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
. 1997 Jan;6(1):13–23. doi: 10.1002/pro.5560060103

Structure of the beta 2 homodimer of bacterial luciferase from Vibrio harveyi: X-ray analysis of a kinetic protein folding trap.

J B Thoden 1, H M Holden 1, A J Fisher 1, J F Sinclair 1, G Wesenberg 1, T O Baldwin 1, I Rayment 1
PMCID: PMC2143504  PMID: 9007973

Abstract

Luciferase, as isolated from Vibrio harveyi, is an alpha beta heterodimer. When allowed to fold in the absence of the alpha subunit, either in vitro or in vivo, the beta subunit of enzyme will form a kinetically stable homodimer that does not unfold even after prolonged incubation in 5 M urea at pH 7.0 and 18 degrees C. This form of the beta subunit, arising via kinetic partitioning on the folding pathway, appears to constitute a kinetically trapped alternative to the heterodimeric enzyme (Sinclair JF, Ziegler MM, Baldwin TO. 1994. Kinetic partitioning during protein folding yields multiple native states. Nature Struct Biol 1: 320-326). Here we describe the X-ray crystal structure of the beta 2 homodimer of luciferase from V. harveyi determined and refined at 1.95 A resolution. Crystals employed in the investigational belonged to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 58.8 A, b = 62.0 A, and c = 218.2 A and contained one dimer per asymmetric unit. Like that observed in the functional luciferase alpha beta heterodimer, the major tertiary structural motif of each beta subunit consists of an (alpha/beta)8 barrel (Fisher AJ, Raushel FM, Baldwin TO, Rayment I. 1995. Three-dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 A resolution. Biochemistry 34: 6581-6586). The root-mean-square deviation of the alpha-carbon coordinates between the beta subunits of the hetero- and homodimers is 0.7 A. This high resolution X-ray analysis demonstrated that "domain" or "loop" swapping has not occurred upon formation of the beta 2 homodimer and thus the stability of the beta 2 species to denaturation cannot be explained in such simple terms. In fact, the subunit:subunit interfaces observed in both the beta 2 homodimer and alpha beta heterodimer are remarkably similar in hydrogen-bonding patterns and buried surface areas.

Full Text

The Full Text of this article is available as a PDF (8.5 MB).

