Structure of the N-terminal domain of the protein Expansion: an 'Expansion' to the Smad MH2 fold

Gene-expression changes observed in Drosophila embryos after inducing the transcription factor Tramtrack led to the identification of the protein Expansion. Expansion contains an N-terminal domain similar in sequence to the MH2 domain characteristic of Smad proteins, which are the central mediators of the effects of the TGF- signalling pathway. Apart from Smads and Expansion, no other type of protein belonging to the known kingdoms of life contains MH2 domains. To compare the Expansion and Smad MH2 domains, the crystal structure of the Expansion domain was determined at 1.6 A˚ resolution, the first structure of a non-Smad MH2 domain to be characterized to date. The structure displays the main features of the canonical MH2 fold with two main differences: the addition of an -helical region and the remodelling of a protein-interaction site that is conserved in the MH2 domain of Smads. Owing to these differences, to the new domain was referred to as N-MH2. Despite the presence of the N-MH2 domain, Expansion does not participate in TGF- signalling; instead, it is required for other activities specific to the protostome phyla. Based on the structural similarities to the MH2 fold, it is proposed that the N-MH2 domain should be classified as a new member of the Smad/FHA superfamily. 1

Structural and Biochemical Studies on ATP Binding and Hydrolysis by the Escherichia coli RNA Chaperone Hfq

In Escherichia coli the RNA chaperone Hfq is involved in riboregulation by assisting base-pairing between small regulatory RNAs (sRNAs) and mRNA targets. Several structural and biochemical studies revealed RNA binding sites on either surface of the donut shaped Hfq-hexamer. Whereas sRNAs are believed to contact preferentially the YKH motifs present on the proximal site, poly(A)15 and ADP were shown to bind to tripartite binding motifs (ARE) circularly positioned on the distal site. Hfq has been reported to bind and to hydrolyze ATP. Here, we present the crystal structure of a C-terminally truncated variant of E. coli Hfq (Hfq65) in complex with ATP, showing that it binds to the distal R-sites. In addition, we revisited the reported ATPase activity of full length Hfq purified to homogeneity. At variance with previous reports, no ATPase activity was observed for Hfq. In addition, FRET assays neither indicated an impact of ATP on annealing of two model oligoribonucleotides nor did the presence of ATP induce strand displacement. Moreover, ATP did not lead to destabilization of binary and ternary Hfq-RNA complexes, unless a vast stoichiometric excess of ATP was used. Taken together, these studies strongly suggest that ATP is dispensable for and does not interfere with Hfq-mediated RNA transactions