Skip to main content
SpringerLink
Log in
Book cover

Calcium-Binding Proteins of the EF-Hand Superfamily pp 275–290Cite as

S100 Proteins in the Innate Immune Response to Pathogens

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1929))

Abstract

S100 proteins are distinct dimeric EF-hand Ca2+-binding proteins that can bind Zn2+, Mn2+, and other transition metals with high affinity at two sites in the dimer interface. Certain S100 proteins, including S100A7, S100A12, S100A8, and S100A9, play key roles in the innate immune response to pathogens. These proteins function via a “nutritional immunity” mechanism by depleting essential transition metals in the infection that are required for the invading organism to grow and thrive. They also act as damage-associated molecular pattern ligands, which activate pattern recognition receptors (e.g., Toll-like receptor 4, RAGE) that mediate inflammation. Here we present protocols for these S100 proteins for high-level production of recombinant protein, measurement of binding affinities using isothermal titration calorimetry, and an assay of antimicrobial activity.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD   219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Nelson MR, Chazin WJ (1998) Structures of EF-hand Ca(2+)-binding proteins: diversity in the organization, packing and response to Ca2+ binding. Biometals 11(4):297–318

    Article  CAS  Google Scholar 

  2. Donato R (2003) Intracellular and extracellular roles of S100 proteins. Microsc Res Tech 15:540–551

    Article  Google Scholar 

  3. Donato R, Cannon BR, Sorci G, Riuzzi F, Hsu K, Weber DJ, Geczy CL (2013) Functions of S100 proteins. Curr Mol Med 13:24–57

    Article  CAS  Google Scholar 

  4. Zackular JP, Chazin WJ, Skaar EP (2015) Nutritional immunity: S100 proteins at the host-pathogen interface. J Biol Chem 290:18991–18998

    Article  CAS  Google Scholar 

  5. Hunter MJ, Chazin WJ (1998) High level expression and dimer characterization of the S100 EF-hand proteins, migration inhibitory factor-related proteins 8 and 14. J Biol Chem 273:12427–12435

    Article  CAS  Google Scholar 

  6. Bhattacharya S, Bunick CG, Chazin WJ (2004) Target selectivity in EF-hand calcium binding proteins. Biochim Biophys Acta 1742(1–3):69–79

    CAS  PubMed  Google Scholar 

  7. Gilston BA, Skaar EP, Chazin WJ (2016) Binding of transition metals to S100 proteins. Science China 59:792–801

    Article  CAS  Google Scholar 

  8. Damo SM, Kehl-Fie TE, Sugitani N, Holt ME, Rathi S, Murphy WJ, Zhang Y, Betz C, Hench L, Fritz G, Skaar EP, Chazin WJ (2013) Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens. Proc Natl Acad Sci U S A 110(10):3841–3846

    Article  CAS  Google Scholar 

  9. Moroz OV, Blagova EV, Wilkinson AJ, Wilson KS, Bronstein IB (2009) The crystal structures of human S100A12 in apo form and in complex with zinc: new insights into S100A12 oligomerisation. J Mol Biol 391:536–551

    Article  CAS  Google Scholar 

  10. Brodersen DE, Nyborg J, Kjeldgaard M (1999) Zinc-binding site of an S100 protein revealed. Two crystal structures of Ca2+−bound human psoriasin (S100A7) in the Zn2+−loaded and Zn2+−free states. Biochemistry 38:1695–1704

    Article  CAS  Google Scholar 

  11. Moroz OV, Burkitt W, Wittkowski H, He W, Ianoul A, Novitskaya V, Xie J, Polyakova O, Lednev IK, Shekhtman A, Derrick PJ, Bjoerk P, Foell D, Bronstein IB (2009) Both Ca2+ and Zn2+ are essential for S100A12 protein oligomerization and function. BMC Biochem 10:1–18

    Article  Google Scholar 

  12. Hood MI, Skaar EP (2012) Nutritional immunity: transition metals at the pathogen-host interface. Nat Rev Microbiol 10(8):525–537

    Article  CAS  Google Scholar 

  13. Palmer LD, Skaar EP (2016) Transition metals and virulence in bacteria. Annu Rev Genet 50:67–91

    Article  CAS  Google Scholar 

  14. Kehl-Fie TE, Chitayat S, Hood MI, Damo S, Restrepo N, Garcia C, Munro KA, Chazin WJ, Skaar EP (2011) Nutrient metal sequestration by calprotectin inhibits bacterial superoxide defense enhancing neutrophil killing of Staphylococcus aureus. Cell Host Microbe 10:158–164

