Abstract
Although a significant number of proteins include bound metals as part of their structure, the identification of amino acid residues coordinated to non-paramagnetic metals by NMR remains a challenge. Metal ligands can stabilize the native structure and/or play critical catalytic roles in the underlying biochemistry. An atom’s chemical shift is exquisitely sensitive to its electronic environment. Chemical shift data can provide valuable insights into structural features, including metal ligation. In this study, we demonstrate that overlapped 13Cβ chemical shift distributions of Zn-ligated and non-metal-ligated cysteine residues are largely resolved by the inclusion of the corresponding 13Cα chemical shift information, together with secondary structural information. We demonstrate this with a bivariate distribution plot, and statistically with a multivariate analysis of variance (MANOVA) and hierarchical logistic regression analysis. Using 287 13Cα/13Cβ shift pairs from 79 proteins with known three-dimensional structures, including 86 13Cα and13Cβ shifts for 43 Zn-ligated cysteine residues, along with corresponding oxidation state and secondary structure information, we have built a logistic regression model that distinguishes between oxidized cystines, reduced (non-metal ligated) cysteines, and Zn-ligated cysteines. Classifying cysteines/cystines with a statisical model incorporating all three phenomena resulted in a predictor of Zn ligation with a recall, precision and F-measure of 83.7%, and an accuracy of 95.1%. This model was applied in the analysis of Bacillus subtilis IscU, a protein involved in iron–sulfur cluster assembly. The model predicts that all three cysteines of IscU are metal ligands. We confirmed these results by (i) examining the effect of metal chelation on the NMR spectrum of IscU, and (ii) inductively coupled plasma mass spectrometry analysis. To gain further insight into the frequency of occurrence of non-cysteine Zn ligands, we analyzed the Protein Data Bank and found that 78% of the Zn ligands are histidine and cysteine (with nearly identical frequencies), and 18% are acidic residues aspartate and glutamate.
Similar content being viewed by others
References
T.B. Acton K.C. Gunsalus R. Xiao L.C. Ma J. Aramini M.C. Baran Y.W. Chiang T. Climent B. Cooper N.G. Denissova S.M. Douglas J.K. Everett C.K. Ho D. Macapagal P.K. Rajan R. Shastry L.Y. Shih G.V.T. Swapna M. Wilson M. Wu M. Gerstein M. Inouye J.F. Hunt G.T. Montelione (2005) Meth. Enzymol. 394 210–243 Occurrence Handle10.1016/S0076-6879(05)94008-1
J.N. Agar C. Krebs J. Frazzon B.H. Huynh D.R. Dean M.K. Johnson (2000) Biochemistry 39 7856–7862 Occurrence Handle10.1021/bi000931n
A. Bateman L. Coin R. Durbin R.D. Finn V. Hollich S. Griffiths-Jones A. Khanna M. Marshall S. Moxon E.L.L. Sonnhammer D.J. Studholme C. Yeats S.R. Eddy (2004) Nucleic Acids Res. 32 138–141 Occurrence Handle10.1093/nar/gkh121
A. Becker I. Schlichting W. Kabsch D. Groche S. Schultz A.F. Wagner (1998) Nat. Struct. Biol. 5 1053–1058 Occurrence Handle10.1038/4162
J.M. Berg Y. Shi (1996) Science 271 1081–1085 Occurrence Handle1996Sci...271.1081B
M. Clamp D. Andrews D. Barker P. Bevan G. Cameron Y. Chen L. Clark T. Cox J. Cuff V. Curwen T. Down R. Durbin E. Eyras J. Gilbert M. Hammond T. Hubbard A. Kasprzyk D. Keefe H. Lehvaslaiho V. Iyer C. Melsopp E. Mongin R. Pettett S. Potter A. Rust E. Schmidt S. Searle G. Slater J. Smith W. Spooner A. Stabenau J. Stalker E. Stupka A. Ureta-Vidal I. Vastrik E. Birney (2003) Nucleic Acids Res. 31 38–42 Occurrence Handle10.1093/nar/gkg083
N.D. Clarke J.M. Berg (1998) Science 282 2018–2022 Occurrence Handle10.1126/science.282.5396.2018 Occurrence Handle1998Sci...282.2018C
T.B. Coplen J.K. Bohlke P. De Bievre T. Ding N.E. Holden J.A. Hopple H.R. Krouse A. Lamberty H.S. Peiser K. Revesz S.E. Rieder K.J.R. Rosman E. Roth P.D.P. Taylor R.D. Vocke Y.K. Xiao (2002) Pure Appl. Chem. 74 1987–2017
