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1.  Analysis of microRNA-target interactions across diverse cancer types 
Nature structural & molecular biology  2013;20(11):1325-1332.
Little is known about the extent to which individual microRNAs (miRNAs) regulate common processes of tumor biology across diverse cancer types. Using molecular profiles of >3,000 tumors from 11 human cancer types in The Cancer Genome Atlas, we systematically analyzed expression of miRNAs and mRNAs across cancer types to infer recurrent cancer-associated mi RNA-target relationships. As we expected, the inferred relationships were consistent with sequence-based predictions and published data from miRNA perturbation experiments. Notably, miRNAs with recurrent target relationships were frequently regulated by genetic and epigenetic alterations across the studied cancer types. We also identify new examples of miRNAs that coordinately regulate cancer pathways, including the miR-29 family, which recurrently regulates active DNA demethylation pathway members TET1 and TDG. The online resource allows exploration and prioritization of miRNA-target interactions that potentially regulate tumorigenesis.
PMCID: PMC3982325  PMID: 24096364
2.  Crystal structure of the trithorax group protein Ash2L reveals a Forkhead-like DNA binding domain 
Human ASH2L is a trithorax group (TrxG) protein and a regulatory subunit of the SET1 family of lysine methyltransferases. Here we report that Ash2L binds DNA employing a Forkhead-like helix-wing-helix (HWH) domain. In vivo, Ash2L HWH domain is required for binding to the β-globin locus control region (LCR), histone H3 Lys4 tri-methylation and maximal expression of the β-globin gene, validating the functional importance of Ash2L DNA binding activity.
PMCID: PMC3983046  PMID: 21642971 CAMSID: cams4090
3.  The structural basis of transferrin sequestration by transferrin-binding protein B 
Neisseria meningitidis, the causative agent of bacterial meningitis, acquires the essential element iron from the host glycoprotein transferrin (Tf) during infection via a surface Tf receptor system composed of proteins TbpA and TbpB. Here in we present the crystal structures of TbpB from N. meningitidis, in its apo form and in complex with human Tf (hTf). The structure reveals how TbpB sequesters hTf and initiates iron release from hTf.
PMCID: PMC3981719  PMID: 22343719 CAMSID: cams4091
4.  Regulation of Mus81-Eme1 Holliday junction resolvase in response to DNA damage 
Structure-specific DNA endonucleases have critical roles during DNA replication, repair and recombination, yet they also harbor the potential for causing genome instability. Controlling these enzymes may be essential to ensure efficient processing of ad hoc substrates and to prevent random, unscheduled processing of other DNA structures, but it is unknown whether structure-specific endonucleases are regulated in response to DNA damage. Here, we uncover DNA damage-induced activation of Mus81-Eme1 Holliday junction resolvase in fission yeast. This novel regulation requires both Cdc2CDK1 and Rad3ATR-dependent phosphorylations of Eme1. Mus81-Eme1 activation prevents gross chromosomal rearrangements in cells lacking the BLM-related DNA helicase Rqh1. We propose that linking Mus81-Eme1 DNA damaged-induced activation to cell cycle progression ensures efficient resolution of Holliday junctions that escape dissolution by Rqh1-TopIII while preventing unnecessary DNA cleavages.
PMCID: PMC3978046  PMID: 23584455
5.  Complexes of HIV-1 RT, NNRTI and RNA/DNA hybrid reveal a structure compatible with RNA degradation 
Structures of type-1 human immunodeficiency virus (HIV-1) reverse transcriptase (RT) have been determined in several forms, but only one contains an RNA/DNA hybrid. Here we report three structures of HIV-1 RT complexed with a non-nucleotide RT inhibitor (NNRTI) and an RNA/DNA hybrid. In the presence of an NNRTI, the RNA/DNA structure differs from all prior nucleic acid bound to RT including the RNA/DNA hybrid. The enzyme structure also differs from all previous RT–DNA complexes. As a result, the hybrid has ready access to the RNase H active site. These observations indicate that an RT–nucleic acid complex may adopt two structural states, one competent for DNA polymerization and the other for RNA degradation. RT mutations that confer drug resistance but are distant from the inhibitor-binding sites often map to the unique RT–hybrid interface that undergoes conformational changes between two catalytic states.
PMCID: PMC3973182  PMID: 23314251
RNase H; p51-p66 interface; drug resistance; bent and underwound helix
6.  A cancer-associated BRCA2 mutation reveals masked nuclear export signals controlling localization 
Nature structural & molecular biology  2013;20(10):1191-1198.