    Article  CAS  Google Scholar 

  15. Dhople AM, Ibanez MA, Poirier TC (1996) Role of iron in the pathogenesis of Mycobacterium avium infection in mice. Microbios 87(351):77–87

    CAS  PubMed  Google Scholar 

  16. McDermid JM, Hennig BJ, van der Sande M, Hill AV, Whittle HC, Jaye A, Prentice AM (2013) Host iron redistribution as a risk factor for incident tuberculosis in HIV infection: an 11-year retrospective cohort study. BMC Infect Dis 13:e48

    Article  Google Scholar 

  17. Zackular JP, Moore JL, Jordan AT, Juttukonda LJ, Noto MJ, Nicholson MR, Crews JD, Semler MW, Zhang Y, Ware LB, Washington MK, Chazin WJ, Caprioli RM, Skaar EP (2016) Dietary zinc alters the microbiota and decreases resistance to Clostridium difficile infection. Nat Med 22(11):1330–1334

    Article  CAS  Google Scholar 

  18. Juttukonda LJ, Berends ETM, Zackular JP, Moore JL, Stier MT, Zhang Y, Schmitz JE, Beavers WN, Wijers CD, Gilston BA, Kehl-Fie TE, Atkinson J, Washington MK, Peebles RS, Chazin WJ, Torres VJ, Caprioli RM, Skaar EP (2017) Dietary manganese promotes staphylococcal infection of the heart. Cell Host Microbe 22(4):531–542

    Article  CAS  Google Scholar 

  19. Besold AN, Gilston BA, Radin JN, Ramsoomair C, Culbertson EM, Li CX, Cormack BP, Chazin WJ, Kehl-Fie TE, Culotta VC (2018) Role of calprotectin in withholding zinc and copper from Candida albicans. Infect Immun 86:e00779–e00717

    CAS  PubMed Central  Google Scholar 

  20. Corbin BD, Seeley EH, Raab A, Feldmann J, Miller MR, Torres VJ, Anderson KL, Dattilo BM, Dunman PM, Gerads R, Caprioli RM, Nacken W, Chazin WJ, Skaar EP (2008) Metal chelation and inhibition of bacterial growth in tissue abscesses. Science 319(5865):962–965

    Article  CAS  Google Scholar 

  21. Gaddy JA, Radin JN, Loh JT, Piazuelo MB, Kehl-Fie TE, Delgado AG, Ilca FT, Peek RM, Cover TL, Chazin WJ, Skaar EP, Scott Algood HM (2014) The host protein calprotectin modulates the Helicobacter pylori cag type IV secretion system via zinc sequestration. PLoS Pathog 10(10):e1004450

    Article  Google Scholar 

  22. Hood MI, Mortensen BL, Moore JL, Zhang Y, Kehl-Fie TE, Sugitani N, Chazin WJ, Caprioli RM, Skaar EP (2012) Identification of an Acinetobacter baumannii zinc acquisition system that facilitates resistance to calprotectin-mediated zinc sequestration. PLoS Pathog 8(12):e1003068

    Article  CAS  Google Scholar 

  23. Kehl-Fie TE, Zhang Y, Moore JL, Farrand AJ, Hood MI, Rathi S, Chazin WJ, Caprioli RM, Skaar EP (2013) MntABC and MntH contribute to systemic Staphylococcus aureus infection by competing with calprotectin for nutrient manganese. Infect Immun 81(9):3395–3405

    Article  CAS  Google Scholar 

  24. Urban CF, Ermert D, Schmid M, Abu-Abed U, Goosmann C, Nacken W, Brinkmann V, Jungblut PR, Zychlinsky A (2009) Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS Pathog 5(10):e1000639

    Article  Google Scholar 

  25. Shank JM, Kelley BR, Jackson JW, Tweedie JL, Franklin D, Damo SM, Gaddy JA, Murphy CN, Johnson JG (2018) The host antimicrobial protein Calgranulin c participates in the control of campylobacter jejuni growth via zinc sequestration. Infect Immun 86(6):e00234–e00218

