Tank-binding kinase I (TBK1) plays a key role in the innate immune system by integrating signals from pattern-recognition receptors. Here, we report the X-ray crystal structures of inhibitor-bound inactive and active TBK1 determined to 2.6 Å and 4.0 Å resolution, respectively. The structures reveal a compact dimer made up of trimodular subunits containing an N-terminal kinase domain (KD), a ubiquitin-like domain (ULD), and an α-helical scaffold dimerization domain (SDD). Activation rearranges the KD into an active conformation while maintaining the overall dimer conformation. Low-resolution SAXS studies reveal that the missing C-terminal domain (CTD) extends away from the main body of the kinase dimer. Mutants that interfere with TBK1 dimerization show significantly reduced trans-autophosphorylation but retain the ability to bind adaptor proteins through the CTD. Our results provide detailed insights into the architecture of TBK1 and the molecular mechanism of activation.

Catalytic heme enzymes carry out a wide range of oxidations in biology. They have in common a mechanism that requires formation of highly oxidized ferryl intermediates. It is these ferryl intermediates that provide the catalytic engine to drive the biological activity. Unravelling the nature of the ferryl species is of fundamental and widespread importance. The essential question is whether the ferryl is best described as a Fe(IV)=O or a Fe(IV)–OH species, but previous spectroscopic and X-ray crystallographic studies have not been able to unambiguously differentiate between the two species. Here we use a different approach. We report a neutron crystal structure of the ferryl intermediate in Compound II of a heme peroxidase; the structure allows the protonation states of the ferryl heme to be directly observed. This, together with pre-steady state kinetic analyses, electron paramagnetic resonance spectroscopy and single crystal X-ray fluorescence, identifies a Fe(IV)–OH species as the reactive intermediate. The structure establishes a precedent for the formation of Fe(IV)–OH in a peroxidase.

Bacteriophage T4 RNase H, a flap endonuclease-1 family nuclease, removes RNA primers from lagging strand fragments. It has both 5 nuclease and flap endonuclease activities. Our previous structure of native T4 RNase H (PDB code 1TFR) revealed an active site composed of highly conserved Asp residues and two bound hydrated magnesium ions. Here, we report the crystal structure of T4 RNase H in complex with a fork DNA substrate bound in its active site. This is the first structure of a flap endonuclease-1 family protein with its complete branched substrate. The fork duplex interacts with an extended loop of the helix-hairpin-helix motif class 2. The 5 arm crosses over the active site, extending below the bridge (helical arch) region. Cleavage assays of this DNA substrate identify a primary cut site 7-bases in from the 5 arm. The scissile phosphate, the first bond in the duplex DNA adjacent to the 5 arm, lies above a magnesium binding site. The less ordered 3 arm reaches toward the C and N termini of the enzyme, which are binding sites for T4 32 protein and T4 45 clamp, respectively. In the crystal structure, the scissile bond is located within the double-stranded DNA, between the first two duplex nucleotides next to the 5 arm, and lies above a magnesium binding site. This complex provides important insight into substrate recognition and specificity of the flap endonuclease-1 enzymes.The flap endonuclease-1 (FEN-1) 3 nuclease family is conserved in sequence and structure from bacteriophage to humans. These nucleases play essential roles in DNA replication by removing the RNA primers from lagging strand fragments. In addition, FEN-1-related nucleases are important in long-patch base excision repair and in maintenance of genomic stability. Homozygous knockouts of FEN-1 in mice are lethal to embryos (for review, see Refs. 1-3).Like other family members, bacteriophage T4-encoded RNase H shows 5Ј to 3Ј exonuclease activity on either RNA/ DNA or DNA/DNA duplexes and endonuclease activity on either flap or fork DNA structures (4, 5). T4 rnh deletion mutants give no phage production and accumulate unligated, lagging strand fragments in Escherichia coli hosts with defective polymerase I 5Ј nuclease (6). In addition, the mutants are hypersensitive to UV irradiation and anti-tumor agents (7).FEN-1 family nuclease activities are modulated by interactions with DNA replication clamps (e.g. eukaryotic proliferating cell nuclear antigen and T4 gene 45 clamp) and single-stranded DNA-binding proteins (e.g. eukaryotic replication protein A and T4 gene 32 protein) (5, 8 -12). Human FEN-1 nuclease is also stimulated by interactions with the Werner (13, 14) and Bloom (15) syndrome helicases as well as the Rad9-Rad1-Hus1 (9-1-1) checkpoint clamp (16).T4 RNase H 5Ј nuclease removes a short oligonucleotide (1-4 bases) each time it binds its substrate. T4 32 protein, binding on single-stranded DNA behind the nuclease, increases its processivity so that about 10 -50 short oligonucleotides are removed in a single binding event (5). On nicked sub...

