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Characterizing metal-binding sites in proteins with X-ray crystallography

Abstract

Metals have crucial roles in many physiological, pathological, toxicological, pharmaceutical, and diagnostic processes. Proper handling of metal-containing macromolecule samples for structural studies is not trivial, and failure to handle them properly is often a source of irreproducibility caused by issues such as pH changes, incorporation of unexpected metals, or oxidization/reduction of the metal. This protocol outlines the guidelines and best practices for characterizing metal-binding sites in protein structures and alerts experimenters to potential pitfalls during the preparation and handling of metal-containing protein samples for X-ray crystallography studies. The protocol features strategies for controlling the sample pH and the metal oxidation state, recording X-ray fluorescence (XRF) spectra, and collecting diffraction data sets above and below the corresponding metal absorption edges. This protocol should allow experimenters to gather sufficient evidence to unambiguously determine the identity and location of the metal of interest, as well as to accurately characterize the coordinating ligands in the metal binding environment within the protein. Meticulous handling of metal-containing macromolecule samples as described in this protocol should enhance experimental reproducibility in biomedical sciences, especially in X-ray macromolecular crystallography. For most samples, the protocol can be completed within a period of 7–190 d, most of which (2–180 d) is devoted to growing the crystal. The protocol should be readily understandable to structural biologists, particularly protein crystallographers with an intermediate level of experience.

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Figure 1: Overview of the protocol depicted in a flowchart.
Figure 2: Representative structures of ESA–Zn2+ complexes showing the dynamic behavior of His247.
Figure 3: The influence of transition metals on the stability of STM1931 protein from S. Typhimuruim, analyzed through TSA experiments.
Figure 4: Fluorescence spectra, fluorescence scan, and electron density maps of a zinc-containing protein dihydroorotase from Y. pestis CO92.
Figure 5: Use of restraints for a zinc-binding site in ESA.
Figure 6

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References

  1. Rötzschke, O., Lau, J.M., Hofstätter, M., Falk, K. & Strominger, J.L. A pH-sensitive histidine residue as control element for ligand release from HLA-DR molecules. Proc. Natl. Acad. Sci. USA 99, 16946–16950 (2002).

    PubMed  PubMed Central  Google Scholar 

  2. Holm, R.H., Kennepohl, P. & Solomon, E.I. Structural and functional aspects of metal sites in biology. Chem. Rev. 96, 2239–2314 (1996).

    CAS  PubMed  Google Scholar 

  3. Pyle, A.M. Metal ions in the structure and function of RNA. J. Biol. Inorg. Chem. 7, 679–690 (2002).

    CAS  PubMed  Google Scholar 

  4. Potter, J.D., Sheng, Z., Pan, B. & Zhao, J. A structural role for the Ca2+-Mg2+ sites on troponin regulation of muscle contraction. J. Biol. Chem. 270, 2557–2562 (1995).

    CAS  PubMed  Google Scholar 

  5. Pace, N. & Weerapana, E. Zinc-binding cysteines: diverse functions and structural motifs. Biomolecules 4, 419–434 (2014).

    PubMed  PubMed Central  Google Scholar 

  6. McCall, K.A., Huang, C. & Fierke, C.A. Function and mechanism of zinc metalloenzymes. J. Nutr. 130, 1455–1458 (2000).

    Google Scholar 

  7. Solomon, E.I. et al. Copper active sites in biology. Chem. Rev. 114, 3659–3853 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. MacPherson, I.S. & Murphy, M.E.P. Type-2 copper-containing enzymes. Cell. Mol. Life Sci. 64, 2887–2899 (2007).

    CAS  PubMed  Google Scholar 

  9. Mccall, K.A., Huang, C.-C. & Fierke, C.A. Zinc and health: current status and future directions function and mechanism of zinc metalloenzymes 1. J. Nutr. 130, 1437–1446 (2000).

    Google Scholar 

  10. Lyons, T. & Eide, D. Transport and storage of metal ions in biology. in Biological Inorganic Chemistry: Structure and Reactivity (eds. Bertini, I. et al.) 57–78 (Univ. Sci. Books, 2007).

  11. Farrell, N. Metal complexes as drugs and chemotherapeutic agents. in Comprehensive Coordination Chemistry II (eds. McCleverty, J.A. & Meyer. T.J.) 9, 809–840 (Elsevier Science, 2003).

    Google Scholar 

  12. Williams, C.J., Whitehouse, J.M.A. & Medical, B. Cis-platinum: a new anticancer agent. Br. Med. J. 1, 1689–1691 (1979).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Crack, J, Green, J., Thomson, A.J. & Le Brun, N.E. Techniques for the production, isolation, and analysis of iron–sulfur proteins. Methods Mol. Biol. 1122, 33–48 (2014).

