two dominant conformations: an extended form that uncouples a regulatory domain from its associated RNA polymerase core and a compact state that sequesters the regulatory region from the site of primer synthesis. FRET studies and priming assays reveal that the regulatory domain of one primase subunit productively associates with nucleic acid that is bound to the polymerase domain of a second protomer in trans. This intersubunit interaction allows primase to select initiation sites on template DNA and implicates the regulatory domain as a "molecular brake" that restricts primer length. Our data suggest that the replisome may cooperatively use multiple primases and this conformational switch to control initiation frequency, processivity, and ultimately, Okazaki fragment synthesis.

Bacteriophage T7↗

PubMed2005

Structure of the nuclease domain of ribonuclease III from M. tuberculosis at 2.1 A.

RNase III enzymes are a highly conserved family of proteins that specifically cleave double-stranded (ds)RNA. These proteins are involved in a diverse group of functions, including ribosomal RNA processing, mRNA maturation and decay, snRNA and snoRNA processing, and RNA interference. Here we report the crystal structure of the nuclease domain of RNase III from the pathogen Mycobacterium tuberculosis. Although globally similar to other RNase III folds, this structure has some features not observed in previously reported models. These include the presence of an additional metal ion near the catalytic site, as well as conserved secondary structural elements that are proposed to have functional roles in the recognition of dsRNAs.

Bacterial Proteins↗

PubMed2005

The structural basis for substrate specificity in DNA topoisomerase IV.

Most bacteria possess two type IIA topoisomerases, DNA gyrase and topo IV, that together help manage chromosome integrity and topology. Gyrase primarily introduces negative supercoils into DNA, an activity mediated by the C-terminal domain of its DNA binding subunit (GyrA). Although closely related to gyrase, topo IV preferentially decatenates DNA and relaxes positive supercoils. Here we report the structure of the full-length Escherichia coli ParC dimer at 3.0 A resolution. The N-terminal DNA binding region of ParC is highly similar to that of GyrA, but the ParC dimer adopts a markedly different conformation. The C-terminal domain (CTD) of ParC is revealed to be a degenerate form of the homologous GyrA CTD, and is anchored to the top of the N-terminal domains in a configuration different from that thought to occur in gyrase. Biochemical assays show that the ParC CTD controls the substrate specificity of topo IV, likely by capturing DNA segments of certain crossover geometries. This work delineates strong mechanistic parallels between topo IV and gyrase, while explaining how structural differences between the two enzyme families have led to distinct activity profiles. These findings in turn explain how the structures and functions of bacterial type IIA topoisomerases have evolved to meet specific needs of different bacterial families for the control of chromosome superstructure.

DNA Topoisomerase IV↗

PubMed2005

The MukF subunit of Escherichia coli condensin: architecture and functional relationship to kleisins.

The Escherichia coli MukB, MukE, and MukF proteins form a bacterial condensin (MukBEF) that contributes to chromosome management by compacting DNA. MukB is an ATPase and DNA-binding protein of the SMC superfamily; however, the structure and function of non-SMC components, such as MukF, have been less forthcoming. Here, we report the crystal structure of the N-terminal 287 amino acids of MukF at 2.9 A resolution. This region folds into a winged-helix domain and an extended coiled-coil domain that self-associate to form a stable, doubly domain-swapped dimer. Protein dissection and affinity purification data demonstrate that the region of MukF C-terminal to this fragment binds to MukE and MukB. Our findings, together with sequence analyses, indicate that MukF is a kleisin subunit for E. coli condensin and suggest a means by which it may organize the MukBEF assembly.

Adenosine Triphosphatases↗

PubMed2005

Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA.

In senescent cells, specialized domains of transcriptionally silent senescence-associated heterochromatic foci (SAHF), containing heterochromatin proteins such as HP1, are thought to repress expression of proliferation-promoting genes. We have investigated the composition and mode of assembly of SAHF and its contribution to cell cycle exit. SAHF is enriched in a transcription-silencing histone H2A variant, macroH2A. As cells approach senescence, a known chromatin regulator, HIRA, enters PML nuclear bodies, where it transiently colocalizes with HP1 proteins, prior to incorporation of HP1 proteins into SAHF. A physical complex containing HIRA and another chromatin regulator, ASF1a, is rate limiting for formation of SAHF and onset of senescence, and ASF1a is required for formation of SAHF and efficient senescence-associated cell cycle exit. These data indicate that HIRA and ASF1a drive formation of macroH2A-containing SAHF and senescence-associated cell cycle exit, via a pathway that appears to depend on flux of heterochromatic proteins through PML bodies.

Amino Acid Sequence↗

PubMed2005

Unraveling the early steps of prokaryotic replication.

In prokaryotes, many of the physical mechanisms governing the process of initiating DNA replication are now emerging. For example, certain organizational features of origins, such as the use of repetitive sequence elements for initiator-binding sites, are found throughout bacteria and many archaea. Common themes in the regulation of initiation, including origin sequestration by trans-acting factors, titration of initiator levels by cis- and trans-acting factors, and control of initiator function by ATP, also exist. Recent studies have shown that prokaryotic initiator structures are both modular and conserved, and have begun to reveal how these proteins specifically recognize target DNA sequences. These properties probably control initiator self-assembly and DNA remodeling to properly fire replication origins.

Adenosine Triphosphatases↗