The Toll/interleukin-1 receptor (TIR) domain is a highly conserved signaling domain found in the intracellular regions of Toll-like receptors (TLRs), in interleukin-1 receptors, and in several cytoplasmic adaptor proteins. TIR domains mediate receptor signal transduction through recruitment of adaptor proteins and play critical roles in the innate immune response and inflammation. This work presents the 2.2 Å crystal structure of the TIR domain of human TLR10, revealing a symmetric dimer in the asymmetric unit. The dimer interaction surface contains residues from the BB-loop, DD-loop, and ␣C-helix, which have previously been identified as important structural motifs for signaling in homologous TLR receptors. The interaction surface is extensive, containing a central hydrophobic patch surrounded by polar residues. The BB-loop forms a tight interaction, where a range of consecutive residues binds in a pocket formed by the reciprocal BB-loop and ␣C-helix. This pocket appears to be well suited for binding peptide substrates, which is consistent with the notion that peptides and peptide mimetics of the BB-loop are inhibitors for TLR signaling. The TLR10 structure is in good agreement with available biochemical data on TLR receptors and is likely to provide a good model for the physiological dimer.The Toll-like receptors (TLRs) 3 are type I transmembrane receptors (1) that play an important role in innate immunity as they recognize conserved pathogen signatures and induce early gene expression responses to infections (2, 3). TLR signaling is mediated through the cytoplasmic Toll/interleukin-1 receptor (TIR) domain, the oligomerization of which initiates the recruitment of TIR domain-containing adaptor proteins. The cytoplasmic adaptor subgroup comprises five members: MyD88, MyD88-adaptor-like (MAL), TIR-domain-containing adaptor protein inducing IFNbeta (TRIF), TRIF-related adaptor molecule (TRAM), and sterile ␣-and armadillo-motif containing protein (SARM) (4), which mediate or modulate the downstream signaling. Subsequent kinase activation results in the induction of transcription factors such as interferon regulatory factor and NF-B followed by the production of numerous regulators and effectors of the innate immune response. In addition to the TLRs and the cytoplasmic TIR adaptor subgroup, the family of TIR domain-containing proteins encompasses the interleukin-1 (IL-1) receptor family (5).The 10 members of the human TLR family recognize a wide variety of pathogen-associated molecular patterns. As no ligand has yet been reported for TLR10, it remains the only orphan family member. Activation of TLRs is believed to involve receptor oligomerization, and the current view is that the receptors are active as either homodimers or heterodimers, depending on the receptor type. The cytoplasmic TIR domains dimerize to form a platform for the recruitment of adaptor proteins and additional signaling molecules. TLR10 has been shown to form homodimers and to interact with TLRs 1 and 2 (6). All of the TLRs, except TLR3, have b...
DEXD/H-box RNA helicases couple ATP hydrolysis to RNA remodeling by an unknown mechanism. We used x-ray crystallography and biochemical analysis of the human DEXD/H-box protein DDX19 to investigate its regulatory mechanism. The crystal structures of DDX19, in its RNA-bound prehydrolysis and free posthydrolysis state, reveal an ␣-helix that inserts between the conserved domains of the free protein to negatively regulate ATPase activity. This finding was corroborated by biochemical data that confirm an autoregulatory function of the N-terminal region of the protein. This is the first study describing crystal structures of a DEXD/H-box protein in its open and closed cleft conformations.RNA helicase activity is involved in all aspects of RNA metabolism, including transcription, pre-mRNA splicing, ribosome biogenesis, nuclear export, translation initiation and termination, RNA degradation, viral replication, and viral RNA detection. The DEXD/H-box RNA helicases couple hydrolysis of ATP to cycles of RNA binding and release that typically result in non-processive RNA duplex unwinding (1) or disruption of RNP 3 complexes (2, 3). These proteins interact in a non-sequence-specific manner with the phosphoribose backbone of single-stranded RNA. DEXD/H-box RNA helicases contain two ␣/-RecA-like domains that both feature conserved sequence motifs involved in RNA binding and ATP hydrolysis (4, 5). Accessory proteins are involved in the regulation of RNA binding and ATPase activities, although no general mechanism has been demonstrated.The DDX19 member of the DEXD/H-box RNA helicase family performs an essential function in mRNA nuclear export by remodeling RNP particles during passage of mRNA through the nuclear pore complex (3, 6). Dbp5, the yeast orthologue of DDX19 (7,8), causes displacement of the RNP constituent, Mex67, thereby preventing re-entry of mRNA into the nucleus (9). Dbp5 is also involved in translation termination (10). A specific function has been assigned to the ADP-bound form of Dbp5, which displaces the RNA-binding protein Nab2, an event that is required for mRNA export (3). In vivo, Dbp5 is activated by the nuclear pore complex-associated protein, Gle1 (11,12). Crystal structures of DEXD/H-box proteins show two-lobed proteins with the nucleotide binding site located in the lower part of the cleft separating the conserved domains and the RNA binding site across the upper cleft opening (13-17). DEXD/Hbox helicases in general share little homology in their coding sequences upstream of the conserved domain-1. The N-terminal extension of DDX19, however, shares significant homology with that of DDX25/GRTH, a testis-specific protein that is essential for spermatogenesis (18), supporting a functional significance for this sequence. Herein, we present a crystal structure of human DDX19 that shows the ADP-bound protein with an ␣-helical segment of the N-terminal extension wedged between the core domains, preventing cleft closure. In the structure of the ADPNP-bound protein in complex with RNA, this ␣-helix has moved...