    CAS  PubMed  Google Scholar 

  14. Volbeda, A. X-ray crystallographic studies of metalloproteins. Methods Mol. Biol. 1122, 189–206 (2014).

    CAS  PubMed  Google Scholar 

  15. Bowman, S.E.J., Bridwell-Rabb, J. & Drennan, C.L. Metalloprotein crystallography: more than a structure. Acc. Chem. Res. 49, 695–702 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Brink, A. & Helliwell, J.R. New leads for fragment-based design of rhenium/technetium radiopharmaceutical agents. IUCrJ 4, 283–290 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Helliwell, J.R. New developments in crystallography: exploring its technology, methods and scope in the molecular biosciences. Biosci. Rep. 37, BSR20170204 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Tanley, S.W.M., Schreurs, A.M.M., Kroon-Batenburg, L.M.J. & Helliwell, J.R. Re-refinement of 4g4a: room-temperature X-ray diffraction study of cisplatin and its binding to His15 of HEWL after 14 months chemical exposure in the presence of DMSO. Acta Crystallogr. F Struct. Biol. Commun. 72, 253–254 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Tanley, S.W.M., Schreurs, A.M.M., Kroon-Batenburg, L.M.J. & Helliwell, J.R. Re-refinement of 4xan: hen egg-white lysozyme with carboplatin in sodium bromide solution. Acta Crystallogr. F, Struct. Biol. Commun. 72, 251–252 (2016).

    CAS  Google Scholar 

  20. Russo Krauss, I. et al. Principles and methods used to grow and optimize crystals of protein-metallodrug adducts, to determine metal binding sites and to assign metal ligands. Metallomics 9, 1534–1547 (2017).

    CAS  PubMed  Google Scholar 

  21. Frier, J.A. & Perutz, M.F. Structure of human foetal deoxyhaemoglobin. J. Mol. Biol. 112, 97–112 (1977).

    CAS  PubMed  Google Scholar 

  22. Reeke, G.N.J., Becker, J.W. & Edelman, G.M. The covalent and three-dimensional structure of concanavalin A. IV. Atomic coordinates, hydrogen bonding, and quaternary structure. J. Biol. Chem. 250, 1525–1548 (1975).

    CAS  PubMed  Google Scholar 

  23. Berman, H.M. et al. The protein data bank. Nucleic Acids Res. 28, 235–242 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Gatzeva-Topalova, P.Z., Warner, L.R., Pardi, A. & Carlos, M. Metalloproteomics: forward and reverse approaches in metalloprotein structural and functional characterization. Curr. Opin. Chem. Biol. 18, 1492–1501 (2011).

    Google Scholar 

  25. Hall, J.F. et al. Towards the high-throughput expression of metalloproteins from the Mycobacterium tuberculosis genome. J. Synchrotron Radiat. 12, 4–7 (2005).

    CAS  PubMed  Google Scholar 

  26. Acton, A.Q. Metalloproteins: Advances in Research and Application: 2013 Edition (ScholaryEditions, 2013).

    Google Scholar 

  27. Nicolini, C. Molecular Manufacturing (Springer Science & Business Media, 2013).

    Google Scholar 

  28. Maret, W. Metalloproteomics, metalloproteomes, and the annotation of metalloproteins. Metallomics 2, 117–125 (2010).

    CAS  PubMed  Google Scholar 

  29. Shi, W. et al. Characterization of metalloproteins by high-throughput X-ray absorption spectroscopy. Genome Res. 21, 898–907 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Macedo, S. et al. Can soaked-in scavengers protect metalloprotein active sites from reduction during data collection? J. Synchrotron Radiat. 16, 191–204 (2009).

    CAS  PubMed  Google Scholar 

  31. Metalloproteins: Theory, Calculations, and Experiments (eds. Cho, A.E. & Goddard, W.A.) (CRC Press, 2015).

  32. Domagalski, M. et al. The quality and validation of structures from structural genomics. Methods Mol. Biol. 1091, 297–314 (2013).

    Google Scholar 

  33. Zheng, H. et al. Validation of metal-binding sites in macromolecular structures with the CheckMyMetal web server. Nat. Protoc. 9, 156–170 (2014).

    CAS  PubMed  Google Scholar 

  34. Riordan, J. & Valee, B. (eds.) Methods in Enzymology: Metallobiochemistry, Part C 226 (Academic Press, 1993).

    Google Scholar 

  35. Riordan, J. & Valee, B. (eds.) Methods in Enzymology: Metallobiochemistry, Part A 158 (Academic Press, 1988).

    Google Scholar 

  36. Riordan, J. & Valee, B. (eds.) Methods in Enzymology: Metallobiochemistry, Part D 227 (Academic Press, 1993).

    Google Scholar 

  37. Papageorgiou, A.C. & Mattsson, J. Protein structure validation and analysis with X-ray crystallography. Methods Mol. Biol. 1129, 397–421 (2014).