Germline mis-sense mutations affecting a single BRCA2 allele predispose humans to cancer. Here, we identify a protein-targeting mechanism disrupted by the cancer-associated mutation, BRCA2D2723H that controls the nuclear localization of BRCA2 and its cargo, the recombination enzyme RAD51. A nuclear export signal (NES) in BRCA2 is masked by its interaction with a partner protein, DSS1, such that point mutations impairing BRCA2-DSS1 binding render BRCA2 cytoplasmic. In turn, cytoplasmic mis-localization of mutant BRCA2 inhibits the nuclear retention of RAD51, by exposing a similar NES in RAD51 usually obscured by the BRCA2-RAD51 interaction. Thus, a series of NES-masking interactions localizes BRCA2 and RAD51 in the nucleus. Interestingly, BRCA2D2723H decreases RAD51 nuclear retention even when wildtype BRCA2 is present. Our findings suggest a mechanism for regulation of the nucleo-cytoplasmic distribution of BRCA2 and RAD51, and for its impairment by a heterozygous disease-associated mutation.
PMCID: PMC3796201  PMID: 24013206
BRCA2; RAD51; nuclear localization; protein targeting; cancer predisposition; germline mutation
7.  Regulation of transcription by the MLL2 complex and MLL complex-associated AKAP95 
Nature structural & molecular biology  2013;20(10):10.1038/nsmb.2656.
Although histone H3 lysine 4 (H3K4) methylation is widely associated with gene activation, direct evidence for its causal role in transcription, through specific MLL family members, is scarce. Here we have purified a human MLL2 (Kmt2b) complex that is highly active in H3K4 methylation and chromatin transcription in a cell-free system. This effect requires SAM and intact H3K4, establishing a direct and causal role for MLL2-mediated H3K4 methylation in transcription. We then show that human AKAP95, a chromatin-associated protein, is physically and functionally associated with the DPY30–MLL complexes and directly enhances their methyltransferase activity. Ectopic AKAP95 stimulates expression of a chromosomal reporter in synergy with MLL1 or MLL2, whereas AKAP95 depletion impairs retinoic acid-mediated gene induction in embryonic stem cells. These results demonstrate an important role for AKAP95 in regulating histone methylation and gene expression, particularly during cell fate transitions.
PMCID: PMC3813012  PMID: 23995757
8.  Mechanism and consequence of the autoactivation p38α Mitogen-activated Protein Kinase promoted by TAB1 
Nature structural & molecular biology  2013;20(10):10.1038/nsmb.2668.
p38α Mitogen-activated Protein Kinase (p38α) is activated by a variety of mechanisms, including autophosphorylation initiated by TGFβ-activated kinase 1 binding protein 1 (TAB1) during myocardial ischemia and other stresses. Chemical genetic approaches and co-expression in mammalian, bacterial and cell-free systems revealed that mouse p38α autophosphorylation occurs in cis by direct interaction with TAB1(371-416). In isolated rat cardiac myocytes and perfused mouse hearts TAT-TAB1(371-416) rapidly activates p38 and profoundly perturbs function. Crystal structures and characterization in solution revealed a bipartite docking site for TAB1 in the p38α C-terminal kinase lobe. TAB1 binding stabilizes active p38α and induces rearrangements within the activation segment by helical extension of the Thr-Gly-Tyr motif that allows auto-phosphorylation in cis. Interference with p38α recognition by TAB1 abolishes its cardiac toxicity. Potentially, such intervention could circumvent the drawbacks seen when pharmacological inhibitors of p38 catalytic activity are used clinically.
PMCID: PMC3822283  PMID: 24037507
9.  The Microprocessor controls the activity of mammalian retrotransposons 
Nature structural & molecular biology  2013;20(10):10.1038/nsmb.2658.
More than half of the human genome is made of Transposable Elements. Their ongoing mobilization is a driving force in genetic diversity; however, little is known about how the host regulates their activity. Here, we show that the Microprocessor (Drosha-DGCR8), which is required for microRNA biogenesis, also recognizes and binds RNAs derived from human LINE-1 (Long INterspersed Element 1), Alu and SVA retrotransposons. Expression analyses demonstrate that cells lacking a functional Microprocessor accumulate LINE-1 mRNA and encoded proteins. Furthermore, we show that structured regions of the LINE-1 mRNA can be cleaved in vitro by Drosha. Additionally, we used a cell culture-based assay to show that the Microprocessor negatively regulates LINE-1 and Alu retrotransposition in vivo. Altogether, these data reveal a new role for the Microprocessor as a post-transcriptional repressor of mammalian retrotransposons acting as a defender of human genome integrity.
