Translation initiation factors eIF4A and eIF4G form, together with the cap-binding factor eIF4E, the eIF4F complex, which is crucial for recruiting the small ribosomal subunit to the mRNA 5 end and for subsequent scanning and searching for the start codon. eIF4A is an ATP-dependent RNA helicase whose activity is stimulated by binding to eIF4G. We report here the structure of the complex formed by yeast eIF4G's middle domain and full-length eIF4A at 2.6-Å resolution. eIF4A shows an extended conformation where eIF4G holds its crucial DEAD-box sequence motifs in a productive conformation, thus explaining the stimulation of eIF4A's activity. A hitherto undescribed interaction involves the amino acid Trp-579 of eIF4G. Mutation to alanine results in decreased binding to eIF4A and a temperature-sensitive phenotype of yeast cells that carry a Trp579Ala mutation as its sole source for eIF4G. Conformational changes between eIF4A's closed and open state provide a model for its RNA-helicase activity.translation initiation ͉ DEAD-box protein ͉ X-ray structure ͉ eIF4F T ranslation initiation in eukarya is usually the rate-limiting and most tightly controlled stage of polypeptide synthesis (reviewed in refs. 1-3). For the majority of eukaryotic mRNAs, the cap-dependent pathway is used for translation initiation (3). It comprises four consecutive steps: (i) formation of the 43S preinitiation complex consisting of the 40S ribosomal subunit, initiation factors (eIF2, eIF3), and Met-tRNA i ; (ii) recruitment of the 43S preinitiation complex to the capped 5Ј end of the mRNA; (iii) scanning of the 5Ј untranslated region of the mRNA and start codon recognition; and (iv) joining of the large 60S ribosomal subunit and assembly of the 80S ribosome.Approximately a dozen eukaryotic translation initiation factors (eIFs) are needed for this process. A central component of the second and third step is eIF4F, a heterotrimeric stable complex consisting of the cap-binding protein eIF4E, the DEAD-box helicase eIF4A, and the central multiscaffold protein eIF4G, which possesses additional binding sites for the poly(A)-binding protein PABP and, in mammalia, for eIF3 (Fig. 1A). Mammalian eIF4G possesses a second eIF4A binding site in its C-terminal region in proximity to a binding site for protein kinase Mnk1 (mitogen-activated protein kinase-interacting kinase), which phosphorylates eIF4E. Crystal structures of the central and the C-terminal region of human eIF4GII reveal the formation of one or two HEAT domains, respectively (4, 5)Saccharomyces cerevisiae possesses two genes encoding for eIF4G, TIF4631 and TIF4632. The gene products, eIF4GI and eIF4GII, are 952 and 914 aa long and share Ϸ50% sequence identity. Deletion of one of these genes is tolerated by yeast cells, but double deletion of both genes causes lethality. Interaction of eIF4G with eIF4A is essential for the cell (6, 7). The 45-kDa initiation factor 4A (eIF4A) is a prototypical DEAD-box helicase (8). Its ATPase activity is RNA-dependent and its activity is substantially enhanced in ...

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.

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.

Poly-ADP-ribose polymerases (PARPs) catalyze transfer of ADP-ribose from NAD(+) to specific residues in their substrate proteins or to growing ADP-ribose chains. PARP activity is involved in processes such as chromatin remodeling, transcription control, and DNA repair. Inhibitors of PARP activity may be useful in cancer therapy. PARP2 is the family member that is most similar to PARP1, and the two can act together as heterodimers. We used X-ray crystallography to determine two structures of the catalytic domain of human PARP2: the complexes with PARP inhibitors 3-aminobenzamide and ABT-888. These results contribute to our understanding of structural features and compound properties that can be employed to develop selective inhibitors of human ADP-ribosyltransferases.

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RNA helicases of the DExD/H-box superfamily are critically involved in all RNA-related processes. No crystal structures of human DExH-box domains had been determined previously, and their structures were difficult to predict owing to the low level of homology among DExH-motif-containing proteins from diverse species. Here we present the crystal structures of the conserved domain 1 of the DEIH-motif-containing helicase DHX9 and of the DEAD-box helicase DDX20. Both contain a RecA-like core, but DHX9 differs from DEAD-box proteins in the arrangement of secondary structural elements and is more similar to viral helicases such as NS3. The N-terminus of the DHX9 core contains two long alpha-helices that reside on the surface of the core without contributing to nucleotide binding. The RNA-polymerase-II-interacting minimal transactivation domain sequence forms an extended loop structure that resides in a hydrophobic groove on the surface of the DEIH domain. DHX9 lacks base-selective contacts and forms an unspecific but important stacking interaction with the base of the bound nucleotide, and our biochemical analysis confirms that the protein can hydrolyze ATP, guanosine 5'-triphosphate, cytidine 5'-triphosphate, and uridine 5'-triphosphate. Together, these findings allow the localization of functional motifs within the three-dimensional structure of a human DEIH helicase and show how these enzymes can bind nucleotide with high affinity in the absence of a Q-motif.

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