Abstract
P1B-type ATPases transport transition metals across biological membranes. The chemical characteristics of these substrates, as well as, physiological requirements have contributed to the evolution of high metal binding affinities (fM) in these enzymes. Metal binding determinations are consequently facilitated by the stable metal–protein interaction, while affinity measurements require careful analysis of metal levels. In the cell, transition metals are associated with chaperone proteins. Metals reach the ATPase transport sites following specific protein–protein interactions and ligand exchange enabling the metal transfer from the chaperone to the transporter. Here, we describe methods for analyzing the binding of Cu+ to Cu+-ATPases, as well as the approach to monitor Cu+ transfer from soluble Cu+-chaperones donors to and from membrane Cu+-ATPases.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Solioz M, Abicht HK, Mermod M, Mancini S (2010) Response of Gram-positive bacteria to copper stress. J Biol Inorg Chem 15(1):3–14
Argüello JM, González-Guerrero M, Raimunda D (2011) Bacterial transition metal P1B-ATPases: transport mechanism and roles in virulence. Biochemistry 50(46):9940–9949
Argüello JM (2003) Identification of ion-selectivity determinants in heavy-metal transport P1B-type ATPases. J Membr Biol 195(2):93–108
Rensing C, Fan B, Sharma R, Mitra B, Rosen BP (2000) CopA: an Escherichia coli Cu(I)-translocating P-type ATPase. Proc Natl Acad Sci U S A 97(2):652–656
Osman D, Cavet JS (2008) Copper homeostasis in bacteria. Adv Appl Microbiol 65:217–247
Argüello JM, Raimunda D, González-Guerrero M (2012) Metal transport across biomembranes: emerging models for a distinct chemistry. J Biol Chem 287(17):13510–13517
Robinson NJ, Winge DR (2010) Copper metallochaperones. Annu Rev Biochem 79:537–562
Padilla-Benavides T, George Thompson AM, McEvoy MM, Argüello JM (2014) Mechanism of ATPase-mediated Cu+ export and delivery to periplasmic chaperones: the interaction of Escherichia coli CopA and CusF. J Biol Chem 289(30):20492–20501
González-Guerrero M, Argüello JM (2008) Mechanism of Cu+-transporting ATPases: soluble Cu+ chaperones directly transfer Cu+ to transmembrane transport sites. Proc Natl Acad Sci U S A 105(16):5992–5997
Argüello JM, Eren E, González-Guerrero M (2007) The structure and function of heavy metal transport P1B-ATPases. Biometals 20(3–4):233–248
González-Guerrero M, Eren E, Rawat S, Stemmler TL, Argüello JM (2008) Structure of the two transmembrane Cu+ transport sites of the Cu+-ATPases. J Biol Chem 283(44):29753–29759
Raimunda D, Subramanian P, Stemmler T, Argüello JM (2012) A tetrahedral coordination of Zinc during transmembrane transport by P-type Zn2+-ATPases. Biochim Biophys Acta 1818(5):1374–1377
Padilla-Benavides T, Long JE, Raimunda D, Sassetti CM, Argüello JM (2013) A novel P1B-type Mn2+-transporting ATPase is required for secreted protein metallation in mycobacteria. J Biol Chem 288(16):11334–11347
Zielazinski EL, Cutsail GE III, Hoffman BM, Stemmler TL, Rosenzweig AC (2012) Characterization of a cobalt-specific P(1B)-ATPase. Biochemistry 51(40):7891–7900
Liu J, Dutta SJ, Stemmler AJ, Mitra B (2006) Metal-binding affinity of the transmembrane site in ZntA: implications for metal selectivity. Biochemistry 45(3):763–772
Glynn IM, Karlish SJ (1990) Occluded cations in active transport. Annu Rev Biochem 59:171–205
Eren E, Kennedy DC, Maroney MJ, Argüello JM (2006) A novel regulatory metal binding domain is present in the C terminus of Arabidopsis Zn2+-ATPase HMA2. J Biol Chem 281(45):33881–33891
Rae TD, Schmidt PJ, Pufahl RA, Culotta VC, O’Halloran TV (1999) Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase. Science 284(5415):805–808
Padilla-Benavides T, McCann CJ, Argüello JM (2013) The mechanism of Cu+ transport ATPases: interaction with Cu+ chaperones and the role of transient metal-binding sites. J Biol Chem 288(1):69–78
González-Guerrero M, Hong D, Argüello JM (2009) Chaperone-mediated Cu+ delivery to Cu+ transport ATPases: requirement of nucleotide binding. J Biol Chem 284(31):20804–20811
Gourdon P et al (2011) Crystal structure of a copper-transporting PIB-type ATPase. Nature 475(7354):59–64
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Brenner AJ, Harris ED (1995) A quantitative test for copper using bicinchoninic acid. Anal Biochem 226(1):80–84
Yatsunyk LA, Rosenzweig AC (2007) Cu(I) binding and transfer by the N terminus of the Wilson disease protein. J Biol Chem 282(12):8622–8631
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Padilla-Benavides, T., Argüello, J.M. (2016). Assay of Copper Transfer and Binding to P1B-ATPases. In: Bublitz, M. (eds) P-Type ATPases. Methods in Molecular Biology, vol 1377. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3179-8_24
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3179-8_24
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3178-1
Online ISBN: 978-1-4939-3179-8
eBook Packages: Springer Protocols