    Article  CAS  Google Scholar 

  26. Haley KP, Delgado AG, Piazuelo MB, Mortensen BL, Correa P, Damo SM, Chazin WJ, Skaar EP, Gaddy JA (2015) The human antimicrobial protein calgranulin C participates in control of Helicobacter pylori growth and regulation of virulence. Infect Immun 83(7):2944–2956

    Article  CAS  Google Scholar 

  27. Moroz OV, Antson AA, Grist SJ, Maitland NJ, Dodson GG, Wilson KS, Lukanidin E, Bronstein IB (2003) Structure of the human S100A12-copper complex: implications for host-parasite defence. Acta Crystallogr D Biol Crystallogr 59(Pt 5):859–867

    Article  CAS  Google Scholar 

  28. Ravasi T, Hsu K, Goyette J, Schroder K, Yang Z, Rahimi F, Miranda LP, Alewood PF, Hume DA, Geczy C (2004) Probing the S100 protein family through genomic and functional analysis. Genomics 84(1):10–22

    Article  CAS  Google Scholar 

  29. Buchau AS, Hassan M, Kukova G, Lewerenz V, Kellermann S, Wurthner JU, Wolf R, Walz R, Walz M, Gallo RL, Ruzicka T (2007) S100A15, an antimicrobial protein of the skin: regulation by E. coli through Toll-like receptor 4. J Invest Dermatol 127:2596–2604

    Article  Google Scholar 

  30. Glaser R, Harder J, Lange H, Bartels J, Christophers E, Schroder JM (2005) Antimicrobial psoriasin (S100A7) protects human skin from Escherichia coli infection. Nat Immunol 6:57–64

    Article  Google Scholar 

  31. Lee KC, Eckert RL (2007) S100A7 (Psoriasin)- mechanism of antibacterial action in wounds. J Invest Dermatol 127:945–957

    Article  CAS  Google Scholar 

  32. Boniface K, Bernard F-X, Garcia M, Gurney AL, Lecron J-C, Morel F (2005) IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes. J Immunol 174:3695–3702

    Article  CAS  Google Scholar 

  33. Wolf R, Mirmohammadsadegh A, Walz M, Lysa B, Tartler U, Remus R, Hengge U, Michel G, Ruzicka T (2003) Molecular cloning and characterization of alternatively spliced mRNA isoforms from psoriatic skin encoding a novel member of the S100 family. FASEB J 17:1969–1971

    Article  CAS  Google Scholar 

  34. Hsu K, Champaiboon C, Guenther BD, Sorenson BS, Khammanivong A, Ross KF, Geczy CL, Herzberg MC (2009) Anti-infective protective properties of S100 calgranulins. Antiinflamm Antiallergy Agents Med Chem 8(4):290–305

    Article  CAS  Google Scholar 

  35. Lagasse E, Clerc RG (1988) Cloning and expression of two human genes encoding calcium-binding proteins that are regulated during myeloid differentiation. Mol Cell Biol 8(6):2402–2410

    Article  CAS  Google Scholar 

  36. Goebeler M, Roth J, van den Bos C, Ader G, Sorg C (1995) Increase of calcium levels in epithelial cells induces translocation of calcium-binding proteins migration inhibitory factor-related protein 8 (MRP8) and MRP14 to keratin intermediate filaments. Biochem J 309(Pt 2):419–424

    Article  CAS  Google Scholar 

  37. Grimbaldeston MA, Geczy CL, Tedla N, Finlay-Jones JJ, Hart PH (2003) S100A8 induction in keratinocytes by ultraviolet A irradiation is dependent on reactive oxygen intermediates. J Invest Dermatol 121(5):1168–1174

    Article  CAS  Google Scholar 

  38. Mork G, Schjerven H, Mangschau L, Soyland E, Brandtzaeg P (2003) Proinflammatory cytokines upregulate expression of calprotectin (L1 protein, MRP-8/MRP-14) in cultured human keratinocytes. Br J Dermatol 149(3):484–491

    Article  CAS  Google Scholar 

  39. Stoll SW, Zhao X, Elder JT (1998) EGF stimulates transcription of CaN19 (S100A2) in HaCaT keratinocytes. J Invest Dermatol 111(6):1092–1097