    CAS  PubMed  Google Scholar 

  38. Shabalin, I., Dauter, Z., Jaskolski, M., Minor, W. & Wlodawer, A. Crystallography and chemistry should always go together: a cautionary tale of protein complexes with cisplatin and carboplatin. Acta Crystallogr. D Biol. Crystallogr. 71, 1965–1979 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Zimmerman, M.D., Proudfoot, M., Yakunin, A. & Minor, W. Structural insight into the mechanism of substrate specificity and catalytic activity of an HD-domain phosphohydrolase: the 5′-deoxyribonucleotidase YfbR from Escherichia coli. J. Mol. Biol. 378, 215–226 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Minor, W. et al. Crystal structure of soybean lipoxygenase L-1 at 1.4 A resolution. Biochemistry 35, 10687–10701 (1996).

    CAS  PubMed  Google Scholar 

  41. Tomchick, D.R., Phan, P., Cymborowski, M., Minor, W. & Holman, T.R. Structural and functional characterization of second-coordination sphere mutants of soybean lipoxygenase-1. Biochemistry 40, 7509–7517 (2001).

    CAS  PubMed  Google Scholar 

  42. Chruszcz, M., Wlodawer, A. & Minor, W. Determination of protein structures: a series of fortunate events. Biophys. J. 95, 1–9 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Saikatendu, K.S. et al. Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center. BMC Struct. Biol. 6, 27 (2006).

    PubMed  PubMed Central  Google Scholar 

  44. Niedzialkowska, E. et al. Molecular basis for phosphospecific recognition of histone H3 tails by Survivin paralogues at inner centromeres. Mol. Biol. Cell 23, 1457–1466 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Majorek, K.A., Kuhn, M.L., Chruszcz, M., Anderson, W.F. & Minor, W. Structural, functional, and inhibition studies of a Gcn5-related N-acetyltransferase (GNAT) superfamily protein PA4794: a new C-terminal lysine protein acetyltransferase from Pseudomonas aeruginosa. J. Biol. Chem. 288, 30223–30235 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Luo, H.-B. et al. Crystal structure and molecular modeling study of N-carbamoylsarcosine amidase Ta0454 from Thermoplasma acidophilum. J. Struct. Biol. 169, 304–311 (2010).

    CAS  PubMed  Google Scholar 

  47. Murphy, T.A. et al. Crystal structure of Pseudomonas aeruginosa SPM-1 provides insights into variable zinc affinity of metallo-beta-lactamases. J. Mol. Biol. 357, 890–903 (2006).

    CAS  PubMed  Google Scholar 

  48. Segraves, E.N. et al. Kinetic, spectroscopic, and structural investigations of the soybean lipoxygenase-1 first-coordination sphere mutant, Asn694Gly. Biochemistry 45, 10233–10242 (2006).

    CAS  PubMed  Google Scholar 

  49. Zheng, H., Chruszcz, M., Lasota, P., Lebioda, L. & Minor, W. Data mining of metal ion environments present in protein structures. J. Inorg. Biochem. 102, 1765–76 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Zheng, H., Shabalin, I.G., Handing, K.B., Bujnicki, J.M. & Minor, W. Magnesium-binding architectures in RNA crystal structures: validation, binding preferences, classification and motif detection. Nucleic Acids Res. 43, 3789–3801 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Handing, K.B. et al. Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins. Chem. Sci. 7, 6635–6648 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. Gabbiani, C. et al. Protein metalation by metal-based drugs: reactions of cytotoxic gold compounds with cytochrome c and lysozyme. J. Biol. Inorg. Chem. 17, 1293 (2012).

    CAS  PubMed  Google Scholar 

  53. Merlino, A., Marzo, T. & Messori, L. Protein metalation by anticancer metallodrugs: a joint ESI MS and XRD investigative strategy. Chemistry 23, 6942–6947 (2017).

    CAS  PubMed  Google Scholar 

  54. Allen, J.P. Biophysical Chemistry (Wiley-Blackwell, 2008).

    Google Scholar 

  55. Ostendorp, T., Diez, J., Heizmann, C.W. & Fritz, G. The crystal structures of human S100B in the zinc- and calcium-loaded state at three pH values reveal zinc ligand swapping. Biochim. Biophys. Acta 1813, 1083–1091 (2011).

    CAS  PubMed  Google Scholar 

  56. Giroux, E.L. & Henkin, R.I. Competition for zinc among serum albumin and amino acids. Biochim. Biophys. Acta 273, 64–72 (1972).

    CAS  PubMed  Google Scholar 

  57. Pantoliano, M.W., Valentine, J.S., Mammone, R.J. & Scholler, D.M. The pH dependence of metal ion binding to the native zinc site of bovine erythrocuprein (superoxide dismutase). J. Am. Chem. Soc. 104, 1717–1723 (1982).

