Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2000 Apr;9(4):671–682. doi: 10.1110/ps.9.4.671

Backbone dynamics measurements on leukemia inhibitory factor, a rigid four-helical bundle cytokine.

S Yao 1, D K Smith 1, M G Hinds 1, J G Zhang 1, N A Nicola 1, R S Norton 1
PMCID: PMC2144613  PMID: 10794409

Abstract

The backbone dynamics of the four-helical bundle cytokine leukemia inhibitory factor (LIF) have been investigated using 15N NMR relaxation and amide proton exchange measurements on a murine-human chimera, MH35-LIF. For rapid backbone motions (on a time scale of 10 ps to 100 ns), as probed by 15N relaxation measurements, the dynamics parameters were calculated using the model-free formalism incorporating the model selection approach. The principal components of the inertia tensor of MH35-LIF, as calculated from its NMR structure, were 1:0.98:0.38. The global rotational motion of the molecule was, therefore, assumed to be axially symmetric in the analysis of its relaxation data. This yielded a diffusion anisotropy D(parallel)/D(perpendicular) of 1.31 and an effective correlation time (4D(perpendicular) + 2D(parallel))(-1) of 8.9 ns. The average values of the order parameters (S2) for the four helices, the long interhelical loops, and the N-terminus were 0.91, 0.84, and 0.65, respectively, indicating that LIF is fairly rigid in solution, except at the N-terminus. The S2 values for the long interhelical loops of MH35-LIF were higher than those of their counterparts in short-chain members of the four-helical bundle cytokine family. Residues involved in LIF receptor binding showed no consistent pattern of backbone mobilities, with S2 values ranging from 0.71 to 0.95, but residues contributing to receptor binding site III had relatively lower S2 values, implying higher amplitude motions than for the backbone of sites I and II. In the relatively slow motion regime, backbone amide exchange measurements showed that a number of amides from the helical bundle exchanged extremely slowly, persisting for several months in 2H2O at 37 degrees C. Evidence for local unfolding was considered, and correlations among various structure-related parameters and the backbone amide exchange rates were examined. Both sets of data concur in showing that LIF is one of the most rigid four-helical bundle cytokines.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bai Y., Milne J. S., Mayne L., Englander S. W. Primary structure effects on peptide group hydrogen exchange. Proteins. 1993 Sep;17(1):75–86. doi: 10.1002/prot.340170110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brüschweiler R., Liao X., Wright P. E. Long-range motional restrictions in a multidomain zinc-finger protein from anisotropic tumbling. Science. 1995 May 12;268(5212):886–889. doi: 10.1126/science.7754375. [DOI] [PubMed] [Google Scholar]
  3. Clore G. M., Driscoll P. C., Wingfield P. T., Gronenborn A. M. Analysis of the backbone dynamics of interleukin-1 beta using two-dimensional inverse detected heteronuclear 15N-1H NMR spectroscopy. Biochemistry. 1990 Aug 14;29(32):7387–7401. doi: 10.1021/bi00484a006. [DOI] [PubMed] [Google Scholar]
  4. Cordier F., Caffrey M., Brutscher B., Cusanovich M. A., Marion D., Blackledge M. Solution structure, rotational diffusion anisotropy and local backbone dynamics of Rhodobacter capsulatus cytochrome c2. J Mol Biol. 1998 Aug 14;281(2):341–361. doi: 10.1006/jmbi.1998.1950. [DOI] [PubMed] [Google Scholar]
  5. Englander S. W., Sosnick T. R., Englander J. J., Mayne L. Mechanisms and uses of hydrogen exchange. Curr Opin Struct Biol. 1996 Feb;6(1):18–23. doi: 10.1016/s0959-440x(96)80090-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Feher V. A., Cavanagh J. Millisecond-timescale motions contribute to the function of the bacterial response regulator protein Spo0F. Nature. 1999 Jul 15;400(6741):289–293. doi: 10.1038/22357. [DOI] [PubMed] [Google Scholar]
  7. Feng Y., Klein B. K., McWherter C. A. Three-dimensional solution structure and backbone dynamics of a variant of human interleukin-3. J Mol Biol. 1996 Jun 14;259(3):524–541. doi: 10.1006/jmbi.1996.0337. [DOI] [PubMed] [Google Scholar]
