Expression and Purification of HPF Protein from Staphylococcus aureus and Analysis of Its Structure by the Method of NMR Spectroscopy

R.Kh. Ayupov^a*, K.S. Usachev^a**, I.Sh. Khusainov^a,b***, B. Kieffer^b****, M.M. Yusupov^a*****

^aKazan Federal University, Kazan, 420008 Russia

^bInstitute of Genetics and Molecular and Cellular Biology (IGBMC), Illkirch-Graffenstaden, 67400 France

E-mail: ^*aurusta@mail.ru, ^**Konstantin.Usachev@kpfu.ru, ^***khusaino@igbmc.fr, ^****bruno.kieffer@igbmc.fr, ^*****marat@igbmc.fr

Received April 7, 2017

Full text PDF

Abstract

Staphylococcus aureus hibernation-promoting factor (SaHPF) is a 22.2 kDa translation factor which under stress conditions (under amino acid starvation or antibiotic pressure) binds with the ribosome and inactivates it, thereby ensuring cell survival under stress. There are many problems with crystallization of this protein which still remain unsolved. Therefore, its analysis by NMR spectroscopy is of great interest. In this paper, we have described expression, purification, and NMR analysis of ¹³C/¹⁵N-labeled SaHPF protein and showed that it is present in a dimeric form in the solution. Notably, two types of signals in the NMR spectra have been observed: with weak intensity and high dispersion from N-terminal domain; with high intensity but low dispersion from a flexible loop between domains. No signals from C-terminal domain have been observed in the NMR spectra, which may indicate possible dimerization of this part of the protein. Protein dimerization has been also detected by the method of electrophoresis under native conditions.

Keywords: SaHPF, Staphylococcus aureus, NMR, ribosome, antibiotic, translation factor

Acknowledgments. This study was funded by the subsidy allocated to Kazan Federal University as part of the state program for increasing its competitiveness among the world's leading centers of science and education and by the Russian Foundation for Basic Research (project no. 16-34-60001_mol_a_dk).

Figure Captions

Fig. 1. ¹H–¹⁵N HSQC NMR spectrum (700 MHz) of SaHPF (0.1 mM) in PBS buffer + 250 mM NH₄Cl (¹³C, ¹⁵N), T = 298 K.

Fig. 2. ¹H–¹⁵N HSQC NMR spectra (700 MHz) of SaHPF at various NH₄Cl salt concentrations, T = 298 K.

Fig. 3. ¹H–¹⁵N HSQC NMR spectra (700 MHz) of SaHPF in the RE buffer with 500 mM NH₄Cl, T = 308 K. On the left – an increase in the intensity of weak signals from residues corresponding to N-terminal domain.

Fig. 4. The structure of SaHPF protein predicted using the ROBETTA program. Color changes designate amino-acid residue number growth (sequence beginning is shown with blue, sequence end is shown with red).

Fig. 5. Electrophoresis of SaHPF protein under native conditions: М – protein ladder, 1 – SaHPF.

References

1. Wada M., Yamazaki Y., Fujita N., Ishihama A. Structure and probable genetic location of a “ribosome modulation factor” associated with 100S ribosomes in stationary-phase Escherichia coli cells. Proc. Natl. Acad. Sci. U. S. A., 1990, vol. 87, no 7, pp. 2657–2661.
2. Ueta M., Yoshida H., Wada C., Baba T., Mori H., Wada A. Ribosome binding proteins YhbH and YfiA have opposite functions during 100S formation in the stationary phase of Escherichia coli. Genes Cells, 2005, vol. 10, no. 12, pp. 1103–1112. doi: 10.1111/j.1365-2443.2005.00903.x.
3. Polikanov Yu.S., Blaha G.M., Steitz T.A. How hibernation factors RMF, HPF, and YfiA turn off protein synthesis. Science, 2012, vol. 336, no. 6083, pp. 915–918. doi: 10.1126/science.1218538.
4. Ueta M., Wada Ch., Wada A. Formation of 100S ribosomes in Staphylococcus aureus by the hibernation promoting factor homolog SaHPF. Genes Cells, 2010, vol. 15, no. 1, pp. 43–58. doi: 10.1111/j.1365-2443.2009.01364.x.
5. Ayupov R.K., Akberova N.I. Prediction of the three-dimensional structure of the protein SaHPF and analysis of its molecular dynamics. Int. J. Pharm. Technol., 2016, vol. 8, no. 2, pp. 14548–14557.
6. Ayupov R.Kh., Akberova N.I. Analysis of the structure of SaHPF Staphylococcus aureus protein. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2016, vol. 158, no. 3, pp. 327–337. (In Russian)
7. Khusainov I., Vicens Q., Bochler A., Grosse F., Myasnikov A., Ménétret J.-F., Chicher J., Marzi S., Romby P., Yusupova G., Yusupov M., Hashem Y. Structure of the 70S ribosome from human pathogen Staphylococcus aureus. Nucleic Acid Res., 2016, vol. 44, no. 21, pp. 10491–10504. doi: 10.1093/nar/gkw933.
8. Ayupov R.K., Khusainov I.Sh., Validov S.Z., Yusupova G.Z., Yusupov M.M. Isolation and purification of staphylococcus aureus hibernationpromoting factor inactivating of the ribosome. Int. J. Pharm. Technol., 2016, vol. 8, no. 2, pp. 14392–14398.
9. Golovanov A.P., Hautbergue G.M., Wilson S.A., Lu-Yun Lian. A simple method for improving protein solubility and long-term stability. J. Am. Chem. Soc., 2004, vol. 126, no. 29, pp. 8933–8939. doi: 10.1021/ja049297h.
10. Hjelmeland L.M. A nondenaturing zwitterionic detergent for membrane biochemistry: Design and synthesis. Proc. Natl. Acad. Sci. U. S. A., 1980, vol. 77, no. 11, pp. 6368–6370.
11. Bachmann M., Trautmann F., Messer R., Zahn R.K., Meyer zum Büschenfelde K.H., Müller W.E.G. Association of a polyuridylate-specific endoribonuclease with small nuclear ribonucleo-proteins which had been isolated by affinity chromatography using antibodies from a patient with systemic lupus erythematosus. Eur. J. Biochem., 1983, vol. 136, no. 3, pp. 447–451. doi: 10.1111/j.1432-1033.1983.tb07762.x.
12. Raman S., Vernon R., Thompson J., Tyka M., Sadreyev R., Pei J., Kim D., Kellogg E., DiMaio F., Lange O., Kinch L., Sheffler W., Kim B-H., Das R., Grishin N.V., Baker D. Structure prediction for CASP8 with all-atom refinement using Rosetta. Proteins, 2009, vol. 77, no. 9, pp. 89–99. doi: 10.1002/prot.22540.
13. Seetharaman J., Neely H., Wang D., Janjua H., Cunningham K., Owens L., Xiao R., Liu J., Baran M.C., Acton T.B., Rost B., Montelione G.T., Hunt J.F., Tong L. Crystal structure of Ribosome-associated protein Y (PSrp-1) from Clostridium acetobutylicum. Northeast Structural Genomics Consortium target id CaR123A. 2009. Available at: http://www.rcsb.org/pdb/explore/explore.do?pdbId=3KA5. doi: 10.2210/pdb3ka5/pdb.

For citation: Ayupov R.Kh., Usachev K.S., Khusainov I.Sh., Kieffer B., Yusupov M.M. Expression and purification of HPF protein from Staphylococcus aureus and analysis of its structure by the method of NMR spectroscopy. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2017, vol. 159, no. 2, pp. 332–341. (In Russian)

The content is available under the license Creative Commons Attribution 4.0 License.