    CAS  Google Scholar 

  58. McPherson, A. Current approaches to macromolecular crystallization. Eur. J. Biochem. 189, 1–23 (1990).

    CAS  PubMed  Google Scholar 

  59. Newman, J., Sayle, R.A. & Fazio, V.J. A universal indicator dye pH assay for crystallization solutions and other high-throughput applications. Acta Crystallogr. D Biol. Crystallogr. 68, 1003–1009 (2012).

    CAS  PubMed  Google Scholar 

  60. Newman, J., Fazio, V.J., Lawson, B. & Peat, T.S. The C6 web tool: a resource for the rational selection of crystallization conditions. Cryst. Growth Des. 10, 2785–2792 (2010).

    CAS  Google Scholar 

  61. Mikol, V., Rodeau, J.L. & Giegé, R. Changes of pH during biomacromolecule crystallization by vapor diffusion using ammonium sulfate as the precipitant. J. Appl. Crystallogr. 22, 155–161 (1989).

    CAS  Google Scholar 

  62. Hampton Research Corporation. PEG Stability (2012).

  63. Orlov, Y.F., Maslov, E.I. & Belkina, E.I. Solubilities of metal hydroxides. Russ. J. Inorg. Chem. 58, 1458–1466 (2013).

    Google Scholar 

  64. Fischer, B.E., Häring, U.K., Tribolet, R. & Sigel, H. Metal ion/buffer interactions: stability of binary and ternary complexes containing 2-amino-2(hydroxymethyl)-1,3-propanediol (Tris) and adenosine 5-triphosphate (ATP). Eur. J. Biochem. 94, 523–530 (1979).

    CAS  PubMed  Google Scholar 

  65. Belviso, B.D. et al. Oxaliplatin binding to human copper chaperone Atox1 and protein dimerization. Inorg. Chem. 55, 6563–6573 (2016).

    CAS  PubMed  Google Scholar 

  66. David, G., Blondeau, K., Schiltz, M., Penel, S. & Lewit-Bentley, A. YodA from Escherichia coli is a metal-binding, lipocalin-like protein. J. Biol. Chem. 278, 43728–43735 (2003).

    CAS  PubMed  Google Scholar 

  67. Cotelesage, J.J.H., Pushie, M.J., Grochulski, P., Pickering, I.J. & George, G.N. Metalloprotein active site structure determination: synergy between X-ray absorption spectroscopy and X-ray crystallography. J. Inorg. Biochem. 115, 127–137 (2012).

    CAS  PubMed  Google Scholar 

  68. Ascone, I. & Strange, R. Biological X-ray absorption spectroscopy and metalloproteomics. J. Synchrotron Radiat. 16, 413–421 (2009).

    CAS  PubMed  Google Scholar 

  69. Yukl, E.T. et al. Diradical intermediate within the context of tryptophan tryptophylquinone biosynthesis. Proc. Natl. Acad. Sci. USA 110, 4569–4573 (2013).

    PubMed  PubMed Central  Google Scholar 

  70. Garman, E.F. Radiation damage in macromolecular crystallography: what is it and why should we care? Acta Crystallogr. D Biol. Crystallogr. 66, 339–351 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Seitlich, T., Kühnel, K., Schulze-Briese, C., Shoeman, R.L. & Schlichting, I. Cryoradiolytic reduction of crystalline heme proteins: analysis by UV-Vis spectroscopy and X-ray crystallography. J. Synchrotron Radiat. 14, 11–23 (2007).

    Google Scholar 

  72. Frankaer, C.G., Mossin, S., Ståhl, K. & Harris, P. Towards accurate structural characterization of metal centres in protein crystals: the structures of Ni and Cu T6 bovine insulin derivatives. Acta Crystallogr. D Biol. Crystallogr. 70, 110–122 (2014).

    CAS  PubMed  Google Scholar 

  73. Trofimov, A.A. et al. Structural study of the X-ray-induced enzymatic reaction of octahaem cytochrome C nitrite reductase. Acta Crystallogr. D 71, 1087–1094 (2015).

    CAS  PubMed  Google Scholar 

  74. Brophy, M.B., Hayden, J.A. & Nolan, E.M. Calcium ion gradients modulate the zinc affinity and antibacterial activity of human calprotectin. J. Am. Chem. Soc. 134, 18089–18100 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Shirran, S., Garnaud, P., Daff, S., McMillan, D. & Barran, P. The formation of a complex between calmodulin and neuronal nitric oxide synthase is determined by ESI-MS. J. R. Soc. Interface 2, 465–476 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  76. Kaltashov, I.A., Zhang, M., Eyles, S.J. & Abzalimov, R.R. Investigation of structure, dynamics and function of metalloproteins with electrospray ionization mass spectrometry. Anal. Bioanal. Chem. 386, 472–481 (2006).