We report two crystal structures of the PARP domain of human tankyrase-2 (TNKS2). Tankyrases are involved in fundamental cellular processes such as telomere homeostasis and Wnt signaling. The complex of TNKS2 with the potent inhibitor XAV939 provides insights into the molecular basis of the strong interaction and suggests routes for further development of tankyrase inhibitors.
A prerequisite for structural genomics and related projects is to standardize the process of gene overexpression and protein solubility screening to enable automation for higher throughput. We have tested a methodology to rapidly subclone a large number of human genes and screen these for expression and protein solubility in Escherichia coli. The methodology, which can be partly automated, was used to compare the effect of six different N-terminal fusion proteins and an N-terminal 6*His tag. As a realistic test set we selected 32 potentially interesting human proteins with unknown structures and sizes suitable for NMR studies. The genes were transferred from cDNA to expression vectors using subcloning by recombination. The subcloning yield was 100% for 27 (of 32) genes for which a PCR fragment of correct size could be obtained. Of these, 26 genes (96%) could be overexpressed at detectable levels and 23 (85%) are detected in the soluble fraction with at least one fusion tag. We find large differences in the effects of fusion protein or tag on expression and solubility. In short, four of seven fusions perform very well, and much better than the 6*His tag, but individual differences motivate the inclusion of several fusions in expression and solubility screening. We also conclude that our methodology and expression vectors can be used for screening of genes for structural studies, and that it should be possible to obtain a large fraction of all NMR-sized and nonmembrane human proteins as soluble fusion proteins in E. coli.
DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections. DEAD-box proteins contain two conserved domains that both contribute to RNA and ATP binding. Despite recent advances the molecular details of how these enzymes convert chemical energy into RNA remodeling is unknown. We present crystal structures of the isolated DEAD-domains of human DDX2A/eIF4A1, DDX2B/eIF4A2, DDX5, DDX10/DBP4, DDX18/myc-regulated DEAD-box protein, DDX20, DDX47, DDX52/ROK1, and DDX53/CAGE, and of the helicase domains of DDX25 and DDX41. Together with prior knowledge this enables a family-wide comparative structural analysis. We propose a general mechanism for opening of the RNA binding site. This analysis also provides insights into the diversity of DExD/H- proteins, with implications for understanding the functions of individual family members.
We have compared four different vectors for expression of proteins with N- or C-terminal hexahistidine (His6) tags in Escherichia coli by testing these on 20 human proteins. We looked at a total recombinant protein production levels per gram dry cell weight, solubility of the target proteins, and yield of soluble and total protein when purified by immobilized metal ion affinity purification. It was found that, in general, both N- and C-terminal His6 tags have a noticeable negative affect on protein solubility, but the effect is target protein specific. A solubilizing fusion tag was able to partly counteract this negative effect. Most target proteins could be purified under denaturing conditions and about half of the proteins could be purified under physiological conditions. The highest protein production levels and yield of purified protein were obtained from a construct with C-terminal His tag. We also observe a large variation in cell growth rate, which we determined to be partly caused by the expression vectors and partly by the targets. This variation was found to be independent of the production level, solubility and tertiary structure content of the target proteins.
Poly(ADP-ribose) polymerases (PARPs) activate DNA repair mechanisms upon stress- and cytotoxin-induced DNA damage, and inhibition of PARP activity is a lead in cancer drug therapy. We present a structural and functional analysis of the PARP domain of human PARP-3 in complex with several inhibitors. Of these, KU0058948 is the strongest inhibitor of PARP-3 activity. The presented crystal structures highlight key features for potent inhibitor binding and suggest routes for creating isoenzyme-specific PARP inhibitors.
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