    Article  CAS  Google Scholar 

  40. Cornish CJ, Devery JM, Poronnik P, Lackmann M, Cook DI, Geczy CL (1996) S100 protein CP-10 stimulates myeloid cell chemotaxis without activation. J Cell Physiol 166(2):427–437

    Article  CAS  Google Scholar 

  41. Lackmann M, Cornish CJ, Simpson RJ, Moritz RL, Geczy CL (1992) Purification and structural analysis of a murine chemotactic cytokine (CP-10) with sequence homology to S100 proteins. J Biol Chem 267(11):7499–7504

    CAS  PubMed  Google Scholar 

  42. Vogl T, Tenbrock K, Ludwig S, Leukert N, Ehrhardt C, van Zoelen MA, Nacken W, Foell D, van der Poll T, Sorg C, Roth J (2007) Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock. Nat Med 13(9):1042–1049

    Article  CAS  Google Scholar 

  43. Foell D, Wittkowski H, Vogl T, Roth J (2007) S100 proteins expressed in phagocytes: a novel group of damage-associated molecular pattern molecules. J Leukoc Biol 81(1):28–37

    Article  CAS  Google Scholar 

  44. Leclerc E, Fritz G, Vetter SW, Heizmann CW (2009) Binding of S100 proteins to RAGE: an update. Biochim Biophys Acta 1793(6):993–1007

    Article  CAS  Google Scholar 

  45. Chen X, Eksioglu EA, Zhou J, Zhang L, Djeu J, Fortenbery N, Epling-Burnette P, Van Bijnen S, Dolstra H, Cannon J, Youn JI, Donatelli SS, Qin D, De Witte T, Tao J, Wang H, Cheng P, Gabrilovich DI, List A, Wei S (2013) Induction of myelodysplasia by myeloid-derived suppressor cells. J Clin Invest 123(11):4595–4611

    Article  CAS  Google Scholar 

  46. Bresnick AR, Weber DJ, Zimmer DB (2015) S100 proteins in cancer. Nat Rev 15:96–109

    Article  CAS  Google Scholar 

  47. Choi IY, Gerlag DM, Holzinger D, Roth J, Tak PP (2014) From synovial tissue to peripheral blood: myeloid related protein 8/14 is a sensitive biomarker for effective treatment in early drug development in patients with rheumatoid arthritis. PLoS One 9(8):e106253

    Article  Google Scholar 

  48. Frosch M, Ahlmann M, Vogl T, Wittkowski H, Wulffraat N, Foell D, Roth J (2009) The myeloid-related proteins 8 and 14 complex, a novel ligand of toll-like receptor 4, and interleukin-1beta form a positive feedback mechanism in systemic-onset juvenile idiopathic arthritis. Arthritis Rheum 60(3):883–891

    Article  CAS  Google Scholar 

  49. Haga HJ, Brun JG, Berntzen HB, Cervera R, Khamashta M, Hughes GR (1993) Calprotectin in patients with systemic lupus erythematosus: relation to clinical and laboratory parameters of disease activity. Lupus 2(1):47–50

    Article  CAS  Google Scholar 

  50. Nordal HH, Brun JG, Halse AK, Madland TM, Fagerhol MK, Jonsson R (2014) Calprotectin (S100A8/A9), S100A12, and EDTA-resistant S100A12 complexes (ERAC) in primary Sjogren’s syndrome. Scand J Rheumatol 43(1):76–78

    Article  CAS  Google Scholar 

  51. Pietzsch J (2011) S100 proteins in health and disease. Amino Acids 41:755–760

    Article  CAS  Google Scholar 

  52. Moore BW (1965) A soluble protein characteristic of the nervous system. Biochem Biophys Res Commun 19:739–744

    Article  CAS  Google Scholar 

  53. Brophy MB, Hayden JA, Nolan EM (2012) Calcium ion gradients modulate the zinc affinity and antibacterial activity of human calprotectin. J Am Chem Soc 134:18089–18100

    Article  CAS  Google Scholar 

  54. Jackson E, Little S, Franklin DS, Gaddy JA, Damo SM (2017) Expression, purification, and antimicrobial activity of S100A12. J Vis Exp 123:6–11

    Google Scholar 

  55. Murray JI, Tonkin ML, Whiting AL, Peng F, Farnell B, Cullen JT, Hof F, Boulanger MJ (2012) Structural characterization of S100A15 reveals a novel zinc coordination site among S100 proteins and altered surface chemistry with functional implications for receptor binding. BMC Struct Biol 12:16–23