  8. Gervais V., Zerial A., Oschkinat H. NMR investigations of the role of the sugar moiety in glycosylated recombinant human granulocyte-colony-stimulating factor. Eur J Biochem. 1997 Jul 1;247(1):386–395. doi: 10.1111/j.1432-1033.1997.00386.x. [DOI] [PubMed] [Google Scholar]
  9. Goodman E. M., Kim P. S. Periodicity of amide proton exchange rates in a coiled-coil leucine zipper peptide. Biochemistry. 1991 Dec 17;30(50):11615–11620. doi: 10.1021/bi00114a002. [DOI] [PubMed] [Google Scholar]
  10. Gryk M. R., Jardetzky O., Klig L. S., Yanofsky C. Flexibility of DNA binding domain of trp repressor required for recognition of different operator sequences. Protein Sci. 1996 Jun;5(6):1195–1197. doi: 10.1002/pro.5560050624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hinds M. G., Maurer T., Zhang J. G., Nicola N. A., Norton R. S. Resonance assignments, secondary structure and topology of leukaemia inhibitory factor in solution. J Biomol NMR. 1997 Feb;9(2):113–126. doi: 10.1023/a:1018636018243. [DOI] [PubMed] [Google Scholar]
  12. Hinds M. G., Maurer T., Zhang J. G., Nicola N. A., Norton R. S. Solution structure of leukemia inhibitory factor. J Biol Chem. 1998 May 29;273(22):13738–13745. doi: 10.1074/jbc.273.22.13738. [DOI] [PubMed] [Google Scholar]
  13. Hudson K. R., Vernallis A. B., Heath J. K. Characterization of the receptor binding sites of human leukemia inhibitory factor and creation of antagonists. J Biol Chem. 1996 May 17;271(20):11971–11978. doi: 10.1074/jbc.271.20.11971. [DOI] [PubMed] [Google Scholar]
  14. Jeng M. F., Englander S. W. Stable submolecular folding units in a non-compact form of cytochrome c. J Mol Biol. 1991 Oct 5;221(3):1045–1061. doi: 10.1016/0022-2836(91)80191-v. [DOI] [PubMed] [Google Scholar]
  15. Kabsch W., Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. doi: 10.1002/bip.360221211. [DOI] [PubMed] [Google Scholar]
  16. Kay L. E., Torchia D. A., Bax A. Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry. 1989 Nov 14;28(23):8972–8979. doi: 10.1021/bi00449a003. [DOI] [PubMed] [Google Scholar]
  17. Kriwacki R. W., Hengst L., Tennant L., Reed S. I., Wright P. E. Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity. Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11504–11509. doi: 10.1073/pnas.93.21.11504. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Layton M. J., Owczarek C. M., Metcalf D., Clark R. L., Smith D. K., Treutlein H. R., Nicola N. A. Conversion of the biological specificity of murine to human leukemia inhibitory factor by replacing 6 amino acid residues. J Biol Chem. 1994 Nov 25;269(47):29891–29896. [PubMed] [Google Scholar]
  19. Lee B., Richards F. M. The interpretation of protein structures: estimation of static accessibility. J Mol Biol. 1971 Feb 14;55(3):379–400. doi: 10.1016/0022-2836(71)90324-x. [DOI] [PubMed] [Google Scholar]
  20. Luginbühl P., Pervushin K. V., Iwai H., Wüthrich K. Anisotropic molecular rotational diffusion in 15N spin relaxation studies of protein mobility. Biochemistry. 1997 Jun 17;36(24):7305–7312. doi: 10.1021/bi963161h. [DOI] [PubMed] [Google Scholar]
  21. Mandel A. M., Akke M., Palmer A. G., 3rd Backbone dynamics of Escherichia coli ribonuclease HI: correlations with structure and function in an active enzyme. J Mol Biol. 1995 Feb 10;246(1):144–163. doi: 10.1006/jmbi.1994.0073. [DOI] [PubMed] [Google Scholar]
  22. Marion D., Wüthrich K. Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem Biophys Res Commun. 1983 Jun 29;113(3):967–974. doi: 10.1016/0006-291x(83)91093-8. [DOI] [PubMed] [Google Scholar]
  23. Maurer T., Smith D. K., Owczarek C. M., Layton M. J., Zhang J. G., Nicola N. A., Norton R. S. NMR studies of a murine-human chimera of leukaemia inhibitory factor (LIF). Comparison with human LIF. Growth Factors. 1994;11(4):271–276. doi: 10.3109/08977199409010999. [DOI] [PubMed] [Google Scholar]
  24. Miller S., Janin J., Lesk A. M., Chothia C. Interior and surface of monomeric proteins. J Mol Biol. 1987 Aug 5;196(3):641–656. doi: 10.1016/0022-2836(87)90038-6. [DOI] [PubMed] [Google Scholar]
  25. Owczarek C. M., Layton M. J., Metcalf D., Lock P., Willson T. A., Gough N. M., Nicola N. A. Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant. EMBO J. 1993 Sep;12(9):3487–3495. doi: 10.1002/j.1460-2075.1993.tb06023.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pedersen T. G., Thomsen N. K., Andersen K. V., Madsen J. C., Poulsen F. M. Determination of the rate constants k1 and k2 of the Linderström-Lang model for protein amide hydrogen exchange. A study of the individual amides in hen egg-white lysozyme. J Mol Biol. 1993 Mar 20;230(2):651–660. doi: 10.1006/jmbi.1993.1176. [DOI] [PubMed] [Google Scholar]