Z. Dauter K.S. Wilson L.C. Sieker J.M. Moulis J. Meyer (1996) Proc. Natl. Acad. Sci. USA 93 8836–8840 Occurrence Handle10.1073/pnas.93.17.8836 Occurrence Handle1996PNAS...93.8836D
A.C. Drohat K. Kwon D.J. Krosky J.T. Stivers (2002) Nat. Struct. Biol. 9 659–664 Occurrence Handle10.1038/nsb829
B.S. Everitt G. Dunn (2001) Applied Multivariate Data Analysis. Arnold London
T. Fujii Y. Hata M. Oozeki H. Moriyama T. Wakagi N. Tanaka T. Oshima (1997) Biochemistry 36 1505–1513 Occurrence Handle10.1021/bi961966j
J.P. Glusker A.K. Katz C.W. Bock (1999) Rigaku 16 8–16
M. Hernick C.A. Fierke (2005) Arch. Biochem. Biophys. 433 71–84 Occurrence Handle10.1016/j.abb.2004.08.006
M. Jansson Y.C. Li L. Jendeberg S. Anderson B.T. Montelione B. Nilsson (1996) J. Biomol. NMR 7 131–141 Occurrence Handle10.1007/BF00203823
W. Kabsch C. Sander (1983) Biopolymers 22 2577–2637 Occurrence Handle10.1002/bip.360221211
Klug, A. and Rhodes, D. (1987) Zinc fingers: a novel protein fold for nucleic acid recognition. Cold Spring Harb. Symp. Quant. Biol 52, 473–482
R. Koradi M. Billeter K. Wuthrich (1996) J. Mol. Graph. 14 29–32
S.S. Krishna I. Majumdar N.V. Grishin (2003) Nucleic Acids Res. 31 532–550 Occurrence Handle10.1093/nar/gkg161
K. Kwon C. Cao J.T. Stivers (2003) J. Biol. Chem. 278 19442–19446 Occurrence Handle10.1074/jbc.M300934200
W.N. Lipscomb N. Strater (1996) Chem. Rev. 96 2375–2433 Occurrence Handle10.1021/cr950042j
J. Liu N. Oganesyan D.H. Shin J. Jancarik H. Yokota R. Kim S.H. Kim (2005) Proteins 59 875–881 Occurrence Handle10.1002/prot.20421
D. Lu M.A. Searles A. Klug (2003) Nature 426 96–100 Occurrence Handle10.1038/nature02088 Occurrence Handle2003Natur.426...96L
R.G. Miller (1997) Beyond ANOVA: Basics of Applied Statistics Chapman & Hall Boca Raton, FL Occurrence Handle0885.62081
H.N. Moseley G. Sahota G.T. Montelione (2004) J. Biomol. NMR 28 341–355 Occurrence Handle10.1023/B:JNMR.0000015420.44364.06
A.G. Murzin S.E. Brenner T. Hubbard C. Chothia (1995) J. Mol. Biol. 247 536–540 Occurrence Handle10.1006/jmbi.1995.0159
D. Neuhaus G. Wagner M. Vasak J.H. Kagi K. Wuthrich (1984) Eur. J. Biochem. 143 659–667 Occurrence Handle10.1111/j.1432-1033.1984.tb08419.x
J.G. Pelton D.A. Torchia N.D. Meadow S. Roseman (1993) Protein Sci. 2 543–558 Occurrence Handle10.1002/pro.5560020406
T.A. Ramelot J.R. Cort S. Goldsmith-Fischman G.J. Kornhaber R. Xiao R. Shastry T.B. Acton B. Honig G.T. Montelione M.A. Kennedy (2004) J. Mol. Biol. 344 567–583 Occurrence Handle10.1016/j.jmb.2004.08.038
P.A. Rea (2003) Nat. Biotechnol. 21 1149–1151 Occurrence Handle10.1038/nbt1003-1149
D. Sharma K. Rajarathnam (2000) J. Biomol. NMR 18 165–171 Occurrence Handle10.1023/A:1008398416292
J.R. Taylor (1997) An introduction to Error Analysis: The Study of Uncertainties in Physical Measurements University Science Books Sausalito, CA
M. Vasak E. Worgotter G. Wagner J.H. Kagi K. Wuthrich (1987) J. Mol. Biol. 196 711–719 Occurrence Handle10.1016/0022-2836(87)90042-8
J.C. Venter M.D. Adams E.W. Myers P.W. Li R.J. Mural G.G. Sutton H.O. Smith M. Yandell C.A. Evans R.A. Holt J.D. Gocayne P. Amanatides R.M. Ballew D.H. Huson J.R. Wortman Q. Zhang C.D. Kodira X.Q.H. Zheng L. Chen M. Skupski et al. (2001) Science 291 1304–1351 Occurrence Handle10.1126/science.1058040 Occurrence Handle2001Sci...291.1304V
H.Y. Zhang S. Neal D.S. Wishart (2003) J. Biomol. NMR 25 173–195 Occurrence Handle10.1023/A:1022836027055
L. Zheng V.L. Cash D.H. Flint D.R. Dean (1998) J. Biol. Chem. 273 13264–13272 Occurrence Handle10.1074/jbc.273.21.13264
Z.S. Zhou K. Peariso J.E. Penner-Hahn R.G. Matthews (1999) Biochemistry 38 15915–15926 Occurrence Handle10.1021/bi992062b
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Kornhaber, G.J., Snyder, D., Moseley, H.N.B. et al. Identification of Zinc-ligated Cysteine Residues Based on 13Cα and 13Cβ Chemical Shift Data. J Biomol NMR 34, 259–269 (2006). https://doi.org/10.1007/s10858-006-0027-5
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/s10858-006-0027-5