Abstract
Activating signal cointegrator-1 homology (ASCH) domains were initially reported in human as a part of the ASC-1 transcriptional regulator, a component of a putative RNA-interacting protein complex; their presence has now been confirmed in a wide range of organisms. Here, we have determined the trigonal and monoclinic crystal structures of an ASCH domain-containing protein from Zymomonas mobilis (ZmASCH), and analyzed the structural determinants of its nucleic acid processing activity. The protein has a central β-barrel structure with several nearby α-helices. Positively charged surface patches form a cleft that runs through the pocket formed between the β-barrel and the surrounding α-helices. We further demonstrate by means of in vitro assays that ZmASCH binds nucleic acids, and degrades single-stranded RNAs in a magnesium ion-dependent manner with a cleavage preference for the phosphodiester bond between the pyrimidine and adenine nucleotides. ZmASCH also removes a nucleotide at the 5′-end. Mutagenesis studies, guided by molecular dynamics simulations, confirmed that three residues (Tyr47, Lys53, and Ser128) situated in the cleft contribute to nucleic acid-binding and RNA cleavage activities. These structural and biochemical studies imply that prokaryotic ASCH may function to control the cellular RNA amount.
Highlights
Human activating signal cointegrator-1 (ASC-1) is a transcriptional regulator and a component of a putative RNA-interacting protein complex[1,2]
Based on minute structural differences, and on sequence variation compared to pseudouridine synthase and archaeosine transglycosylase’ (PUA) and Activating signal cointegrator-1 homology (ASCH) domains, the authors of that study concluded that EVE proteins might be functionally closer to eukaryotic YT521-B homology (YTH) domains rather than PUA or ASCH domains
In vitro assays showed that ZmASCH bound nucleic acids and possessed cleavage activity towards single-stranded RNAs
Summary
The crystal structures of the wild-type protein in the monoclinic system was solved by molecular replacement using the trigonal structure as a search model. (d) 32P-labelled RNA or DNA probes (0.3 μM) were incubated with wild-type and mutant proteins of ZmASCH (3 μM) for 30 min at 310 K in the presence of 10 mM MgCl2. Labeled DNA or RNA probes were mixed with ZmASCH for 30 min at 310 K, in either the presence or absence of divalent metal ions, in a buffer solution consisting of 20 mM Tris-HCl (pH 8.0), 100 mM potassium chloride, and 100 ng/μL bovine serum albumin. All molecular dynamics simulations (ZmASCH in water, ZmASCH:RNA in water) were carried out with Gromacs 4.6.5 using the AMBER03 protein, AMBER94 nucleic acid, and the TIP3P water model[20]. Analyses were performed with Gromacs tools and automated plots generated with Grace (http://plasma-gate.weizmann.ac.il/Grace/)
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