Structural dynamics of a spinlabeled ribosome elongation factor P (EF-P) from Staphylococcus aureus by EPR spectroscopy

Abstract

Elongation factor P (EF-P) is a three domain protein that binds to the ribosome between P and E sites.The EF-P involved in a specialized translation of stalling amino acid motifs such as (PPP or APP). Proteins with stalling motifs are involved in various cell processes, including stress resistance and virulence of bacteria. EF-P stabilizes the P-tRNA and increases the entropy of stalled ribosome complex to compensate for rigid nature of proline residue, thus providing adequate protein synthesis rate. Detailed structural mechanisms of this effect are still poorly understood. It was shown that most of bacteria needs a special post-translational modification in a conservative region of the loop in the domain I of the EF-P located near the CCA-end of P-tRNA and the peptidyl transferase center. In present paper by EPR spectroscopy we investigated a spinlabeled EF-P from Staphylococcus aureus-a pathogenic bacteria which causes various human diseases. Addition of MTSL ((S-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)methyl methanesulfonothioate)) label covalently bound to 32Cys in the highly conservative part of EF-P loop in the domain I of the EF-P allows us to analyze protein dynamic by EPR spectroscopy of the high mobility region which was not observed in NMR spectra due to fast proton exchange leading to the absence of the corresponding amide NMR resonances. We suppose that our result could be extended in future for the analysis of EF-P-ribosome complex formation process by advanced EPR techniques.

Posttranslational modification of Elongation Factor P from Staphylococcus aureus

“…subtilis , and Bs EF‐P has a conservative KPG K G motif where the 32nd lysine residue carries the modification [11]. Sa EF‐P has a typical three‐domain structure with unstructured loop in domain I and KPG K G motif with lysine residue in the 32nd position [17,25,26]. To reveal modification in S .…”

“…The third modification, 5-aminopentanolation, was found in B. subtilis, and BsEF-P has a conservative KPGKG motif where the 32nd lysine residue carries the modification [11]. SaEF-P has a typical three-domain structure with unstructured loop in domain I and KPGKG motif with lysine residue in the 32nd position [17,25,26]. To reveal modification in S. aureus, we carried out homologous expression of SaEF-P tagged with six histidine residues.…”