No abstract

The interior of living cells is a dense and polydisperse suspension of macromolecules. Such a complex system challenges an understanding in terms of colloidal suspensions. As a fundamental test we employ neutron spectroscopy to measure the diffusion of tracer proteins (immunoglobulins) in a cell-like environment (cell lysate) with explicit control over crowding conditions. In combination with Stokesian dynamics simulation, we address protein diffusion on nanosecond time scales where hydrodynamic interactions dominate over negligible protein collisions. We successfully link the experimental results on these complex, flexible molecules with coarse-grained simulations providing a consistent understanding by colloid theories. Both experiments and simulations show that tracers in polydisperse solutions close to the effective particle radius R eff = ⟨R i 3⟩1/3 diffuse approximately as if the suspension was monodisperse. The simulations further show that macromolecules of sizes R > R eff (R < R eff) are slowed more (less) effectively even at nanosecond time scales, which is highly relevant for a quantitative understanding of cellular processes.

Cytokine signaling through the JAK/STAT pathway controls multiple cellular responses including growth, survival, differentiation, and pathogen resistance. An expansion in the gene regulatory repertoire controlled by JAK/STAT signaling occurs through the interaction of STATs with IRF transcription factors to form ISGF3, a complex that contains STAT1, STAT2, and IRF9 and regulates expression of IFN-stimulated genes. ISGF3 function depends on selective interaction between IRF9, through its IRF-association domain (IAD), with the coiled-coil domain (CCD) of STAT2. Here, we report the crystal structures of the IRF9-IAD alone and in a complex with STAT2-CCD. Despite similarity in the overall structure among respective paralogs, the surface features of the IRF9-IAD and STAT2-CCD have diverged to enable specific interaction between these family members. We derive a model for the ISGF3 complex bound to an ISRE DNA element and demonstrate that the observed interface between STAT2 and IRF9 is required for ISGF3 function in cells.JAK/STAT signaling | IRF transcription factor | STAT2 | innate immunity | crystal structure C ytokine signaling via the JAK-STAT pathway controls the development, differentiation, and regulation of cells in the immune system and is frequently dysregulated in disease (1). JAK-STAT signaling is mediated by four structurally related JAK kinases (JAK1, JAK2, JAK3, TYK2) and seven STAT (1-4, 5a, 5b, 6) proteins (2). A hallmark of cytokine signaling is functional redundancy and extensive pleiotropy, the ability of multiple cytokines to exert overlapping biological activities (3, 4). A critical question is how a limited number of JAK and STAT molecules enable such extensive redundancy and pleiotropy and how gene duplication and divergence among STATs contributes to specificity in cytokine signaling.JAK-mediated tyrosine phosphorylation of STATs induces dimerization and translocation to the nucleus, where STATs bind the gamma-activated sequence (GAS), a palindromic 9-11 base pair (bp) DNA element, 5′-TTCN 2-4 GAA-3′ in the promoter of target genes (2). An exception occurs in the response to type I and type III IFNs: These cytokines are rapidly induced during viral infection and stimulate activation of a complex termed ISGF3 (IFN-stimulated gene factor 3). ISGF3 contains a STAT1/ STAT2 heterodimer that interacts with IRF9, a member of the IRF family of transcription factors (5-8). Mammals contain 10 IRF paralogs that typically bind to the consensus DNA sequence 5′-AANNGAAA-3′ (9-13). As a result of STAT and IRF complex formation, ISGF3 binds to a ∼12-15-bp composite IFN-stimulated response DNA element (ISRE) 5′-G/ANGAAAN 2 GAAACT-3′. Thus, the physical association of STATs with IRFs contributes to functional specificity in cytokine signaling and enables expression of ISGs (6,14).IRFs contain a conserved N-terminal DNA-binding domain (DBD) and a C-terminal IRF-association domain (IAD; Fig. 1A). The IAD belongs to the SMAD/FHA domain superfamily (15)(16)(17). IRF3 is the best understood IRF family member. Sign...

The flagellated Gram-negative bacteriumEscherichia coliis one of the most studied microorganisms. Despite extensive studies as a model prokaryotic cell, the ultrastructure of the cell envelope at the nanometre scale has not been fully elucidated. Here, a detailed structural analysis of the bacterium using a combination of small-angle X-ray and neutron scattering (SAXS and SANS, respectively) and ultra-SAXS (USAXS) methods is presented. A multiscale structural model has been derived by incorporating well established concepts in soft-matter science such as a core-shell colloid for the cell body, a multilayer membrane for the cell wall and self-avoiding polymer chains for the flagella. The structure of the cell envelope was resolved by constraining the model by five different contrasts from SAXS, and SANS at three contrast match points and full contrast. This allowed the determination of the membrane electron-density profile and the inter-membrane distances on a quantitative scale. The combination of USAXS and SAXS covers size scales from micrometres down to nanometres, enabling the structural elucidation of cells from the overall geometry down to organelles, thereby providing a powerful method for a non-invasive investigation of the ultrastructure. This approach may be applied for probingin vivothe effect of detergents, antibiotics and antimicrobial peptides on the bacterial cell wall.

The first implementation and use of anin situsize exclusion chromatography (SEC) system on a small-angle neutron scattering instrument (SANS) is described. The possibility of deploying such a system for biological solution scattering at the Institut Laue–Langevin (ILL) has arisen from the fact that current day SANS instruments at ILL now allow datasets to be acquired using small sample volumes with exposure times that are often shorter than a minute. This capability is of particular importance for the study of unstable biological macromolecules where aggregation or denaturation issues are a major problem. The first use of SEC-SANS on ILL's instrument D22 is described for a variety of proteins including one particularly aggregation-prone system.