PMCID: PMC3836241  PMID: 23995758
10.  Structure of a pseudokinase domain switch that controls oncogenic activation of Jak kinases 
Nature structural & molecular biology  2013;20(10):10.1038/nsmb.2673.
The V617F mutation in the Jak2 pseudokinase domain causes myeloproliferative neoplasms, and the equivalent mutation in Jak1 (V658F) is found in T-cell leukemias. Crystal structures of wild type and V658F mutant human Jak1 pseudokinase reveal a conformational switch that remodels a linker segment encoded by exon 12, which is also a site of mutations in Jak2. This switch is required for V617F-mediated Jak2 activation, and possibly for physiologic Jak activation.
PMCID: PMC3863620  PMID: 24013208
11.  Reconstitution of the 26S proteasome reveals functional asymmetries in its AAA+ unfoldase 
Nature structural & molecular biology  2013;20(10):10.1038/nsmb.2659.
The 26S proteasome is the major eukaryotic ATP-dependent protease, yet the detailed mechanisms utilized by the proteasomal heterohexameric AAA+ unfoldase to drive substrate degradation remain poorly understood. To perform systematic mutational analyses of individual ATPase subunits, we heterologously expressed unfoldase subcomplex from Saccharomyces cerevisiae in Escherichia coli and reconstituted the proteasome in vitro. Our studies demonstrate that the six ATPases play distinct roles in degradation, corresponding to their positions in spiral staircases adopted by the AAA+ domains in the absence and presence of substrate. ATP hydrolysis in subunits at the top of the staircases is critical for substrate engagement and translocation. While the unfoldase relies on this vertical asymmetry for substrate processing, interaction with the peptidase exhibits three-fold symmetry with contributions from every other subunit. These diverse functional asymmetries highlight how the 26S proteasome deviates from simpler, homomeric AAA+ proteases.
PMCID: PMC3869383  PMID: 24013205
12.  Cycles in spatial and temporal chromosomal organization driven by the circadian clock 
Nature structural & molecular biology  2013;20(10):10.1038/nsmb.2667.
Dynamic transitions in the epigenome have been associated with regulated patterns of nuclear organization. The accumulating evidence that chromatin remodeling is implicated in circadian function prompted us to explore whether the clock may control nuclear architecture. We applied the 3C-derived 4C technology (Chromosome Conformation Capture on Chip) in mouse embryonic fibroblasts (MEFs) to demonstrate the presence of circadian long-range interactions, using the clock-controlled Dbp gene as bait. The circadian genomic interactions with Dbp are highly specific and are absent in MEFs whose clock is disrupted by ablation of the Bmal1 gene. We establish that the Dbp circadian interactome contains a wide variety of genes and clock-related DNA elements. These findings reveal a previously unappreciated circadian and clock-dependent shaping of the nuclear landscape.
PMCID: PMC3885543  PMID: 24056944
13.  mRNA–mRNA duplexes that auto-elicit Staufen1-mediated mRNA decay 
Nature structural & molecular biology  2013;20(10):1214-1220.
We report a new mechanism by which human mRNAs crosstalk: an Alu element in the 3'-untranslated region (3' UTR) of one mRNA can base-pair with a partially complementary Alu element in the 3' UTR of a different mRNA thereby creating a Staufen1 (STAU1)-binding site (SBS). STAU1 binding to a 3' UTR SBS was previously shown to trigger STAU1-mediated mRNA decay (SMD) by directly recruiting the ATP-dependent RNA helicase UPF1, which is also a key factor in the mechanistically related nonsense-mediated mRNA decay (NMD) pathway. In the case of a 3' UTR SBS created via mRNA–mRNA base-pairing, we show that SMD targets both mRNAs in the duplex provided that both mRNAs are translated. If only one mRNA is translated, then it alone is targeted for SMD. We demonstrate the importance of mRNA–mRNA-triggered SMD to the processes of cell migration and invasion.
PMCID: PMC3947523  PMID: 24056942
14.  MicroRNA-based discovery of barriers to dedifferentiation of fibroblasts to pluripotent stem cells 
Nature structural & molecular biology  2013;20(10):1227-1235.
Individual microRNAs (miRNAs) can target hundreds of messenger RNAs forming networks of presumably cooperating genes. To test this presumption, we functionally screened miRNAs and their targets in the context of de-differentiation of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Along with the miR-302/miR-294 family, the miR-181 family arose as a novel enhancer of the initiation phase of reprogramming. Endogenous miR-181 miRNAs were transiently elevated with introduction of Oct4, Sox2, and Klf4 (OSK), and their inhibition diminished iPSC colony formation. We tested the functional contribution of 114 individual targets of the two families, revealing twenty-five genes that normally suppress initiation. Co-inhibition of targets cooperatively promoted both the frequency and kinetics of OSK reprogramming. These data establish two of the largest functionally defined networks of miRNA-mRNA interactions, elucidating novel relationships among genes that act together to suppress early stages of reprogramming.
PMCID: PMC3955211  PMID: 24037508
15.  Centromere chromatin: a loose grip on the nucleosome? 
PMCID: PMC3970403  PMID: 24389544
16.  DNA secondary structures and epigenetic determinants of cancer genome evolution 
An unstable genome is a hallmark of many cancers. It is unclear, however, whether some mutagenic features driving somatic alterations in cancer are encoded in the genome sequence and whether they can operate in a tissue-specific manner. We performed a genome-wide analysis of 663,446 DNA breakpoints associated with somatic copy-number alterations (SCNAs) from 2,792 cancer samples classified into 26 cancer types. Many SCNA breakpoints are spatially clustered in cancer genomes. We observed a significant enrichment for G-quadruplex sequences (G4s) in the vicinity of SCNA breakpoints and established that SCNAs show a strand bias consistent with G4-mediated structural alterations. Notably, abnormal hypomethylation near G4s-rich regions is a common signature for many SCNA breakpoint hotspots. We propose a mechanistic hypothesis that abnormal hypomethylation in genomic regions enriched for G4s acts as a mutagenic factor driving tissue-specific mutational landscapes in cancer.
PMCID: PMC3963273  PMID: 21725294
17.  Antidiabetic Phospholipid – Nuclear Receptor Complex Reveals the Mechanism for Phospholipid Driven Gene Regulation 
The nuclear receptor Liver Receptor Homolog-1, LRH-1, plays an important role in controlling lipid and cholesterol homeostasis and is a potential target for treatment of diabetes and hepatic diseases. LRH-1 is known to bind phospholipids (PLs) but the role of PLs in controlling LRH-1 activation remains highly debated. Here we describe the structure of both apo LRH-1 and the protein in complex with the antidiabetic dilauroylphosphatidylcholine (DLPC). Our studies show that DLPC binding is a novel dynamic process that alters coregulator selectivity and that the lipid-free receptor interacts with widely expressed corepressors. These observations greatly enhance our understating of LRH-1 regulation and highlight its importance as a novel therapeutic target for controlling diabetes.
PMCID: PMC3960984  PMID: 22504882
nuclear receptor; stem cells; phospholipids; NR5A; Diabetes; phosphatidylcholine
18.  CFTR regulatory region interacts with NBD1 predominantly via multiple transient helices 
The regulatory (R) region of the cystic fibrosis transmembrane conductance regulator (CFTR) is intrinsically disordered and must be phosphorylated at multiple sites for full CFTR channel activity, with no one specific phosphorylation site required. In addition, nucleotide binding and hydrolysis at the nucleotide-binding domains (NBDs) of CFTR are required for channel gating. We report NMR studies in the absence and presence of NBD1 that provide structural details for the isolated R region and its interaction with NBD1 at residue-level resolution. Several sites in the R region with measured fractional helical propensity mediate interactions with NBD1. Phosphorylation reduces the helicity of many R-region sites and reduces their NBD1 interactions. This evidence for a dynamic complex with NBD1 that transiently engages different sites of the R region suggests a structural explanation for the dependence of CFTR activity on multiple PKA phosphorylation sites.
PMCID: PMC3943242  PMID: 17660831
19.  Structurally encoded intraclass differences in EphA clusters drive distinct cell responses 
The functional outcomes of ephrin binding to Eph receptors (Ephs) range from cell repulsion to adhesion. Here we used cell collapse and stripe assays to show contrasting effects of human ephrinA5 binding to EphA2 and EphA4. Despite equivalent ligand-binding affinities EphA4 triggered greater cell collapse, while EphA2-expressing cells adhered better to ephrinA5-coated surfaces. Chimeric receptors showed the ectodomain is a major determinant of cell response. We report crystal structures of EphA4 ectodomain alone and in complexes with ephrinB3 and ephrinA5. These revealed closed clusters with a dimeric or circular arrangement in the crystal lattice, contrasting with extended arrays previously observed for EphA2 ectodomain. Localization microscopy-based analyses showed ligand-stimulated EphA4 induces smaller clusters than EphA2. Mutant Ephs link these characteristics to interactions observed in the crystal lattices, suggesting a mechanism by which distinctive ectodomain surfaces determine clustering, and thereby signalling, properties.
PMCID: PMC3941021  PMID: 23812375
cell adhesion; cell repulsion; receptor clustering; receptor cis interaction; Eph–ephrin crystal structures; Eph ectodomain
20.  E2 ubiquitin conjugating enzymes regulate the deubiquitinating activity of OTUB1 
OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin conjugating enzymes including UBC13 and UBCH5. OTUB1 binds to E2~Ub thioester intermediates and prevent ubiquitin transfer, thereby non-catalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin, and that this stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1-E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin.
PMCID: PMC3941643  PMID: 23955022
21.  Host versus flu: antibodies win a round? 
Structural and functional analyses of three neutralizing antibodies against influenza virus H2 HA may explain why this HA subtype has disappeared from circulation in the human population and point to a potential new avenue for antiflu therapeutics.
PMCID: PMC3762974  PMID: 23463305
22.  Molecular Basis for Amino-Terminal Acetylation by the Heterodimeric NatA Complex 
Amino-terminal acetylation is ubiquitous among eukaryotic proteins and controls a myriad of biological processes. Of the N-terminal acetyltransferases (NATs) that facilitate this co-translational modification, the heterodimeric NatA complex harbors the most diversity for substrate selection and modifies the majority of all amino-terminally acetylated proteins. Here, we report the X-ray crystal structure of the 100 kDa holo-NatA complex from Schizosaccharomyces pombe in the absence and presence of a bisubstrate peptide-CoA conjugate inhibitor, as well as the structure of the uncomplexed Naa10p catalytic subunit. The NatA-Naa15p auxiliary subunit contains 13 TPR motifs and adopts a ring-like topology that wraps around the NatA-Naa10p subunit, an interaction that alters the Naa10p active site for substrate-specific acetylation. These studies have implications for understanding the mechanistic details of other NAT complexes and how regulatory subunits modulate the activity of the broader family of protein acetyltransferases.
PMCID: PMC3766382  PMID: 23912279
23.  Bi-directional processing of pri-miRNAs with branched terminal loops by Arabidopsis Dicer-like1 
miRNAs originate from primary transcripts (pri-miRNAs) with characteristic stem-loop structures. Accurate processing of pri-miRNAs is required for functional miRNAs. Here, using pri-miR166 family as a paradigm, we report the decisive role of pri-miRNA terminal loops in miRNA biogenesis. We found that multi-branched terminal loops in pri-miR166s substantially suppressed miR166 expression in vivo. Unlike canonical processing of pri-miRNAs, terminal-loop-branched (TLBed) pri-miRNAs can be processed by Dicer-like1 (DCL1) complexes bi-directionally: from base to loop and from loop to base, resulting in productive and abortive processing of miRNAs, respectively. In either case, DCL1 complexes canonically cut pri-miRNAs at a distance of 16-17 base pairs (bp) from a reference single-stranded loop region. DCL1 also adjusts processing sites toward an internal loop through its helicase domain. Thus, these results provide new insight into the poorly understood processing mechanism of pri-miRNAs with complicated secondary structures.
PMCID: PMC3766402  PMID: 23934148
24.  Structural basis for regulation of Arp2/3 complex by GMF 
Arp2/3 complex mediates formation of complex cellular structures such as lamellapodia by nucleating branched actin filaments. Arp2/3 complex activity is precisely controlled by more than a dozen regulators, yet the structural mechanism by which regulators interact with the complex is unknown. GMF is a recently discovered regulator of Arp2/3 complex that can inhibit nucleation and dissemble branches. We solved the structure of the 240 kDa complex of Mus musculus GMF and Bos taurus Arp2/3 and found GMF binds to the barbed end of Arp2, overlapping with the proposed binding site of WASP family proteins. The structure suggests GMF can bind branch junctions like cofilin binds filament sides, consistent with a modified cofilin-like mechanism for debranching by GMF. The GMF-Arp2 interface reveals how the ADF-H actin-binding domain in GMF is exploited to specifically recognize Arp2/3 complex and not actin.
PMCID: PMC3766443  PMID: 23893131
25.  Allosteric mechanism of water channel gating by Ca2+–calmodulin 
Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits.
PMCID: PMC3766450  PMID: 23893133
aquaporin (AQP); gating; calmodulin (CaM); electron microscopy (EM); molecular dynamics (MD); calcium regulation; water channel; membrane protein complex

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