Advertisement

Generation and characterization of functional phosphoserine-incorporated neuronal nitric oxide synthase holoenzyme

  • Huayu Zheng
  • Jingxuan He
  • Jinghui Li
  • Jing Yang
  • Martin L. Kirk
  • Linda J. Roman
  • Changjian Feng
Original Paper
  • 96 Downloads

Abstract

Phosphorylation is an important pathway for the regulation of nitric oxide synthase (NOS) at the posttranslational level. However, the molecular underpinnings of NOS regulation by phosphorylations remain unclear to date, mainly because of the problems in making a good amount of active phospho-NOS proteins. Herein, we have established a system in which recombinant rat nNOS holoprotein can be produced with site-specific incorporation of phosphoserine (pSer) at residue 1412, using a specialized bacterial host strain for pSer incorporation. The pSer1412 nNOS protein demonstrates UV–Vis, far-UV CD and fluorescence spectral properties that are identical to those of nNOS overexpressed in other bacterial strains. The protein is also functional, possessing normal NO production and NADPH oxidation activities in the presence of abundant substrate l-Arg. Conversely, the rate of FMN–heme interdomain electron transfer (IET) in pSer1412 nNOS is considerably lower than that of wild-type (wt) nNOS, while the phosphomimetic S1142E mutant possesses similar electron transfer kinetics to that of wt. The successful incorporation and high yield of pSer1412 into rat nNOS and the significant change in the IET kinetics upon the phosphorylation demonstrate a highly useful method for incorporating native phosphorylation sites as a substantial improvement to commonly used phosphomimetics.

Keywords

Nitric oxide synthase Phosphoserine Phosphorylation Electron transfer Phosphomimetic 

Notes

Acknowledgements

We thank Dr. Charles Melancon for helpful discussions. This work was supported by the National Institutes of Health (Grant no. GM-081811 to CF, GM-057378 to MLK and GM-081568 to LR). Mass spectrometry data were acquired by the University of Ari-zona Analytical and Biological Mass Spectrometry Facility supported by NIH/NCI Grant CA023074 to the University of Arizona Cancer Center and by the BIO5 Institute of the University of Arizona.

Supplementary material

775_2018_1621_MOESM1_ESM.pdf (2.5 mb)
Supplementary material 1 (PDF 2598 kb)