  27. Purvis D. H., Mabbutt B. C. Solution dynamics and secondary structure of murine leukemia inhibitory factor: a four-helix cytokine with a rigid CD loop. Biochemistry. 1997 Aug 19;36(33):10146–10154. doi: 10.1021/bi970665m. [DOI] [PubMed] [Google Scholar]
  28. Radford S. E., Buck M., Topping K. D., Dobson C. M., Evans P. A. Hydrogen exchange in native and denatured states of hen egg-white lysozyme. Proteins. 1992 Oct;14(2):237–248. doi: 10.1002/prot.340140210. [DOI] [PubMed] [Google Scholar]
  29. Raschke T. M., Marqusee S. Hydrogen exchange studies of protein structure. Curr Opin Biotechnol. 1998 Feb;9(1):80–86. doi: 10.1016/s0958-1669(98)80088-8. [DOI] [PubMed] [Google Scholar]
  30. Redfield C., Boyd J., Smith L. J., Smith R. A., Dobson C. M. Loop mobility in a four-helix-bundle protein: 15N NMR relaxation measurements on human interleukin-4. Biochemistry. 1992 Nov 3;31(43):10431–10437. doi: 10.1021/bi00158a003. [DOI] [PubMed] [Google Scholar]
  31. Robinson R. C., Grey L. M., Staunton D., Vankelecom H., Vernallis A. B., Moreau J. F., Stuart D. I., Heath J. K., Jones E. Y. The crystal structure and biological function of leukemia inhibitory factor: implications for receptor binding. Cell. 1994 Jul 1;77(7):1101–1116. doi: 10.1016/0092-8674(94)90449-9. [DOI] [PubMed] [Google Scholar]
  32. Rose T. M., Bruce A. G. Oncostatin M is a member of a cytokine family that includes leukemia-inhibitory factor, granulocyte colony-stimulating factor, and interleukin 6. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8641–8645. doi: 10.1073/pnas.88.19.8641. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schurr J. M., Babcock H. P., Fujimoto B. S. A test of the model-free formulas. Effects of anisotropic rotational diffusion and dimerization. J Magn Reson B. 1994 Nov;105(3):211–224. doi: 10.1006/jmrb.1994.1127. [DOI] [PubMed] [Google Scholar]
  34. Stone M. J., Fairbrother W. J., Palmer A. G., 3rd, Reizer J., Saier M. H., Jr, Wright P. E. Backbone dynamics of the Bacillus subtilis glucose permease IIA domain determined from 15N NMR relaxation measurements. Biochemistry. 1992 May 12;31(18):4394–4406. doi: 10.1021/bi00133a003. [DOI] [PubMed] [Google Scholar]
  35. Tjandra N., Wingfield P., Stahl S., Bax A. Anisotropic rotational diffusion of perdeuterated HIV protease from 15N NMR relaxation measurements at two magnetic fields. J Biomol NMR. 1996 Oct;8(3):273–284. doi: 10.1007/BF00410326. [DOI] [PubMed] [Google Scholar]
  36. Wand A. J., Roder H., Englander S. W. Two-dimensional 1H NMR studies of cytochrome c: hydrogen exchange in the N-terminal helix. Biochemistry. 1986 Mar 11;25(5):1107–1114. doi: 10.1021/bi00353a025. [DOI] [PubMed] [Google Scholar]
  37. Wyss D. F., Dayie K. T., Wagner G. The counterreceptor binding site of human CD2 exhibits an extended surface patch with multiple conformations fluctuating with millisecond to microsecond motions. Protein Sci. 1997 Mar;6(3):534–542. doi: 10.1002/pro.5560060303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yao S, Hinds MG, Norton RS. Improved Estimation of Protein Rotational Correlation Times from 15N Relaxation Measurements. J Magn Reson. 1998 Apr;131(2):347–350. doi: 10.1006/jmre.1998.1382. [DOI] [PubMed] [Google Scholar]
  39. Zink T., Ross A., Lüers K., Cieslar C., Rudolph R., Holak T. A. Structure and dynamics of the human granulocyte colony-stimulating factor determined by NMR spectroscopy. Loop mobility in a four-helix-bundle protein. Biochemistry. 1994 Jul 19;33(28):8453–8463. doi: 10.1021/bi00194a009. [DOI] [PubMed] [Google Scholar]
  40. van Tilborg P. J., Mulder F. A., de Backer M. M., Nair M., van Heerde E. C., Folkers G., van der Saag P. T., Karimi-Nejad Y., Boelens R., Kaptein R. Millisecond to microsecond time scale dynamics of the retinoid X and retinoic acid receptor DNA-binding domains and dimeric complex formation. Biochemistry. 1999 Feb 16;38(7):1951–1956. doi: 10.1021/bi982526q. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES