Skip to main content

Advertisement

SpringerLink
Log in

Phosphorylation of Leghemoglobin at S45 is Most Effective to Disrupt the Molecular Environment of Its Oxygen Binding Pocket

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

In leguminous plants, nitrogenase that catalyzes anaerobic symbiotic nitrogen fixation is protected by the sequestration of O2 by Leghemoglobin (LegH). The modulation of the oxygen binding capacity of Hemoglobin (Hb) by different post-translational modifications is well studied; whereas similar studies on LegH’s O2 binding are not yet benchmarked. Our results show that in vitro serine phosphorylation of recombinant LegH from Lotus japonicus, a model legume, by a homologous kinase caused a reduction in its oxygen consumption as determined by Clark type electrode. Although mass spectrometry revealed a few phosphorylated serine residues in the LegH, molecular modeling study showed that particularly S45 is the most critical one, along with S55, however the latter with lesser impact on its molecular environment responsible for oxygen consumption. Separate S45D and S55D mutants of recombinant LegH also corroborated the results obtained from molecular modeling study. Thus, this work lays groundwork for further investigation of structural and functional role of serine phosphorylation as one of the mechanisms by which oxygen consumption by LegH may possibly be regulated during nodulation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

Hb:

Hemoglobin

LegH:

Leghemoglobin

2, 3 BPG:

2, 3- Bisphosphoglycerate

LjCCamK:

Lotus japonicus Ca2+/Calmodulin dependent kinase

MD:

Molecular dynamics

References

  1. Kubo H (1939) Überhämoproteinaus den wurzelknöllchen von leguminosen. Acta Phytochim (Tokyo) 11:195–200

    CAS  Google Scholar 

  2. Ross EJH, Lira-Ruan V, Arredondo-Peter R, Klucas RV, Sarath G (2002) Recent insights into plant hemoglobins. Rev Plant Biochem Biotechnol 1:173–189

    Google Scholar 

  3. Oldroyd GED (2001) Dissecting symbiosis: developments in nod factor signal transduction. Annals Bot 87:709–718

    Article  CAS  Google Scholar 

  4. LimpensE BisselingT (2003) Signaling in symbiosis. Curr Opn Plant Biol 6:343–350

    Article  Google Scholar 

  5. Hargrove MS (2003) Plants, humans and hemoglobins. Trends Plant Sci 8(8):387–393

    Article  Google Scholar 

  6. Marroquí S, Zorreguieta A, Santamaría C, Temprano F, Soberón M, Megías M, Downie JA (2001) Enhanced symbiotic performance by Rhizobium tropici glycogen synthase mutants. J Bacteriol 183(3):854–864

    Article  Google Scholar 

  7. Cheng Q (2008) Perspectives in biological nitrogen fixation research. J Int Plant Biol 50(7):786–798

    Article  CAS  Google Scholar 

  8. Wittenberg JB (1973) Nicotinic acid as a ligand affecting leghemoglobin structure and oxygen reactivity. Proc Nat Acad Sci USA 70(2):564–568

    Article  Google Scholar 

  9. Harutyunyan EH, Safonova TN, Kuranova IP, Popov AN, Teplyakov AV, Obmolova GV, Rusakov AA, Vainshtein BK, Dodson GG, Wilson JC, Perutz MF (1995) The structure of deoxy- and oxy-Leghaemoglobin from Lupin. J Mol Biol 251:104–115

    Article  CAS  Google Scholar 

  10. Martí MA, Capece L, Bikiel DE, Falcone B, Estrin DA (2007) Oxygen affinity controlled by dynamical distal conformations: the soybean Leghemoglobin and the Paramecium caudatum hemoglobin cases. Proteins 68(2):480–487

    Article  Google Scholar 

  11. Kawashima K, Suganuma N, Tamaoki M, Kouchi H (2001) Two types of pea leghemoglobin genes showing different O2-binding affinities and distinct patterns of spatial expression in nodules. Plant Physiol 125:641–651