    CAS  PubMed  Google Scholar 

  77. Czernusxewicx, R.S. in Spectroscopic Methods and Analyses: NMR, Mass Spectrometry, and Metalloprotein Techniques (eds. Jones, C. et al.) 345–374 (Humana Press, 1993).

    Google Scholar 

  78. Vincent, K. Triggered infrared spectroscopy for investigating metalloprotein chemistry. Philos. Trans. A Math. Phys. Eng. Sci. 368, 3713–3731 (2010).

    CAS  PubMed  Google Scholar 

  79. Ronda, L., Bruno, S., Bettati, S., Storici, P. & Mozzarelli, A. From protein structure to function via single crystal optical spectroscopy. Front. Mol. Biosci. 2, 12 (2015).

    PubMed  PubMed Central  Google Scholar 

  80. Stoner-Ma, D. et al. Single-crystal Raman spectroscopy and X-ray crystallography at beamline X26-C of the NSLS. J. Synchrotron Radiat. 18, 37–40 (2011).

    CAS  PubMed  Google Scholar 

  81. Merlino, A. et al. Crystallization, preliminary X-ray diffraction studies and Raman microscopy of the major haemoglobin from the sub-Antarctic fish Eleginops maclovinus in the carbomonoxy form. Acta Crystallogr. F Struct. Biol. Cryst. Commun. 66, 1536–1540 (2010).

    CAS  Google Scholar 

  82. Vergara, A., Merlino, A., Pizzo, E., D'Alessio, G. & Mazzarella, L. A novel method for detection of selenomethionine incorporation in protein crystals via Raman microscopy. Acta Crystallogr. D 64, 167–171 (2008).

    CAS  PubMed  Google Scholar 

  83. Hall, J.P. et al. Monitoring one-electron photo-oxidation of guanine in DNA crystals using ultrafast infrared spectroscopy. Nat. Chem. 7, 961–967 (2015).

    CAS  PubMed  Google Scholar 

  84. Knape, M.J. et al. Divalent metal ions Mg2+ and Ca2+ have distinct effects on protein kinase A activity and regulation. ACS Chem. Biol. 10, 2303–2315 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Lu, C.H., Lin, Y.F., Lin, J.J. & Yu, C.S. Prediction of metal ion-binding sites in proteins using the fragment transformation method. PLoS One 7, 1–12 (2012).

    Google Scholar 

  86. Ebert, J.J.C. & Altman, R.R.B. Robust recognition of zinc binding sites in proteins. Protein Sci. 17, 54–65 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. Zhao, W. et al. Structure-based de novo prediction of zinc-binding sites in proteins of unknown function. Bioinformatics 27, 1262–1268 (2011).

    CAS  PubMed  Google Scholar 

  88. Sodhi, J.S. et al. Predicting metal-binding site residues in low-resolution structural models. J. Mol. Biol. 342, 307–320 (2004).

    CAS  PubMed  Google Scholar 

  89. Passerini, A., Lippi, M. & Frasconi, P. MetalDetector v2.0: predicting the geometry of metal binding sites from protein sequence. Nucleic Acids Res. 39, 288–292 (2011).

    Google Scholar 

  90. Ferrè, F. & Clote, P. DiANNA 1.1: an extension of the DiANNA web server for ternary cysteine classification. Nucleic Acids Res. 34, W182–W185 (2006).

    PubMed  PubMed Central  Google Scholar 

  91. Brylinski, M. & Skolnick, J. FINDSITE-metal: integrating evolutionary information and machine learning for structure-based metal binding site prediction at the proteome level. Proteins 79, 735–751 (2011).

    CAS  PubMed  Google Scholar 

  92. Alpi, E. et al. Analysis of the tryptic search space in UniProt databases. Proteomics 15, 48–57 (2015).

    CAS  PubMed  Google Scholar 

  93. Chitale, M., Hawkins, T., Park, C. & Kihara, D. ESG: extended similarity group method for automated protein function prediction. Bioinformatics 25, 1739–1745 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    CAS  PubMed  Google Scholar 

  95. Leuthner, B. et al. Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism. Mol. Microbiol. 28, 615–628 (1998).

    CAS  PubMed  Google Scholar 

  96. Echavarri, C., Arragain, S. & Rubio, L.M. Purification of O2-sensitive metalloproteins. Methods Mol. Biol. 1122, 5–18 (2014).

    Google Scholar 

  97. Pestov, N.B. & Rydstro, J. Purification of recombinant membrane proteins tagged with calmodulin-binding domains by affinity chromatography on calmodulin-agarose: example of nicotinamide nucleotide transhydrogenase. Nat. Protoc. 2, 198–202 (2007).

