Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1999 Feb;8(2):307–317. doi: 10.1110/ps.8.2.307

Comparison of the backbone dynamics of the apo- and holo-carboxy-terminal domain of the biotin carboxyl carrier subunit of Escherichia coli acetyl-CoA carboxylase.

X Yao 1, C Soden Jr 1, M F Summers 1, D Beckett 1
PMCID: PMC2144255  PMID: 10048324

Abstract

The biotin carboxyl carrier protein (BCCP) is a subunit of acetyl-CoA carboxylase, a biotin-dependent enzyme that catalyzes the first committed step of fatty acid biosynthesis. In its functional cycle, this protein engages in heterologous protein-protein interactions with three distinct partners, depending on its state of post-translational modification. Apo-BCCP interacts specifically with the biotin holoenzyme synthetase, BirA, which results in the post-translational attachment of biotin to a single lysine residue on BCCP. Holo-BCCP then interacts with the biotin carboxylase subunit of acetyl-CoA carboxylase, which leads to the addition of the carboxylate group of bicarbonate to biotin. Finally, the carboxy-biotinylated form of BCCP interacts with transcarboxylase in the transfer of the carboxylate to acetyl-CoA to form malonyl-CoA. The determinants of protein-protein interaction specificity in this system are unknown. The NMR solution structure of the unbiotinylated form of an 87 residue C-terminal domain fragment (residue 70-156) of BCCP (holoBCCP87) and the crystal structure of the biotinylated form of a C-terminal fragment (residue 77-156) of BCCP from Escherichia coli acetyl-CoA carboxylase have previously been determined. Comparative analysis of these structures provided evidence for small, localized conformational changes in the biotin-binding region upon biotinylation of the protein. These structural changes may be important for regulating specific protein-protein interactions. Since the dynamic properties of proteins are correlated with local structural environments, we have determined the relaxation parameters of the backbone 15N nuclear spins of holoBCCP87, and compared these with the data obtained for the apo protein. The results indicate that upon biotinylation, the inherent mobility of the biotin-binding region and the protruding thumb, with which the biotin group interacts in the holo protein, are significantly reduced.

Full Text

The Full Text of this article is available as a PDF (952.4 KB).

Selected References

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

  1. Athappilly F. K., Hendrickson W. A. Structure of the biotinyl domain of acetyl-coenzyme A carboxylase determined by MAD phasing. Structure. 1995 Dec 15;3(12):1407–1419. doi: 10.1016/s0969-2126(01)00277-5. [DOI] [PubMed] [Google Scholar]
  2. Barbato G., Ikura M., Kay L. E., Pastor R. W., Bax A. Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible. Biochemistry. 1992 Jun 16;31(23):5269–5278. doi: 10.1021/bi00138a005. [DOI] [PubMed] [Google Scholar]
  3. Cronan J. E., Jr Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins. J Biol Chem. 1990 Jun 25;265(18):10327–10333. [PubMed] [Google Scholar]
  4. Delaglio F., Grzesiek S., Vuister G. W., Zhu G., Pfeifer J., Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995 Nov;6(3):277–293. doi: 10.1007/BF00197809. [DOI] [PubMed] [Google Scholar]
  5. Fall R. R. Analysis of microbial biotin proteins. Methods Enzymol. 1979;62:390–398. doi: 10.1016/0076-6879(79)62246-2. [DOI] [PubMed] [Google Scholar]
  6. Farrow N. A., Muhandiram R., Singer A. U., Pascal S. M., Kay C. M., Gish G., Shoelson S. E., Pawson T., Forman-Kay J. D., Kay L. E. Backbone dynamics of a free and phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry. 1994 May 17;33(19):5984–6003. doi: 10.1021/bi00185a040. [DOI] [PubMed] [Google Scholar]
  7. Jones B. E., Rajagopal P., Klevit R. E. Phosphorylation on histidine is accompanied by localized structural changes in the phosphocarrier protein, HPr from Bacillus subtilis. Protein Sci. 1997 Oct;6(10):2107–2119. doi: 10.1002/pro.5560061006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Knowles J. R. The mechanism of biotin-dependent enzymes. Annu Rev Biochem. 1989;58:195–221. doi: 10.1146/annurev.bi.58.070189.001211. [DOI] [PubMed] [Google Scholar]
  10. Li S. J., Cronan J. E., Jr The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. J Biol Chem. 1992 Jan 15;267(2):855–863. [PubMed] [Google Scholar]
  11. 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]
  12. Marion D., Driscoll P. C., Kay L. E., Wingfield P. T., Bax A., Gronenborn A. M., Clore G. M. Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta. Biochemistry. 1989 Jul 25;28(15):6150–6156. doi: 10.1021/bi00441a004. [DOI] [PubMed] [Google Scholar]
  13. Nenortas E., Beckett D. Purification and characterization of intact and truncated forms of the Escherichia coli biotin carboxyl carrier subunit of acetyl-CoA carboxylase. J Biol Chem. 1996 Mar 29;271(13):7559–7567. doi: 10.1074/jbc.271.13.7559. [DOI] [PubMed] [Google Scholar]
  14. Piotto M., Saudek V., Sklenár V. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR. 1992 Nov;2(6):661–665. doi: 10.1007/BF02192855. [DOI] [PubMed] [Google Scholar]
  15. Reddy D. V., Shenoy B. C., Carey P. R., Sönnichsen F. D. Absence of observable biotin-protein interactions in the 1.3S subunit of transcarboxylase: an NMR study. Biochemistry. 1997 Dec 2;36(48):14676–14682. doi: 10.1021/bi971674y. [DOI] [PubMed] [Google Scholar]
  16. Samols D., Thornton C. G., Murtif V. L., Kumar G. K., Haase F. C., Wood H. G. Evolutionary conservation among biotin enzymes. J Biol Chem. 1988 May 15;263(14):6461–6464. [PubMed] [Google Scholar]
  17. Stone M. J., Chandrasekhar K., Holmgren A., Wright P. E., Dyson H. J. Comparison of backbone and tryptophan side-chain dynamics of reduced and oxidized Escherichia coli thioredoxin using 15N NMR relaxation measurements. Biochemistry. 1993 Jan 19;32(2):426–435. doi: 10.1021/bi00053a007. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Yao X., Wei D., Soden C., Jr, Summers M. F., Beckett D. Structure of the carboxy-terminal fragment of the apo-biotin carboxyl carrier subunit of Escherichia coli acetyl-CoA carboxylase. Biochemistry. 1997 Dec 9;36(49):15089–15100. doi: 10.1021/bi971485f. [DOI] [PubMed] [Google Scholar]
  20. Zhang O., Kay L. E., Olivier J. P., Forman-Kay J. D. Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques. J Biomol NMR. 1994 Nov;4(6):845–858. doi: 10.1007/BF00398413. [DOI] [PubMed] [Google Scholar]

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

RESOURCES