    Article  CAS  Google Scholar 

  12. Coates ML (1975) Hemoglobin function in the vertebrates: an evolutionary model. J Mol Evol 6:285–307

    Article  CAS  Google Scholar 

  13. ArnoneA Perutz MF (1974) Structure of inositol hexaphosphate–human deoxyhaemoglob in complex. Nature 249:34–36

    Article  Google Scholar 

  14. Benesch R, Benesch RE (1967) The effect of organic phosphates from the human erythrocyte on the allosteric properties of haemoglobin. Biochem Biophys Res Commun 26(2):162–167

    Article  CAS  Google Scholar 

  15. Zinchuk VV (2006) Nitric oxide effect on the hemoglobin-oxygen affinity. J Physiol Pharma 57(1):29–38

    Google Scholar 

  16. Hess DT, Stamler JS (2012) Regulation by S-nitrosylation of protein post-translational modification. J Biol Chem 287(7):4411–4418

    Article  CAS  Google Scholar 

  17. Jones RL, Peterson CM (1981) Hematologic alterations in diabetes mellitus. Am J Med 70(2):339–352

    Article  CAS  Google Scholar 

  18. Giardina B, Ascenzi P, Clementi ME, Sanctis GD, Rizzi M, Coletta M (1996) Functional modulation by lactate of myoglobin: a monomeric allosteric hemoprotein. J Biol Chem 271:16999–17001

    Article  CAS  Google Scholar 

  19. Traylo TG, Deardurff LA, Coletta M, Ascenzi P, Antonini E, Brunori M (1983) Reactivity of ferrous heme proteins at low pH. J Biol Chem 258:12147–12148

    Google Scholar 

  20. Jayaraman T, Tejero J, Chen BB, Blood AB, Frizzell S, Shapiro C, Tiso M, Hood BL, Wang X, Zhao X, Conrads TP, Mallampalli RK, Gladwin MT (2011) 14-3-3 binding and phosphorylation of neuroglobin during hypoxia modulate six-to-five heme pocket coordination and rate of nitrite reduction to nitric oxide. J Biol Chem 286(49):42679–42689

    Article  CAS  Google Scholar 

  21. Handberg K, Stougaard J (1992) Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics. Plant J 2:487–496

    Article  Google Scholar 

  22. Becana M, Salin ML, Ji L, Klucas RV (1991) Flavin-mediated reduction of ferric leghemoglobin from soybean nodules. Planta 183(4):575–583

    Article  CAS  Google Scholar 

  23. Tirichine L, Imaizumi-Anraku H, Yoshida S, Murakami Y, Madsen LH, Miwa H, Nakagawa T, Sandal N, Albrektsen AS, Kawaguchi M, Downie A, Sato S, Tabata S, Kouchi H, Parniske M, Kawasaki S, Stougaard J (2006) Deregulation of a Ca2+/calmodulin dependent kinase leads to spontaneous nodule development. Nature 441(7097):1153–1156

    Article  CAS  Google Scholar 

  24. Hargrove MS, Barry JK, Brucker EA, Berry MB, Phillips GN Jr (1997) Characterization of recombinant soybean leghemoglobin a and apolar distal histidine mutants. J Mol Biol 266:1032–1042

    Article  CAS  Google Scholar 

  25. Eswar N, Webb B, Marti-Renom MA, Madhusudhan MS, Eramian D, Shen MY, Pieper U, Sali A (2006) Comparative protein structure modeling with modeller. Curr Protoc Bioinformatics, Wiley Supplement 15 5.6.1–5.6.30

  26. Blom N, Gammeltoft S, Brunak S (1999) Sequence- and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294(5):1351–1362

    Article  CAS  Google Scholar 

  27. Swainsbury DJ, Zhou L, Oldroyd GE, Bornemann S (2012) Calcium ion binding properties of Medicago truncatula calcium/calmodulin dependent protein kinase. Biochemistry 51(35):6895–6907

    Article  CAS  Google Scholar 

  28. Singh S, Parniske M (2012) Activation of calcium- and calmodulin-dependent protein kinase (CCaMK), the central regulator of plant root endosymbiosis. Curr Opn Plant Biol 15:444–453

    Article  CAS  Google Scholar 

  29. White RR, Kwon YG, Taing M, Lawrence DS, Edelman AM (1998) Definition of optimal substrate recognition motifs of Ca2+-calmodulin-dependent protein kinases IV and II reveals shared and distinctive features. J Biol Chem 273(6):3166–3172