Selected References

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

  1. Anfinsen C. B. Principles that govern the folding of protein chains. Science. 1973 Jul 20;181(4096):223–230. doi: 10.1126/science.181.4096.223. [DOI] [PubMed] [Google Scholar]
  2. Baker D., Agard D. A. Kinetics versus thermodynamics in protein folding. Biochemistry. 1994 Jun 21;33(24):7505–7509. doi: 10.1021/bi00190a002. [DOI] [PubMed] [Google Scholar]
  3. Baker D., Sohl J. L., Agard D. A. A protein-folding reaction under kinetic control. Nature. 1992 Mar 19;356(6366):263–265. doi: 10.1038/356263a0. [DOI] [PubMed] [Google Scholar]
  4. Baldwin T. O., Berends T., Bunch T. A., Holzman T. F., Rausch S. K., Shamansky L., Treat M. L., Ziegler M. M. Cloning of the luciferase structural genes from Vibrio harveyi and expression of bioluminescence in Escherichia coli. Biochemistry. 1984 Jul 31;23(16):3663–3667. doi: 10.1021/bi00311a014. [DOI] [PubMed] [Google Scholar]
  5. Baldwin T. O., Devine J. H., Heckel R. C., Lin J. W., Shadel G. S. The complete nucleotide sequence of the lux regulon of Vibrio fischeri and the luxABN region of Photobacterium leiognathi and the mechanism of control of bacterial bioluminescence. J Biolumin Chemilumin. 1989 Jul;4(1):326–341. doi: 10.1002/bio.1170040145. [DOI] [PubMed] [Google Scholar]
  6. Baldwin T. O., Ziegler M. M., Chaffotte A. F., Goldberg M. E. Contribution of folding steps involving the individual subunits of bacterial luciferase to the assembly of the active heterodimeric enzyme. J Biol Chem. 1993 May 25;268(15):10766–10772. [PubMed] [Google Scholar]
  7. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  8. Choi H., Tang C. K., Tu S. C. Catalytically active forms of the individual subunits of Vibrio harveyi luciferase and their kinetic and binding properties. J Biol Chem. 1995 Jul 14;270(28):16813–16819. doi: 10.1074/jbc.270.28.16813. [DOI] [PubMed] [Google Scholar]
  9. Clark A. C., Sinclair J. F., Baldwin T. O. Folding of bacterial luciferase involves a non-native heterodimeric intermediate in equilibrium with the native enzyme and the unfolded subunits. J Biol Chem. 1993 May 25;268(15):10773–10779. [PubMed] [Google Scholar]
  10. Cohn D. H., Mileham A. J., Simon M. I., Nealson K. H., Rausch S. K., Bonam D., Baldwin T. O. Nucleotide sequence of the luxA gene of Vibrio harveyi and the complete amino acid sequence of the alpha subunit of bacterial luciferase. J Biol Chem. 1985 May 25;260(10):6139–6146. [PubMed] [Google Scholar]
  11. Farber G. K., Petsko G. A. The evolution of alpha/beta barrel enzymes. Trends Biochem Sci. 1990 Jun;15(6):228–234. doi: 10.1016/0968-0004(90)90035-a. [DOI] [PubMed] [Google Scholar]
  12. Fedorov A. N., Baldwin T. O. Contribution of cotranslational folding to the rate of formation of native protein structure. Proc Natl Acad Sci U S A. 1995 Feb 14;92(4):1227–1231. doi: 10.1073/pnas.92.4.1227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fisher A. J., Thompson T. B., Thoden J. B., Baldwin T. O., Rayment I. The 1.5-A resolution crystal structure of bacterial luciferase in low salt conditions. J Biol Chem. 1996 Sep 6;271(36):21956–21968. doi: 10.1074/jbc.271.36.21956. [DOI] [PubMed] [Google Scholar]
  14. Friedland J., Hastings J. W. Nonidentical subunits of bacterial luciferase: their isolation and recombination to form active enzyme. Proc Natl Acad Sci U S A. 1967 Dec;58(6):2336–2342. doi: 10.1073/pnas.58.6.2336. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gething M. J., McCammon K., Sambrook J. Expression of wild-type and mutant forms of influenza hemagglutinin: the role of folding in intracellular transport. Cell. 1986 Sep 12;46(6):939–950. doi: 10.1016/0092-8674(86)90076-0. [DOI] [PubMed] [Google Scholar]
  16. Johnston T. C., Thompson R. B., Baldwin T. O. Nucleotide sequence of the luxB gene of Vibrio harveyi and the complete amino acid sequence of the beta subunit of bacterial luciferase. J Biol Chem. 1986 Apr 15;261(11):4805–4811. [PubMed] [Google Scholar]
  17. Jones T. A. Diffraction methods for biological macromolecules. Interactive computer graphics: FRODO. Methods Enzymol. 1985;115:157–171. doi: 10.1016/0076-6879(85)15014-7. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Li Z., Szittner R., Meighen E. A. Subunit interactions and the role of the luxA polypeptide in controlling thermal stability and catalytic properties in recombinant luciferase hybrids. Biochim Biophys Acta. 1993 Oct 3;1158(2):137–145. doi: 10.1016/0304-4165(93)90007-u. [DOI] [PubMed] [Google Scholar]
  20. Mottonen J., Strand A., Symersky J., Sweet R. M., Danley D. E., Geoghegan K. F., Gerard R. D., Goldsmith E. J. Structural basis of latency in plasminogen activator inhibitor-1. Nature. 1992 Jan 16;355(6357):270–273. doi: 10.1038/355270a0. [DOI] [PubMed] [Google Scholar]
  21. Navaza J. On the computation of the fast rotation function. Acta Crystallogr D Biol Crystallogr. 1993 Nov 1;49(Pt 6):588–591. doi: 10.1107/S0907444993005141. [DOI] [PubMed] [Google Scholar]
  22. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  23. Ramakrishnan C., Ramachandran G. N. Stereochemical criteria for polypeptide and protein chain conformations. II. Allowed conformations for a pair of peptide units. Biophys J. 1965 Nov;5(6):909–933. doi: 10.1016/S0006-3495(65)86759-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Rodgers D. W. Cryocrystallography. Structure. 1994 Dec 15;2(12):1135–1140. doi: 10.1016/s0969-2126(94)00116-2. [DOI] [PubMed] [Google Scholar]
  25. Sinclair J. F., Waddle J. J., Waddill E. F., Baldwin T. O. Purified native subunits of bacterial luciferase are active in the bioluminescence reaction but fail to assemble into the alpha beta structure. Biochemistry. 1993 May 18;32(19):5036–5044. doi: 10.1021/bi00070a010. [DOI] [PubMed] [Google Scholar]
  26. Sinclair J. F., Ziegler M. M., Baldwin T. O. Kinetic partitioning during protein folding yields multiple native states. Nat Struct Biol. 1994 May;1(5):320–326. doi: 10.1038/nsb0594-320. [DOI] [PubMed] [Google Scholar]
  27. Waddle J. J., Johnston T. C., Baldwin T. O. Polypeptide folding and dimerization in bacterial luciferase occur by a concerted mechanism in vivo. Biochemistry. 1987 Aug 11;26(16):4917–4921. doi: 10.1021/bi00390a004. [DOI] [PubMed] [Google Scholar]
  28. Waddle J., Baldwin T. O. Individual alpha and beta subunits of bacterial luciferase exhibit bioluminescence activity. Biochem Biophys Res Commun. 1991 Aug 15;178(3):1188–1193. doi: 10.1016/0006-291x(91)91018-8. [DOI] [PubMed] [Google Scholar]
  29. Ziegler M. M., Goldberg M. E., Chaffotte A. F., Baldwin T. O. Refolding of luciferase subunits from urea and assembly of the active heterodimer. Evidence for folding intermediates that precede and follow the dimerization step on the pathway to the active form of the enzyme. J Biol Chem. 1993 May 25;268(15):10760–10765. [PubMed] [Google Scholar]

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

RESOURCES