Biophysical analysis of SECIS binding protein 2 (SBP2) from Naegleria gruberi

Abstract

The Selenocysteine (Sec - U) biosynthesis pathway is present in eukaryotes and prokaryotes, in which its incorporation is directed by the stop codon UGA and the structural mRNA element named Sec Insertion Sequence (SECIS) that contain an essential kink-turn motif recognized by specific RNA-binding proteins. SBP2 is the key player in the interaction with the SECIS element in eukaryotes, and it is essential for the biosynthesis pathway. Free-living amoebas are part of the Heterolobosea phylum, and several species, including Naegleria fowleri, are known human pathogens. In 2013 it was reported that Naegleria gruberi (NgSBP2), which is non-pathogenic, had a divergent SBP2 sequence and all the genes essential for Sec synthesis. The identity of NgSBP2 is confirmed experimentally and its binding affinity to the SECIS element is demonstrated. The N-terminal and the C-terminal domains (NgSBP2-NT and NgSBP2-CT, respectively) of NgSBP2 contain disordered regions, particularly in the N-terminal domain. The SECIS element is bonded to NgSBP2-CT, which results in a decrease in the disordered sequence of the domain, and the NgSBP2-NT domain interacts with NgSBP2-CT.SECIS complex, as we present here. The findings reveal the molecular interaction patterns underlying the selenocysteine incorporation pathway in an early-branching eukaryote, which is influenced by multiple protein-RNA interactions.