    Article  CAS  Google Scholar 

  30. Yan DJ, Li W, Xiang Y, Wen GB, Lin YW, Tan X (2015) A novel tyrosine-heme C–O covalent linkage in F43Y myoglobin: a new post-translational modification of heme proteins. ChemBioChem 16(1):47–50

    Article  CAS  Google Scholar 

  31. Lin YW, Shu XG, Du KJ, Nie CM, Wen GB (2014) Computational insight into nitration of human myoglobin. Comput Biol Chem 52:60–65

    Article  Google Scholar 

  32. Dastidar SG, Raghunathan D, Nicholson J, Hupp TR, Lane DP, Verma CS (2011) Chemical states of the N-terminal “lid” of MDM2 regulate p53 binding: simulations reveal complexities of modulation. Cell Cycle 10(1):82–89

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We greatly appreciate the gift of Lotus japonicus late nodule (21 day old) cDNA library from Dr. J. Stougaard, Dept. of Molecular Biology, Aarhus University, Denmark. SGD acknowledges the infrastructural support provided by Bioinformatics Centre of Excellence and the start-up funding provided by Bose Institute. This research was supported by the Centre for Modern Biology under ‘University Potential for Excellence (UPE) and Center for Advanced Studies in Biochemistry awarded by the University Grants Commission (UGC) India. The financial assistance from Departments of Science and Technology and Biotechnology, Government of India (SR/SO/PS/82/2010; SR/SO/HS-51/2008; BT/PR 11415/BRB/10/656/2008); and Interdisciplinary Program in Life science (IPLS), DBT, Govt. of India also have supported this work. Kaushik Bhar is supported by research fellowship under the scheme for ‘University Potential for Excellence (UPE). Atanu Maity is Senior Research Fellow awarded by Council of Scientific and Industrial Research (Government of India). Amit Ghosh receives his fellowship from IPLS program awarded by DBT, Govt. of India.

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animals rights statements

This article does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

This article does not contain any studies that involve individual participant; therefore, ‘Informed consent’ is not applicable.

Author information

Author notes

    Authors and Affiliations

    1. Department of Biochemistry, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, 700019, India

      Kaushik Bhar, Amit Ghosh & Anirban Siddhanta

    2. BioinformaticsCenter, Bose Institute, A.J.C. Bose Centenary Campus, P-1/12, C.I.T. Scheme VII M, Kolkata, 700054, India

      Atanu Maity & Shubhra Ghosh Dastidar

    3. Infectious Diseases and Immunology Division, Indian Institute of Chemical Biology (CSIR), 4, Raja S.C. Mullick Road, Kolkata, 700032, India

      Tanusree Das

    Authors
    1. Kaushik Bhar
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Atanu Maity
      View author publications

      You can also search for this author in PubMed Google Scholar

    3. Amit Ghosh
      View author publications

      You can also search for this author in PubMed Google Scholar

    4. Tanusree Das
      View author publications

      You can also search for this author in PubMed Google Scholar

    5. Shubhra Ghosh Dastidar
      View author publications

      You can also search for this author in PubMed Google Scholar

    6. Anirban Siddhanta
      View author publications

      You can also search for this author in PubMed Google Scholar

    Corresponding authors

    Correspondence to Shubhra Ghosh Dastidar or Anirban Siddhanta.

    Additional information

    Kaushik Bhar, Atanu Maity and Amit Ghosh have contributed equally to the work.

    Electronic supplementary material

    Below is the link to the electronic supplementary material.

    Supplementary material 1 (PDF 723 kb)

    Supplementary material 2 (MPEG 738 kb)

    Rights and permissions

    Reprints and permissions

    About this article

    Check for updates. Verify currency and authenticity via CrossMark

    Cite this article

    Bhar, K., Maity, A., Ghosh, A. et al. Phosphorylation of Leghemoglobin at S45 is Most Effective to Disrupt the Molecular Environment of Its Oxygen Binding Pocket. Protein J 34, 158–167 (2015). https://doi.org/10.1007/s10930-015-9608-z

    Download citation

    • Published:

    • Issue Date:

    • DOI: https://doi.org/10.1007/s10930-015-9608-z

    Keywords

    Search

    Navigation