Characterization Of Aif5a Protein: A Multifunctional Translation Factor In The Hyperthermophilic Archaeon S. Solfataricus

Translation is a key step in gene expression, thus a complete characterization of the effectors of this process is fundamental. Many translation factors have been studied in eukaryotes and bacteria, whereas in the archaeal kingdom there are significant gaps in the knowledge of this process. In this context, this research deals with one of the universally conserved translation factors, called e/aIF5A in Eukarya and Archaea and EF-P in Bacteria. eIF5A and EF-P perform the essential task to rescue ribosome stalling at poly-proline stretches. Indispensable for this action is a unique post-translation modification: hypusination in eIF5A and β-lysinylation in EF-P. In Eukarya the modification involves two enzymes: deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Conversely, a complete knowledge of the function and the modification pathway of archaeal aIF5A is still lacking. For this reason, we have undertaken a structural and functional characterization of recombinant aIF5A and aDHS from the hyperthermophile Sulfolobus solfataricus. We found that Sso aIF5A is hypusinated, like its eukaryal counterpart. We performed SAXS analysis, showing that aIF5A is a very stable and compact protein. The protein is mainly under monomeric conformation in solution, although with a tendency to form dimers, explored also with Molecular Docking. The recombinant aDHS enzyme forms a tetramer and is able to modify its substrate in vitro resulting in deoxyhypusinated aIF5A, as in Eukarya. Functionally, our data confirm a role of aIF5A as a translation factor, but they also suggest the hypothesis of a multitasking protein involved in RNA metabolism. Hence, through a multidisciplinary approach, we have tried to shed light on an essential translation factor, in a field like Archaea, still little studied but very intriguing in evolutionary terms and for the thermostability of their proteins.