References

  1. 1.
    Förstermann U, Sessa WC (2012) Eur Heart J 33:829–837CrossRefGoogle Scholar
  2. 2.
    Feng C (2012) Coord Chem Rev 256:393–411CrossRefGoogle Scholar
  3. 3.
    Alderton WK, Cooper CE, Knowles RG (2001) Biochem J 357:593–615CrossRefGoogle Scholar
  4. 4.
    Fulton DJR (2016) In: Raouf AK (ed) Adv pharmacol. Academic Press, pp 29-64Google Scholar
  5. 5.
    Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC (1999) Nature 399:597–601CrossRefGoogle Scholar
  6. 6.
    Chen CA, Druhan LJ, Varadharaj S, Chen YR, Zweier JL (2008) J Biol Chem 283:27038–27047CrossRefGoogle Scholar
  7. 7.
    Haque MM, Ray SS, Stuehr DJ (2016) J Biol Chem 291:23047–23057CrossRefGoogle Scholar
  8. 8.
    Tran QK, Leonard J, Black DJ, Nadeau OW, Boulatnikov IG, Persechini A (2009) J Biol Chem 284:11892–11899CrossRefGoogle Scholar
  9. 9.
    Adak S, Santolini J, Tikunova S, Wang Q, Johnson JD, Stuehr DJ (2001) J Biol Chem 276:1244–1252CrossRefGoogle Scholar
  10. 10.
    McCabe TJ, Fulton D, Roman LJ, Sessa WC (2000) J Biol Chem 275:6123–6128CrossRefGoogle Scholar
  11. 11.
    Xie Y, Jiang Y, Ben-Amotz D (2005) Anal Biochem 343:223–230CrossRefGoogle Scholar
  12. 12.
    Mount PF, Kemp BE, Power DA (2007) J Mol Cell Cardiol 42:271–279CrossRefGoogle Scholar
  13. 13.
    Wu PR, Chen BR, Hsieh CC, Lin WC, Wu KK, Hwu Y, Chen PF (2014) Biosci Rep.  https://doi.org/10.1042/bsr20140079 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Pirman NL, Barber KW, Aerni HR, Ma NJ, Haimovich AD, Rogulina S, Isaacs FJ, Rinehart J (2015) Nat Commun 6:8130CrossRefGoogle Scholar
  15. 15.
    Rameau GA, Tukey DS, Garcin-Hosfield ED, Titcombe RF, Misra C, Khatri L, Getzoff ED, Ziff EB (2007) J Neurosci 27:3445–3455CrossRefGoogle Scholar
  16. 16.
    Panda SP, Li W, Venkatakrishnan P, Chen L, Astashkin AV, Masters BSS, Feng C, Roman LJ (2013) FEBS Lett 587:3973–3978CrossRefGoogle Scholar
  17. 17.
    Van Veldhoven PP, Mannaerts GP (1987) Anal Biochem 161:45–48CrossRefGoogle Scholar
  18. 18.
    Carter SG, Karl DW (1982) J Biochem Biophys Methods 7:7–13CrossRefGoogle Scholar
  19. 19.
    Chen GC, Yang JT (1977) Anal Lett 10:1195–1207CrossRefGoogle Scholar
  20. 20.
    Feng CJ, Tollin G, Hazzard JT, Nahm NJ, Guillemette JG, Salerno JC, Ghosh DK (2007) J Am Chem Soc 129:5621–5629CrossRefGoogle Scholar
  21. 21.
    Feng CJ, Roman LJ, Hazzard JT, Ghosh DK, Tollin G, Masters BSS (2008) FEBS Lett 582:2768–2772CrossRefGoogle Scholar
  22. 22.
    Rogerson DT, Sachdeva A, Wang K, Haq T, Kazlauskaite A, Hancock SM, Huguenin-Dezot N, Muqit MMK, Fry AM, Bayliss R, Chin JW (2015) Nat Chem Biol 11:496–503CrossRefGoogle Scholar
  23. 23.
    Guo X, Niemi NM, Hutchins PD, Condon SGF, Jochem A, Ulbrich A, Higbee AJ, Russell JD, Senes A, Coon JJ, Pagliarini DJ (2017) Cell Reports 18:307–313CrossRefGoogle Scholar
  24. 24.
    Lv Z, Rickman KA, Yuan L, Williams K, Selvam SP, Woosley AN, Howe PH, Ogretmen B, Smogorzewska A, Olsen SK (2017) Mol Cell 65:699–714.e696CrossRefGoogle Scholar
  25. 25.
    Steinfeld JB, Aerni HR, Rogulina S, Liu Y, Rinehart J (2014) ACS Chem Biol 9:1104–1112CrossRefGoogle Scholar
  26. 26.
    Shrestha A, Hamilton G, O’Neill E, Knapp S, Elkins JM (2012) Protein Expr Purif 81:136–143CrossRefGoogle Scholar
  27. 27.
    Solari FA, Dell’Aica M, Sickmann A, Zahedi RP (2015) Mol BioSyst 11:1487–1493CrossRefGoogle Scholar
  28. 28.
    Cabrera-Pastor A, Llansola M, Felipo V (2016) ACS Chemical Neuroscience 7:1753–1759CrossRefGoogle Scholar
  29. 29.
    Garbincius JF, Michele DE (2015) Proc Natl Acad Sci 112:13663–13668CrossRefGoogle Scholar
  30. 30.
    Brüne B, Lapetina EG (1991) Biochem Biophys Res Commun 181:921–926CrossRefGoogle Scholar
  31. 31.
    Bredt DS, Ferris CD, Snyder SH (1992) J Biol Chem 267:10976–10981PubMedGoogle Scholar
  32. 32.
    Klatt P, Schmidt K, Lehner D, Glatter O, Bächinger HP, Mayer B (1995) The EMBO journal 14:3687–3695CrossRefGoogle Scholar
  33. 33.
    Richards MK, Clague MJ, Marletta MA (1996) Biochemistry 35:7772–7780CrossRefGoogle Scholar
  34. 34.
    Adak S, Ghosh S, Abu-Soud HM, Stuehr DJ (1999) J Biol Chem 274:22313–22320CrossRefGoogle Scholar
  35. 35.
    Li J, Zheng H, Wang W, Miao Y, Sheng Y, Feng C (2018) FEBS Lett 592:2425–2431CrossRefGoogle Scholar
  36. 36.
    Li J, Zheng H, Feng C (2018) Front Biosci (Landmark edition) 23:1803–1821CrossRefGoogle Scholar
  37. 37.
    Stuehr DJ, Santolini J, Wang ZQ, Wei CC, Adak S (2004) J Biol Chem 279:36167–36170CrossRefGoogle Scholar
  38. 38.
    Arnett DC, Persechini A, Tran Q-K, Black DJ, Johnson CK (2015) FEBS Lett 589:1173–1178CrossRefGoogle Scholar
  39. 39.
    Katakam PV, Snipes JA, Steed MM, Busija DW (2012) J Cereb Blood Flow Metab 32:792–804CrossRefGoogle Scholar
  40. 40.
    Osuka K, Watanabe Y, Usuda N, Atsuzawa K, Takayasu M (2013) Neurochem Int 63:269–274CrossRefGoogle Scholar
  41. 41.
    Astashkin AV, Li J, Zheng H, Miao Y, Feng C (2018) J Inorg Biochem 184:146–155CrossRefGoogle Scholar
  42. 42.
    Astashkin AV, Feng C (2015) J Phys Chem A 119:11066–11075CrossRefGoogle Scholar
  43. 43.
    Hanson QM, Carley JR, Gilbreath TJ, Smith BC, Underbakke ES (2018) J Mol Biol 430:935–947CrossRefGoogle Scholar

Copyright information

© SBIC 2018

Authors and Affiliations

  • Huayu Zheng
    • 1
    • 2
  • Jingxuan He
    • 2
    • 4
  • Jinghui Li
    • 1
  • Jing Yang
    • 2
  • Martin L. Kirk
    • 2
  • Linda J. Roman
    • 3
  • Changjian Feng
    • 1
    • 2
  1. 1.College of PharmacyUniversity of New MexicoAlbuquerqueUSA
  2. 2.Department of Chemistry and Chemical BiologyUniversity of New MexicoAlbuquerqueUSA
  3. 3.Department of Biochemistry and Structural BiologyUniversity of Texas Health Science Center in San AntonioSan AntonioUSA
  4. 4.Department of ChemistryPenn State UniversityUniversity ParkUSA

Personalised recommendations