Abstract
The bacterial translational GTPase EF4/LepA is structurally similar to the canonical elongation factor EF-G. While sharing core structural features with other translational GTPases, the function of EF4 remains unknown. Recent structural data locates the unique C-terminal domain (CTD) of EF4 in proximity to the ribosomal peptidyl transferase center (PTC). To investigate the functional role of EF4's CTD we have constructed three C-terminal truncation variants. These variants are fully functional with respect to binding mant-GTP and mant-GDP as determined by rapid kinetics, as well as their intrinsic multiple turnover GTPase activity. Furthermore, they are able to form stable complexes with the 70S ribosome and 50S/30S ribosomal subunits. However, successive removal of the C-terminus impairs ribosome-dependent multiple turnover GTPase activity of EF4, which for the full-length protein is very similar to EF-G. Our findings suggest that the last 44 C-terminal amino acids of EF4 form a sub-domain within the C-terminal domain that is important for GTP-dependent function on the ribosome. Additionally, we show that efficient nucleotide hydrolysis by EF4 on the ribosome depends on a conserved histidine (His 81), similar to EF-G and EF-Tu.
Highlights
Alberta RNA Research and Training Institute, Department of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, AB, T1K 3M4, Canada
The obtained association and dissociation rate constants governing the interaction between EF4 and mant-GDP/mant-GTP, together with the derived micromolar binding constants (KD), reveal that EF4 has a slightly higher affinity for GDP than for GTP (5 mM and 23 mM, respectively). This is different from the translational GTPase EF-G (KD, GDP 17 mM and KD, GTP 7 mM), but is similar to EF-Tu21 and RF314,15
Given the 10-fold higher concentration of GTP in the cell over GDP22, we estimate that during mid-log phase in vivo, 66% of free EF4 will be bound to GTP and 33% bound to GDP. This is again different than what is predicted for other translational GTPases, such as EF-G13 and RF314,15, and suggests that EF4 is likely sensitive to the guanine nucleotide triphosphate-diphosphate ratio and to the overall energy state of the cell, similar to what has been reported for EttA
Summary
EF4 possesses a unique C-terminal domain (CTD) that spatially occupies a position between domains III and V (Supplemental Figure S1) Both EF4 and EF-G bind to the A site of the ribosome and the available structures of the respective ribosomal complexes reveal that domains I, II, III and V of EF-G and EF4 contact the ribosome in a similar fashion[9,10,11]. In order to investigate the catalytic mechanism of GTP hydrolysis and the role of EF4’s CTD, we have constructed a GTPase inactive, as well as three sequential C-terminal truncation variants These variants are compared to the full-length EF4 with respect to their ability to bind guanine nucleotides, associate with the ribosome and to hydrolyze GTP. Our www.nature.com/scientificreports findings suggest that the last 44 C-terminal residues constitute a C-terminal sub-domain that likely has important regulatory roles during EF4’s functional cycle
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