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. 1996 Nov;5(11):2175–2183. doi: 10.1002/pro.5560051104

A missing link in cupredoxins: crystal structure of cucumber stellacyanin at 1.6 A resolution.

P J Hart 1, A M Nersissian 1, R G Herrmann 1, R M Nalbandyan 1, J S Valentine 1, D Eisenberg 1
PMCID: PMC2143285  PMID: 8931136

Abstract

Stellacyanins are blue (type I) copper glycoproteins that differ from other members of the cupredoxin family in their spectroscopic and electron transfer properties. Until now, stellacyanins have eluded structure determination. Here we report the three-dimensional crystal structure of the 109 amino acid, non-glycosylated copper binding domain of recombinant cucumber stellacyanin refined to 1.6 A resolution. The crystallographic R-value for all 18,488 reflections (sigma > 0) between 50-1.6 A is 0.195. The overall fold is organized in two beta-sheets, both with four beta-stands. Two alpha-helices are found in loop regions between beta-strands. The beta-sheets form a beta-sandwich similar to those found in other cupredoxins, but some features differ from proteins such as plastocyanin and azurin in that the beta-barrel is more flattened, there is an extra N-terminal alpha-helix, and the copper binding site is much more solvent accessible. The presence of a disulfide bond at the copper binding end of the protein confirms that cucumber stellacyanin has a phytocyanin-like fold. The ligands to copper are two histidines, one cysteine, and one glutamine, the latter replacing the methionine typically found in mononuclear blue copper proteins. The Cu-Gln bond is one of the shortest axial ligand bond distances observed to date in structurally characterized type I copper proteins. The characteristic spectroscopic properties and electron transfer reactivity of stellacyanin, which differ significantly from those of other well-characterized cupredoxins, can be explained by its more exposed copper site, its distinctive amino acid ligand composition, and its nearly tetrahedral ligand geometry. Surface features on the cucumber stellacyanin molecule that could be involved in interactions with putative redox partners are discussed.