    CAS  PubMed  Google Scholar 

  98. Majorek, K.A., Kuhn, M.L., Chruszcz, M., Anderson, W.F. & Minor, W. Double trouble-buffer selection and His-tag presence may be responsible for nonreproducibility of biomedical experiments. Protein Sci. 23, 1359–1368 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  99. Van Dam, M.E., Wuenschell, G.E. & Arnold, F.H. Metal affinity precipitation of proteins. Biotechnol. Appl. Biochem. 11, 492–502 (1989).

    CAS  PubMed  Google Scholar 

  100. Ju, T. et al. One protein, two enzymes revisited: a structural entropy switch interconverts the two isoforms of acireductone dioxygenase. J. Mol. Biol. 4, 823–834 (2011).

    Google Scholar 

  101. Smith, R.M., Martell, A.E. & Motekaitis, R.J. NIST critically selected stability constants of metal complexes database (Standard Reference Data Program, National Institute of Standards and Technology, U.S. Dept. of Commerce 2004).

    Google Scholar 

  102. Bijelic, A., Theiner, S., Keppler, B.K. & Rompel, A. X-ray structure analysis of indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) bound to human serum albumin reveals two ruthenium binding sites and provides insights into the drug binding mechanism. J. Med. Chem. 59, 5894–5903 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  103. Yu, Q., Kandegedara, A., Xu, Y. & Rorabacher, D.B. Avoiding interferences from Good's buffers: a contiguous series of noncomplexing tertiary amine buffers covering the entire range of pH 3-11. Anal. Biochem. 253, 50–56 (1997).

    CAS  PubMed  Google Scholar 

  104. Nakon, R. & Krishnamoorthy, C.R. Free-metal ion depletion by 'Good's' buffers. Science 221, 749–750 (1983).

    CAS  PubMed  Google Scholar 

  105. Sokołowska, M. & Bal, W. Cu(II) complexation by 'non-coordinating' N-2- hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES buffer). J. Inorg. Biochem. 99, 1653–1660 (2005).

    PubMed  Google Scholar 

  106. Ferreira, C.M.H., Pinto, I.S.S., Soares, E.V. & Soares, H.M.V.M. (Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ions: a review. RSC Adv. 5, 30989–31003 (2015).

    CAS  Google Scholar 

  107. Lundblad, R.L. Chemical Reagents for Protein Modification (CRC Press, 2014).

    Google Scholar 

  108. Satyanarayana, U. Biochemistry (Elsevier, 2014).

    Google Scholar 

  109. Phillips, C.M., Schreiter, E.R., Stultz, C.M. & Drennan, C.L. Structural basis of low-affinity nickel binding to the nickel-responsive transcription factor NikR from Escherichia coli. Biochemistry 49, 7830–7838 (2010).

    CAS  PubMed  Google Scholar 

  110. Champloy, F., Gruber, K., Jogl, G. & Kratky, C. XAS spectroscopy reveals X-ray-induced photoreduction of free and protein-bound B12 cofactors. J. Synchrotron Radiat. 7, 267–273 (2000).

    CAS  PubMed  Google Scholar 

  111. Huang, Q. & Szebenyi, D.M.E. Improving diffraction resolution using a new dehydration method. Acta Crystallogr. F Struct. Biol. Commun. 72, 152–159 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  112. Newman, J. A review of techniques for maximizing diffraction from a protein crystal in stilla. Acta Crystallogr. D 62, 27–31 (2006).

    PubMed  Google Scholar 

  113. Kim, C.U., Wierman, J.L., Gillilan, R., Lima, E. & Gruner, S.M. A high-pressure cryocooling method for protein crystals and biological samples with reduced background X-ray scatter. J. Appl. Crystallogr. 46, 234–241 (2013).

    CAS  PubMed  Google Scholar 

  114. Vernède, X. & Fontecilla-Camps, J.C. A method to stabilize reduced and/or gas-treated protein crystals by flash-cooling under a controlled atmosphere. J. Appl. Crystallogr. 32, 505–509 (1999).

    Google Scholar 

  115. Bearden, J.A. X-ray wavelengths. Rev. Mod. Phys. 39, 78–124 (1967).

    CAS  Google Scholar 

  116. Nakanishi, T. et al. Lower limits of detection of synchrotron radiation high-energy X-ray fluorescence spectrometry and its possibility for the forensic application for discrimination of glass fragments. Forensic Sci. Int. 175, 227–234 (2008).

    CAS  PubMed  Google Scholar 

  117. Awaji, N. et al. Detection limits of trace elements for wavelength dispersive total X-ray fluorescence under high flux synchrotron radiation. Jpn. J. Appl. Phys. 43, 1644–1648 (2004).

    CAS  Google Scholar 

  118. Sutton, S.R., Rivers, M.L. & Smith, J.V. Synchrotron X-ray fluorescence: diffraction interference. Anal. Chem. 58, 2167–2171 (1986).

    CAS  Google Scholar 

  119. Brock, C.P. et al. (eds.) International Tables for Crystallography. Vol. C(Wiley, 1999).

  120. Srivastava, U.C. & Nigam, H.L. X-ray absorption edge spectrometry (XAES) as applied to coordination chemistry. Coord. Chem. Rev. 9, 275–310 (1973).

    CAS  Google Scholar 

  121. Sathyanarayana, D.N. Electronic Absorption Spectroscopy and Related Techniques (Universities Press, 2001).

    Google Scholar 

  122. Cotelesage, J.J.H., Grochulski, P., Pickering, I.J., George, G.N. & Fodje, M.N. X-ray absorption spectroscopy at a protein crystallography facility: the Canadian Light Source beamline 08B1-1. J. Synchrotron Radiat. 19, 887–891 (2012).

    CAS  PubMed  Google Scholar 

  123. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  124. Murshudov, G.N. et al. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 67, 355–367 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  125. Zheng, H. et al. CheckMyMetal: macromolecular metal binding validation tool. Acta Crystallogr. D 73, 223–233 (2017).

    CAS  Google Scholar 

  126. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  PubMed  Google Scholar 

  127. Gore, S., Velankar, S. & Kleywegt, G.J. Implementing an X-ray validation pipeline for the Protein Data Bank. Acta Crystallogr. D Biol. Crystallogr. 68, 478–483 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  128. Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. HKL-3000: the integration of data reduction and structure solution: from diffraction images to an initial model in minutes. Acta Crystallogr. D Biol. Crystallogr. 62, 859–866 (2006).

    PubMed  Google Scholar 

  129. Stepanov, S. et al. JBluIce: EPICS control system for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 67, 176–188 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  130. Kabsch, W. XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  131. Adams, P.D. et al. PHENIX: a comprehensive python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  132. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    PubMed  Google Scholar 

  133. Bruvold, W.H. A meta-analysis of the California school-based risk reduction program. J. Drug Educ. 20, 139–152 (1990).

    CAS  PubMed  Google Scholar 

  134. Kimple, M.E., Brill, A.L. & Pasker, R.L. Overview of affinity tags for protein purification. Curr. Protoc. Protein Sci. 36 9.9 (2013).

  135. Bonner, P.L.R. Protein Purification (Taylor & Francis, 2007).

    Google Scholar 

  136. Scopes, R.K. Protein Purification: Principles and Practice (Springer, 1993).

    Google Scholar 

  137. Ammann, A.A. Inductively coupled plasma mass spectrometry (ICP MS): a versatile tool. J. Mass Spectrom. 42, 419–427 (2007).

    CAS  PubMed  Google Scholar 

  138. McPherson, A. Crystallization of Biological Macromolecules (Cold Spring Harbor Laboratory Press, 1999).

    Google Scholar 

  139. Pflugrath, J.W. Macromolecular cryocrystallography: methods for cooling and mounting protein crystals at cryogenic temperatures. Methods 34, 415–423 (2004).

    CAS  PubMed  Google Scholar 

  140. Tereshko, V. et al. Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments. Nucleic Acids Res. 29, 1208–15 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  141. Niedzialkowska, E. et al. Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization. Protein Expr. Purif. 134, 47–62 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  142. Lion, T. et al. The translocation t(1;22)(p13;q13) is a nonrandom marker specifically associated with acute megakaryocytic leukemia in young children. Blood 79, 3325–3330 (1992).

    CAS  PubMed  Google Scholar 

  143. Blindauer, C.A. et al. Structure, properties, and engineering of the major zinc binding site on human albumin. J. Biol. Chem. 284, 23116–23124 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  144. Newman, J. Expanding screening space through the use of alternative reservoirs in vapor-diffusion experiments. Acta Crystallogr. D Biol. Crystallogr. 61, 490–493 (2005).

    PubMed  Google Scholar 

  145. Till, M. et al. Improving the success rate of protein crystallization by random microseed matrix screening. J. Vis. Exp. http://doi.org/10.3791/50548 (2013).

  146. Benvenuti, M. & Mangani, S. Crystallization of soluble proteins in vapor diffusion for X-ray crystallography. Nat. Protoc. 2, 1633–1651 (2007).

    CAS  PubMed  Google Scholar 

  147. Malawski, G.A. et al. Identifying protein construct variants with increased crystallization propensity: a case study. Protein Sci. 15, 2718–2728 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  148. Vedadi, M. et al. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc. Natl. Acad. Sci. USA 103, 15835–15840 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  149. McPherson, A. & Cudney, B. Optimization of crystallization conditions for biological macromolecules. Acta Crystallogr. F Struct. Biol. Commun. 70, 1445–1467 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  150. Dong, A., Xu, X. & Edwards, A. In situ proteolysis for protein crystallization and structure determination. Nat. Methods 4, 1019–1021 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  151. Holdgate, G. Isothermal titration calorimetry and differential scanning calorimetry. Methods Mol. Biol. 572, 101–133 (2009).

    PubMed  Google Scholar 

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Acknowledgements

This work was supported by federal funds awarded to W.M. from the National Institute of General Medical Sciences under grant numbers GM117325 and GM117080, and NIH BD2K grant HG008424, as well as from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, and the Department of Health and Human Services under contract nos. HHSN272201200026C and HHSN272201700060C. E.N. was supported by the Foundation for Polish Science (FNP) and received funding from the Marian Smoluchowski Krakow Research Consortium—a Leading National Research Centre KNOW supported by the Polish Ministry of Science and Higher Education. We thank J. Lipowska for providing the fluorescence and diffraction data for dihydroorotase from Y. pestis CO92. We thank M.P. Czub for providing Thermofluor shift data for STM1931 protein from S. Typhimurium. We thank R. Alkire (Structural Biology Center at Argonne National Laboratory) for providing the fluorescence spectrum data for the zinc foil. We thank D.R. Cooper and M. Cymborowski for help with XRF data collection and interpretation. We thank M. Grabowski, W.-S. Tzou, and B. Venkataramany for valuable discussions and critical reading of the manuscript.

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Authors and Affiliations

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Contributions

K.B.H. performed essential experiments to identify the pH-dependent conformational changes of residues coordinating the transition metal in albumin; E.N. provided important data and experience in ICP–OES and TSA experiments; K.B.H., I.G.S., E.N., M.L.K., and H.Z. provided critical experience in metal-binding-protein production and purification; K.B.H., I.G.S., E.N., and W.M. provided critical experience in metal-binding-protein crystallization and data collection; H.Z. and I.G.S. provided extensive experience in characterization of metal-binding sites in protein structures; H.Z. and I.G.S. laid out the structural framework of the manuscript; M.L.K. provided constructive comments and extensively edited the manuscript; K.B.H., E.N., I.G.S., M.L.K., H.Z., and W.M. wrote and approved the manuscript; and H.Z. and W.M. supervised the project.

Corresponding authors

Correspondence to Heping Zheng or Wladek Minor.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Influence of ZnCl2 concentration on pH of 50 (gray) and 100 mM (orange) Tris-HCl buffer solutions.

The green line represents the initial value of the pH of Tris buffers.

Supplementary Figure 2 Fluorescence spectra and fluorescence scans for ESA complexed with four different metals.

Data for ESA complexed with copper (A), gold (B), mercury (C), and platinum (D) are shown. The figure represents screenshots generated by the data collection program JBluIce-EPICS as implemented on APS GM/CA-CAT beamlines. For each panel, the top screenshot shows the fluorescence emission spectrum collected with the excitation energy on or slightly above the theoretical value of the metal absorption edge (+10-20 eV). This spectrum displays the characteristic emission peak for the metal of interest (bracketed by the red boundaries) as well as the incident beam peak. Note that there are no significant peaks for other metals in these spectra. The middle screenshot shows the fluorescence emission spectrum collected with the excitation energy at 30-50 eV below the metal absorption edge; the characteristic peak for the metal of interest is absent on this spectrum. The bottom screenshot shows the fluorescence absorption scan collected with the excitation energy in the range ±30 eV of the tabulated metal absorption edge; note that the emitted fluorescence is measured, which is proportional to the absorbed energy. The energy of the absorption edge, approximated as the inflection f” point (indicated by the orange vertical line and listed in the table below each graph as “infl”), is close to the table values for each metal. Note that the width of the absorption edges (the energy difference between the absorbance inflection point and its peak; these values coincide with f” inflection point and its peak) for both Cu K edge and Hg L-III edge is much wider than those for Au L-III and Pt L-III. In the case of Hg L-III edge, the range of the excitation energy could be increased since the typical range of ±30 eV does not fully cover the absorption edge. The optimal energy for collecting X-ray diffraction data above the absorption edge is the maximum of f” (i.e. the maximum of absorption and fluorescence), which is located at the top of the fluorescence scan and indicated by the green vertical line. The optimal energy for collecting X-ray diffraction data below the absorption edge is the highest energy below the absorption edge that gives only background fluorescence signal (virtually flat area of f”).

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Supplementary Figures 1 and 2, and Supplementary Tables 1–4 (PDF 1048 kb)

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Handing, K., Niedzialkowska, E., Shabalin, I. et al. Characterizing metal-binding sites in proteins with X-ray crystallography. Nat Protoc 13, 1062–1090 (2018). https://doi.org/10.1038/nprot.2018.018

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