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 Jun;6(6):1237–1247. doi: 10.1002/pro.5560060612

Secondary and tertiary structural changes in gamma delta resolvase: comparison of the wild-type enzyme, the I110R mutant, and the C-terminal DNA binding domain in solution.

B Pan 1, Z Deng 1, D Liu 1, S Ghosh 1, G P Mullen 1
PMCID: PMC2143726  PMID: 9194184

Abstract

gamma delta Resolvase is a site-specific DNA recombinase (M(r) 20.5 kDa) in Escherichia coli that shares homology with a family of bacterial resolvases and invertases. We have characterized the secondary and tertiary structural behavior of the cloned DNA binding domain (DBD) and a dimerization defective mutant in solution. Low-salt conditions were found to destabilize the tertiary structure of the DBD dramatically, with concomitant changes in the secondary structure that were localized near the hinge regions between the helices. The molten tertiary fold appears to contribute significantly to productive DNA interactions and supports a mechanism of DNA-induced folding of the tertiary structure, a process that enables the DBD to adapt in conformation for each of the three imperfect palindromic sites. At high salt concentrations, the monomeric I110R resolvase shows a minimal perturbation to the three helices of the DBD structure and changes in the linker segment in comparison to the cloned DBD containing the linker. Comparative analysis of the NMR spectra suggest that the I110R mutant contains a folded catalytic core of approximately 60 residues and that the segment from residues 100 to 149 are devoid of regular structure in the I110R resolvase. No increase in the helicity of the linker region of I110R resolvase occurs on binding DNA. These results support a subunit rotation model of strand exchange that involves the partial unfolding of the catalytic domains.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

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

  1. Abdel-Meguid S. S., Grindley N. D., Templeton N. S., Steitz T. A. Cleavage of the site-specific recombination protein gamma delta resolvase: the smaller of two fragments binds DNA specifically. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2001–2005. doi: 10.1073/pnas.81.7.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blake D. G., Boocock M. R., Sherratt D. J., Stark W. M. Cooperative binding of Tn3 resolvase monomers to a functionally asymmetric binding site. Curr Biol. 1995 Sep 1;5(9):1036–1046. doi: 10.1016/s0960-9822(95)00208-9. [DOI] [PubMed] [Google Scholar]
  3. Boocock M. R., Zhu X., Grindley N. D. Catalytic residues of gamma delta resolvase act in cis. EMBO J. 1995 Oct 16;14(20):5129–5140. doi: 10.1002/j.1460-2075.1995.tb00195.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Hatfull G. F., Sanderson M. R., Freemont P. S., Raccuia P. R., Grindley N. D., Steitz T. A. Preparation of heavy-atom derivatives using site-directed mutagenesis. Introduction of cysteine residues into gamma delta resolvase. J Mol Biol. 1989 Aug 20;208(4):661–667. doi: 10.1016/0022-2836(89)90156-3. [DOI] [PubMed] [Google Scholar]
  6. Hughes R. E., Rice P. A., Steitz T. A., Grindley N. D. Protein-protein interactions directing resolvase site-specific recombination: a structure-function analysis. EMBO J. 1993 Apr;12(4):1447–1458. doi: 10.1002/j.1460-2075.1993.tb05788.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Rice P. A., Steitz T. A. Model for a DNA-mediated synaptic complex suggested by crystal packing of gamma delta resolvase subunits. EMBO J. 1994 Apr 1;13(7):1514–1524. doi: 10.2210/pdb1gdr/pdb. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Sanderson M. R., Freemont P. S., Rice P. A., Goldman A., Hatfull G. F., Grindley N. D., Steitz T. A. The crystal structure of the catalytic domain of the site-specific recombination enzyme gamma delta resolvase at 2.7 A resolution. Cell. 1990 Dec 21;63(6):1323–1329. doi: 10.1016/0092-8674(90)90427-g. [DOI] [PubMed] [Google Scholar]
  9. Stark W. M., Grindley N. D., Hatfull G. F., Boocock M. R. Resolvase-catalysed reactions between res sites differing in the central dinucleotide of subsite I. EMBO J. 1991 Nov;10(11):3541–3548. doi: 10.1002/j.1460-2075.1991.tb04918.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Stark W. M., Sherratt D. J., Boocock M. R. Site-specific recombination by Tn3 resolvase: topological changes in the forward and reverse reactions. Cell. 1989 Aug 25;58(4):779–790. doi: 10.1016/0092-8674(89)90111-6. [DOI] [PubMed] [Google Scholar]
  11. Watson M. A., Boocock M. R., Stark W. M. Rate and selectively of synapsis of res recombination sites by Tn3 resolvase. J Mol Biol. 1996 Mar 29;257(2):317–329. doi: 10.1006/jmbi.1996.0165. [DOI] [PubMed] [Google Scholar]
  12. Weber D. J., Gittis A. G., Mullen G. P., Abeygunawardana C., Lattman E. E., Mildvan A. S. NMR docking of a substrate into the X-ray structure of staphylococcal nuclease. Proteins. 1992 Aug;13(4):275–287. doi: 10.1002/prot.340130402. [DOI] [PubMed] [Google Scholar]
  13. Wishart D. S., Sykes B. D. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J Biomol NMR. 1994 Mar;4(2):171–180. doi: 10.1007/BF00175245. [DOI] [PubMed] [Google Scholar]
  14. Yang W., Steitz T. A. Crystal structure of the site-specific recombinase gamma delta resolvase complexed with a 34 bp cleavage site. Cell. 1995 Jul 28;82(2):193–207. doi: 10.1016/0092-8674(95)90307-0. [DOI] [PubMed] [Google Scholar]
  15. 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