Domain structure, GTP-hydrolyzing activity and 7S RNA binding of Acidianus ambivalens Ffh-homologous protein suggest an SRP-like complex in archaea

In this study we provide, for the first time, experimental evidence that a protein homologous to bacterial Ffh is part of an SRP-like ribonucleoprotein complex in hyperthermophilic archaea. The gene encoding the Ffh homologue in the hyperthermophilic archaeote Acidianus ambivalens has been cloned and sequenced. Recombinant Ffh protein was expressed in E. coli and subjected to biochemical and functional studies. A. ambivalens Ffh encodes a 50.4-kDa protein that is structured by three distinct regions: the N-terminal hydrophilic N-region (N), the GTP/GDP-binding domain (G) and a C-terminal located C-domain (C). The A. ambivalens Ffh sequence shares 44-46% sequence similarity with Ffh of methanogenic archaea, 34-36% similarity with eukaryal SRP54 and 30-34% similarity with bacterial Ffh. A polyclonal antiserum raised against the first two domains of A. ambivalens Ffh reacts specifically with a single protein (apparent molecular mass: 46 kDa, termed p46) present in cytosolic and in plasmamembrane cell fractions of A. ambivalens. Recombinant Ffh has a melting point of t(m) = 89 degrees C. Its intrinsic GTPase activity obviously depends on neutral pH and low ionic strength with a preference for chloride and acetate salts. Highest rates of GTP hydrolysis have been achieved at 81 degrees C in presence of 0.1-1 mM Mg2+. GTP hydrolysis is significantly inhibited by high glycerol concentrations, and the GTP hydrolysis rate also markedly decreases by addition of detergents. The K-m for GTP is 13.7 mu M at 70 degrees C and GTP hydrolysis is strongly inhibited by GDP (K-i = 8 mu M). A. ambivalens Ffh, which includes an RNA-binding motif in the C-terminal domain, is shown to bind specifically to 7S RNA of the related crenarchaeote Sulfolobus solfataricus. Comparative sequence analysis reveals the presence of typical signal sequences in plasma membrane as well as extracellular proteins of hyperthermophilic crenarchaea which strongly supposes recognition events by an Ffh containing SRP-like particle in these organisms.