    Article  CAS  Google Scholar 

  56. Froger A, Hall JE (2007) Transformation of plasmid DNA into E. coli using the heat shock method. J Vis Exp 6:e253

    Google Scholar 

  57. Sivaraja V, Kumar TK, Rajalingam D, Graziani I, Prudovsky I, Yu C (2006) Copper binding affinity of S100A13, a key component of the FGF-1 nonclassical copper-dependent release complex. Biophys J 91:1832–1843

    Article  CAS  Google Scholar 

  58. Wilder PT, Varney KM, Weiss MB, Gitti RK, Weber DJ (2005) Solution structure of zinc- and calcium-bound rat S100B as determined by nuclear magnetic resonance spectroscopy. Biochemistry 44:5690–5702

    Article  CAS  Google Scholar 

  59. Koch M, Bhattacharya S, Kehl T, Gimona M, Vasak M, Chazin W, Heizmann CW, Kroneck PM, Fritz G (2007) Implications on zinc binding to S100A2. Biochim Biophys Acta 1773:457–470

    Article  CAS  Google Scholar 

  60. Schäfer BW, Fritschy JM, Murmann P, Troxler H, Durussel I, Heizmann CW, Cox JA (2000) Brain S100A5 is a novel calcium-, zinc-, and copper ion-binding protein of the EF-hand superfamily. J Biol Chem 275:30623–30630

    Article  Google Scholar 

  61. Sturchler E, Cox JA, Durussel I, Weibel M, Heizmann CW (2006) S100A16, a novel calcium-binding protein of the EF-hand superfamily. J Biol Chem 281:38905–38917

    Article  CAS  Google Scholar 

  62. Cuden LS, Gaillard A, Nolan EM (2016) Calcium ions tune the zinc-sequestering properties and antimicrobial activity of human S100A12. Chem Sci 7:1338–1348

    Article  Google Scholar 

  63. Ecsédi P, Kiss B, Gógl G, Radnai L, Buday L, Koprivanacz K, Liliom K, Leveles I, Vértessy B, Jeszenői N, Hetényi C, Schlosser G, Katona G, Nyitray L (2017) Regulation of the equilibrium between closed and open conformations of Annexin A2 by N-terminal phosphorylation and S100A4-binding. Structure 25:1195–1207

    Article  Google Scholar 

  64. Studier FW (2014) Chapter 2: Stable expression clones and auto-induction for protein production in E. coli. Methods Mol Biol 1091:17–32

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Institutes of Health R01AI101171 (EPS and WJC) and the National Science Foundation HRD1547757 (SMD).

Author information

Author notes

  1. Natalia Kozlyuk, Andrew J. Monteith, and Velia Garcia are contributed equally to the chapter.

Authors and Affiliations

  1. Department of Biochemistry, Vanderbilt University, Nashville, TN, USA

    Natalia Kozlyuk, Steven M. Damo & Walter J. Chazin

  2. Center for Structural Biology, Vanderbilt University, Nashville, TN, USA

    Natalia Kozlyuk, Velia Garcia, Steven M. Damo & Walter J. Chazin

  3. Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN, USA

    Andrew J. Monteith & Eric P. Skaar

  4. Department of Chemistry, Vanderbilt University, Nashville, TN, USA

    Velia Garcia & Walter J. Chazin

  5. Department of Life and Physical Sciences, Fisk University, Nashville, TN, USA

    Steven M. Damo

Authors
  1. Natalia Kozlyuk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andrew J. Monteith
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Velia Garcia
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Steven M. Damo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Eric P. Skaar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Walter J. Chazin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Walter J. Chazin .

Editor information

Editors and Affiliations

  1. Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zürich, Zürich, Switzerland

    Claus W. Heizmann

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Kozlyuk, N., Monteith, A.J., Garcia, V., Damo, S.M., Skaar, E.P., Chazin, W.J. (2019). S100 Proteins in the Innate Immune Response to Pathogens. In: Heizmann, C. (eds) Calcium-Binding Proteins of the EF-Hand Superfamily. Methods in Molecular Biology, vol 1929. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9030-6_18

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9030-6_18

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9029-0

  • Online ISBN: 978-1-4939-9030-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation