Carlos R. Escalante

Structure of IRF-1 with bound DNA reveals determinants of interferon regulation

The family of interferon regulatory factor (IRF) transcription factors is important in the regulation of interferons in response to infection by virus and in the regulation of interferon-inducible genes,. The IRF family is characterized by a unique ‘tryptophan cluster’ DNA-binding region. Here we report the crystal structure of the IRF-1 region bound to the natural positive regulatory domain I (PRD I) DNA element from the interferon-β promoter. The structure provides the first three-dimensional view of a member of the growing IRF family, revealing a new helix–turn–helix motif that latches onto DNA through three of the five conserved tryptophans. The motif selects a short GAAA core sequence through an obliquely angled recognition helix, with an accompanying bending of the DNA axis in the direction of the protein. Together, these features suggest a basis for the occurrence of GAAA repeats within IRF response elements and provide clues to the assembly of the higher-order interferon-β enhancesome.

Transcription Corepressor CtBP Is an NAD+-Regulated Dehydrogenase

Transcriptional repression is based on the selective actions of recruited corepressor complexes, including those with enzymatic activities. One well-characterized developmentally important corepressor is the C-terminal binding protein (CtBP). Although intriguingly related in sequence to D2 hydroxyacid dehydrogenases, the mechanism by which CtBP functions remains unclear. We report here biochemical and crystallographic studies which reveal that CtBP is a functional dehydrogenase. In addition, both a cofactor-dependent conformational change, with NAD(+) and NADH being equivalently effective, and the active site residues are linked to the binding of the PXDLS consensus recognition motif on repressors, such as E1A and RIP140. Together, our data suggest that CtBP is an NAD(+)-regulated component of critical complexes for specific repression events in cells.

Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors

Summary Background Studies on the fusion-inhibitory peptides derived from the heptad repeat 1 and 2 (HR1 and HR2) regions of the HIV-1 envelope glycoprotein gp41 provided crucial information on the viral fusogenic mechanism. We used a similar approach to study the fusogenic mechanism of severe-acute-respiratory-syndrome-associated coronavirus (SARS-CoV). Methods We tested the inhibitory activity against infection of two sets of peptides corresponding to sequences of SARS-CoV spike protein HR1 and HR2 regions and investigated the interactions between the HR1 and HR2 peptides by surface plasmon resonance, sedimentation equilibration analysis, circular dichroism, native polyacrylamide-gel electrophoresis, size exclusion high-performance liquid chromatography, and computer-aided homology modelling and molecule docking analysis. Findings One peptide, CP-1, derived from the HR2 region, inhibited SARS-CoV infection in the micromolar range. CP-1 bound with high affinity to a peptide from the HR1 region, NP-1. CP-1 alone had low -helicity and self-associated to form a trimer in phosphate buffer (pH 7·2). CP-1 and NP-1 mixed in equimolar concentrations formed a six-helix bundle, similar to the fusogenic core structure of HIV-1 gp41. Interpretation After binding to the target cell, the transmembrane spike protein might change conformation by association between the HR1 and HR2 regions to form an oligomeric structure, leading to fusion between the viral and target-cell membranes. At the prefusion intermediate state, CP-1 could bind to the HR1 region and interfere with the conformational changes, resulting in inhibition of SARS-CoV fusion with the target cells. CP-1 might be modifiable to increase its anti-SARS-CoV activity and could be further developed as an antiviral agent for treatment or prophylaxis of SARS-CoV infection.

A Genomic Regulatory Element That Directs Assembly and Function of Immune-Specific AP-1–IRF Complexes

Helping T Helper Transcription Members of the interferon response family of transcription factors (IRFs) are specifically expressed in immune cells and are known to regulate their differentiation. IRF4 and IRF8 regulate gene expression by binding to other transcription factors, which results in their recruitment to composite motifs in the genome. Although the specific mechanism of how this regulation works in some immune cells is understood, how it occurs in T cells is not clear because the transcription factors that normally partner with IRFs are absent. Using genomic analysis, Glasmacher et al. (p. 975, published online 13 September; see the Perspective by Martinez and Rao) now identify IRF4–AP-1 composite elements in T helper 17 (TH17) cells and show that IRF4 and the AP-1 factor Batf cooperatively assemble on a large array of genes required for TH17 cell differentiation and function. Assembly of such heterodimers was also observed in TH2 cells, B cells, and dendritic cells, which suggests the general importance of this motif in immune cell differentiation. Cooperative binding of transcription factors to composite genomic elements regulates T helper 17 cell differentiation. Interferon regulatory factor 4 (IRF4) and IRF8 regulate B, T, macrophage, and dendritic cell differentiation. They are recruited to cis-regulatory Ets-IRF composite elements by PU.1 or Spi-B. How these IRFs target genes in most T cells is enigmatic given the absence of specific Ets partners. Chromatin immunoprecipitation sequencing in T helper 17 (TH17) cells reveals that IRF4 targets sequences enriched for activating protein 1 (AP-1)–IRF composite elements (AICEs) that are co-bound by BATF, an AP-1 factor required for TH17, B, and dendritic cell differentiation. IRF4 and BATF bind cooperatively to structurally divergent AICEs to promote gene activation and TH17 differentiation. The AICE motif directs assembly of IRF4 or IRF8 with BATF heterodimers and is also used in TH2, B, and dendritic cells. This genomic regulatory element and cognate factors appear to have evolved to integrate diverse immunomodulatory signals.

Crystal structure of PU.1/IRF-4/DNA ternary complex

The Ets and IRF transcription factor families contain structurally divergent members, PU.1, Spi-B and IRF-4 (Pip), IRF-8 (ICSBP), respectively, which have evolved to cooperatively assemble on composite DNA elements and regulate gene expression in the immune system. Whereas PU.1 recruits IRF-4 or IRF-8 to DNA, it exhibits an anticooperative interaction with IRF-1 and IRF-2. We report here the structure of the ternary complex formed with the DNA binding domains of PU.1 and IRF-4 on a composite DNA element. The DNA in the complex contorts into an unusual S shape that juxtaposes PU.1 and IRF-4 for selective electrostatic and hydrophobic interactions across the central minor groove. Together, the protein-protein and protein-DNA interactions provide insights into the stereochemical basis of cooperativity and anti-cooperativity between Ets and IRF factors.

Gene repression by coactivator repulsion.

We show that the IRF-2 oncoprotein represses virus-induced IFN-beta gene transcription via a novel mechanism. Virus infection induces recruitment of IRF-2 to some of the endogenous IFN-beta enhancers as part of the enhanceosome. Enhanceosomes bearing IRF-2 cannot activate transcription, due to the presence of a domain in IRF-2 that prevents enhanceosome-dependent recruitment of the CBP-Pol II holoenzyme complex. As a consequence, IRF-2 incorporation into enhanceosomes restricts the number of IFN-beta promoters directing transcription. Remarkably, deletion of the IRF-2 gene increases IFN-beta expression by expanding the number of cells capable of inducing IFN-beta gene transcription in response to virus infection.

DPO4 IS HINDERED IN EXTENDING A G T MISMATCH BY A REVERSE WOBBLE

The ability or inability of a DNA polymerase to extend a mispair directly affects the establishment of genomic mutations. We report here kinetic analyses of the ability of Dpo4, a Y-family polymerase from Sulfolobus solfataricus, to extend from all mispairs opposite a template G or T. Dpo4 is equally inefficient at extending these mispairs, which include, surprisingly, a G·T mispair expected to conform closely to Watson-Crick geometry. To elucidate the basis of this, we solved the structure of Dpo4 bound to G·T-mispaired primer template in the presence of an incoming nucleotide. As a control, we also determined the structure of Dpo4 bound to a matched A-T base pair at the primer terminus. The structures offer a basis for the low efficiency of Dpo4 in extending a G·T mispair: a reverse wobble that deflects the primer 3′-OH away from the incoming nucleotide.

Structure of NF-κB p50/p65 Heterodimer Bound to the PRDII DNA Element from the Interferon-β Promoter

Upon viral infection, NF-kappaB translocates to the nucleus and activates the IFN-beta gene by binding to the PRDII element. Strikingly, NF-kappaB loses its ability to activate the IFN-beta gene when the PRDII element is substituted by closely related sites. We report here the crystal structure of NF-kappaB p50/p65 heterodimer bound to the PRDII element from the IFN-beta promoter. The structure reveals an unexpected alteration in configuration, in which the p50 specificity domain moves by as much as approximately 9 A when compared to NF-kappaB heterodimer bound to the immunoglobulin kappaB site (Ig-kappaB) while maintaining the same base-specific contacts with the DNA. Taken together, the structure offers new insights into the allosteric effects of closely related DNA sites on the configuration of NF-kappaB and its transcriptional selectivity.

Artemis C-terminal region facilitates V(D)J recombination through its interactions with DNA Ligase IV and DNA-PKcs

Interactions of Artemis with DNA Ligase IV and DNA-PKcs are required for efficient coding joint formation.

Residues within the B ' motif are critical for DNA binding by the superfamily 3 helicase Rep40 of adeno-associated virus type 2

We have recently published the crystal structure of the adeno-associated virus type 2 superfamily 3 (SF3) helicase Rep40. Although based on its biochemical properties it is unlikely that Rep40 plays a central role as a replicative helicase the involvement of this motor protein in DNA packaging has recently been demonstrated. Here we focused our attention on residues that fall within and adjacent to the B′ motif of SF3 helicases that directly interact with single-stranded DNA during translocation of the motor protein. In vitro, alanine substitution at positions Lys-404 or Lys-406 abrogated the ability of the protein to interact with single-stranded DNA as demonstrated by electrophoretic mobility shift assay and fluorescence anisotropy, and accordingly these mutants could not unwind a partially duplex DNA substrate. Despite this loss of helicase activity, basal ATPase activity in these mutants remained intact. However, unlike the wild-type protein, K404A and K406A ATPase activity was not stimulated by DNA. As predicted, disruption of motor activity through interference with DNA binding resulted in an inability of Rep40 to package adeno-associated virus DNA in a tissue culture-based assay. Taken together, we characterized, for the first time in an SF3 helicase family member, residues that are directly involved in single-stranded DNA binding and that are critical for the Rep motor activity. Based on our findings we propose B′ as the signature motif of SF3 helicases that is responsible for the complex interactions required for the coupling of DNA binding and ATP hydrolysis.