Structural dynamics of a spinlabeled ribosome elongation factor P (EF-P) from Staphylococcus aureus by EPR spectroscopy
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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.
KeywordsElongation factor P Staphylococcus aureus Ribosome EPR NMR Chemical exchange
In general, the translation process is produced by ribosomes—complex molecular machines that provide peptide bond synthesis in an accordance with the mRNA nucleotide sequence. The mechanisms of translation in eukaryotes and prokaryotes may be different in many aspects, but fundamentally basic processes are uniform. To ensure an accuracy and an efficiency of the synthesis, in addition to a ribosome, special protein factors are required that are involved at various stages of translation. In prokaryotes one of such factors is three domain protein elongation factor P (EF-P) , eukaryotes have a two domain analogue of this factor—eIF5A . There is still no integral comprehensive theory that includes the full mechanism of all the functions of EF-P and its regulation.
There are four known types of possible EF-P PTM of highly conservative loop I of domain I: b-lysinilation , rhamnosilation , 5-aminopentilation  and hypusine in case of eIF5A . Approximately one-third of all bacteria possess modifying enzymes corresponding to the known modification . Modification status of other bacteria is unknown. Highly possible modification of S.aureus EF-P could be 5-aminopentanolitation of 34 K, the same type modification as for B.subtillus EF-P .
Bacteria with mutant forms of the EF-P loop, with the absence of EF-P or the absence of EF-P modifying enzymes have phenotypes that can be correlated with specific cell processes such as stress resistance, cell motility and virulence—that could be due to involvement in this processes proteins containing stalling motifs, regulated by EF-P . Proteins with such sites are often involved in secretion processes, including secretion pathogenicity factors of microorganisms. Last study of EF-P complex with stalled ribosome made by CryoEM  proposed some structural mechanism of B-lysinilation modification action for polyproline antistalling, but there are still few details how this modification could work. Also there is no understanding how another types of modification could function and what is the reason for their structural difference.
2 Materials and methods
2.1 Synthesis and purification of EF-P‑SL
Elongation Factor P from S. aureus (SaEF-P) protein with mutation K32C and histidine tag at its C-terminus was expressed in Escherichia coli BL21star(DE3) transformed with a pGS21A plasmid. The cells were dissolved in the lysis buffer (20 mM Tris–HCl, 200 mM NH4Cl, pH 7.6) with addition of PIC and PMSF. Lisate was clarified by centrifugation at 13.000 rpm, 4 °C for 30 min (Beckman 25.50 rotor). SaEF-P-HIS was purified by gravity flow MAC chromatography (Ni–NTA). After a final step of a gel-filtration chromatography on Superdex 75 10/300, the purified protein was dissolved in phosphate buffer 50 mM phosphate buffer pH 7.4, 250 mM NH4Cl).
2.2 MTSL labeling of EF-P
MTSL spin label, 2,2,5,5-tetramethyl-1-oxyl-3-methyl methanethiosulfonate (O875000, from Toronto Research Chemicals) was dissolved at 10 mg/mL concentration in DMSO. For spin labeling, the SaEF-P(K32C) solution was mixed with ×10 ammount of MTSL and incubated 18 h at 4 °C. The excess spin label was removed by gel exclusion chromatography (gel filtration). Final sample of SaEF-P labeled protein was concentrated to 1 mM with Amicon Ultra Centrifugal Filter Device (10,000 Da cut off) to final volume equal to 500 μl.
2.3 Electron paramagnetic resonance spectroscopy
EPR measurements were done in continuous wave mode by using the abilities of Bruker X-band Elexsys 580 spectrometer at room temperature (Centre of the shared facilities at Kazan Federal University). The solutions were placed into the 0.8 mm inner diameter quartz tubes. Registration parameters were chosen to be 100 µW for the microwave power and 0.2 G at 100 kHz modulation to avoid saturation and overmodulation effects. EPR parameters (isotropic g-factor and hyperfine constant A) and rotational correlation times (by using the values of g- and A- components for MTSL in water listed in  were extracted from the EPR spectra fitting in Easyspin module for Matlab .
3 Results and discussion
Addition of MTSL label covalently bound to 32Cys in the highly conservative part of EF-P loop in the domain I of the EF-P located near the CCA-end of P-tRNA and the peptidyl transferase center (PTC) 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 resonances. We believe that our result could be extended in future for the analysis of EF-P—ribosome complex formation process by EPR spectroscopy.
This work was supported by the Russian Science Foundation (Grant 17-74-20009).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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