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Selected References

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  1. Adman E. T. Copper protein structures. Adv Protein Chem. 1991;42:145–197. doi: 10.1016/s0065-3233(08)60536-7. [DOI] [PubMed] [Google Scholar]
  2. Baker E. N. Structure of azurin from Alcaligenes denitrificans refinement at 1.8 A resolution and comparison of the two crystallographically independent molecules. J Mol Biol. 1988 Oct 20;203(4):1071–1095. doi: 10.1016/0022-2836(88)90129-5. [DOI] [PubMed] [Google Scholar]
  3. Cho K. C., Blair D. F., Banerjee U., Hopfield J. J., Gray H. B., Pecht I., Chan S. I. NMR spectroscopic identification of a hexacyanochromate(III) binding site on Pseudomonas azurin. Biochemistry. 1984 Apr 10;23(8):1858–1862. doi: 10.1021/bi00303a042. [DOI] [PubMed] [Google Scholar]
  4. Durley R., Chen L., Lim L. W., Mathews F. S., Davidson V. L. Crystal structure analysis of amicyanin and apoamicyanin from Paracoccus denitrificans at 2.0 A and 1.8 A resolution. Protein Sci. 1993 May;2(5):739–752. doi: 10.1002/pro.5560020506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Evans S. V. SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. J Mol Graph. 1993 Jun;11(2):134-8, 127-8. doi: 10.1016/0263-7855(93)87009-t. [DOI] [PubMed] [Google Scholar]
  6. Farver O., Pecht I. Identification of an electron transfer locus in plastocyanin by chromium(II) affinity labeling. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4190–4193. doi: 10.1073/pnas.78.7.4190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Farver O., Shahak Y., Pecht I. Electron uptake and delivery sites on plastocyanin in its reactions with the photosynthetic electron transport system. Biochemistry. 1982 Apr 13;21(8):1885–1890. doi: 10.1021/bi00537a028. [DOI] [PubMed] [Google Scholar]
  8. Fields B. A., Guss J. M., Freeman H. C. Three-dimensional model for stellacyanin, a "blue" copper-protein. J Mol Biol. 1991 Dec 20;222(4):1053–1065. doi: 10.1016/0022-2836(91)90593-u. [DOI] [PubMed] [Google Scholar]
  9. Guss J. M., Freeman H. C. Structure of oxidized poplar plastocyanin at 1.6 A resolution. J Mol Biol. 1983 Sep 15;169(2):521–563. doi: 10.1016/s0022-2836(83)80064-3. [DOI] [PubMed] [Google Scholar]
  10. Haehnel W., Jansen T., Gause K., Klösgen R. B., Stahl B., Michl D., Huvermann B., Karas M., Herrmann R. G. Electron transfer from plastocyanin to photosystem I. EMBO J. 1994 Mar 1;13(5):1028–1038. doi: 10.1002/j.1460-2075.1994.tb06351.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Holwerda R. A., Wherland S., Gray H. B. Electron transfer reactions of copper proteins. Annu Rev Biophys Bioeng. 1976;5:363–396. doi: 10.1146/annurev.bb.05.060176.002051. [DOI] [PubMed] [Google Scholar]
  12. Ikeda O., Sakurai T. Electron transfer reaction of stellacyanin at a bare glassy carbon electrode. Eur J Biochem. 1994 Feb 1;219(3):813–819. doi: 10.1111/j.1432-1033.1994.tb18562.x. [DOI] [PubMed] [Google Scholar]
  13. Malmström B. G. Rack-induced bonding in blue-copper proteins. Eur J Biochem. 1994 Aug 1;223(3):711–718. doi: 10.1111/j.1432-1033.1994.tb19044.x. [DOI] [PubMed] [Google Scholar]
  14. Mims W. B., Davis J. L., Peisach J. The accessibility of type I Cu(II) centers in laccase, azurin, and stellacyanin to exchangeable hydrogen and ambient water. Biophys J. 1984 Apr;45(4):755–766. doi: 10.1016/S0006-3495(84)84219-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nersissian A. M., Mehrabian Z. B., Nalbandyan R. M., Hart P. J., Fraczkiewicz G., Czernuszewicz R. S., Bender C. J., Peisach J., Herrmann R. G., Valentine J. S. Cloning, expression, and spectroscopic characterization of Cucumis sativus stellacyanin in its nonglycosylated form. Protein Sci. 1996 Nov;5(11):2184–2192. doi: 10.1002/pro.5560051105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nersissian A. M., Nalbandyan R. M. Particles of photosystem 2 contain plantacyanin. Biochem Biophys Res Commun. 1990 Apr 16;168(1):232–239. doi: 10.1016/0006-291x(90)91698-r. [DOI] [PubMed] [Google Scholar]
  17. Obokata J., Mikami K., Hayashida N., Nakamura M., Sugiura M. Molecular heterogeneity of photosystem I. psaD, psaE, psaF, psaH, and psaL are all present in isoforms in Nicotiana spp. Plant Physiol. 1993 Aug;102(4):1259–1267. doi: 10.1104/pp.102.4.1259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Peisach J., Levine W. G., Blumberg W. E. Structural properties of stellacyanin, a copper mucoprotein from Rhus vernicifera, the Japanese lac tree. J Biol Chem. 1967 Jun 25;242(12):2847–2858. [PubMed] [Google Scholar]
  19. Peisach J., Powers L., Blumberg W. E., Chance B. Stellacyanin. Studies of the metal-binding site using x-ray absorption spectroscopy. Biophys J. 1982 Jun;38(3):277–285. doi: 10.1016/S0006-3495(82)84559-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Petratos K., Dauter Z., Wilson K. S. Refinement of the structure of pseudoazurin from Alcaligenes faecalis S-6 at 1.55 A resolution. Acta Crystallogr B. 1988 Dec 1;44(Pt 6):628–636. [PubMed] [Google Scholar]
  21. Ramachandran G. N., Sasisekharan V. Conformation of polypeptides and proteins. Adv Protein Chem. 1968;23:283–438. doi: 10.1016/s0065-3233(08)60402-7. [DOI] [PubMed] [Google Scholar]
  22. Romero A., Hoitink C. W., Nar H., Huber R., Messerschmidt A., Canters G. W. X-ray analysis and spectroscopic characterization of M121Q azurin. A copper site model for stellacyanin. J Mol Biol. 1993 Feb 20;229(4):1007–1021. doi: 10.1006/jmbi.1993.1101. [DOI] [PubMed] [Google Scholar]
  23. Satow Y., Cohen G. H., Padlan E. A., Davies D. R. Phosphocholine binding immunoglobulin Fab McPC603. An X-ray diffraction study at 2.7 A. J Mol Biol. 1986 Aug 20;190(4):593–604. doi: 10.1016/0022-2836(86)90245-7. [DOI] [PubMed] [Google Scholar]
  24. Tollin G., Meyer T. E., Cheddar G., Getzoff E. D., Cusanovich M. A. Transient kinetics of reduction of blue copper proteins by free flavin and flavodoxin semiquinones. Biochemistry. 1986 Jun 3;25(11):3363–3370. doi: 10.1021/bi00359a041. [DOI] [PubMed] [Google Scholar]

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