N-Terminal region of CusB is sufficient for metal binding and metal transfer with the metallochaperone CusF

Tiffany D. Mealman, Mowei Zhou, Trisiani Affandi, Kelly N. Chacón, Mariana E. Aranguren, Ninian Blackburn, Vicki H. Wysocki, Megan M. McEvoy

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Gram-negative bacteria, such as Escherichia coli, utilize efflux resistance systems in order to expel toxins from their cells. Heavy-metal resistance is mediated by resistance nodulation cell division (RND)-based efflux pumps composed of a tripartite complex that includes an RND-transporter, an outer-membrane factor (OMF), and a membrane fusion protein (MFP) that spans the periplasmic space. MFPs are necessary for complex assembly and have been hypothesized to play an active role in substrate efflux. Crystal structures of MFPs are available, however incomplete, as large portions of the apparently disordered N- and C-termini are unresolved. Such is the case for CusB, the MFP of the E. coli Cu(I)/Ag(I) efflux pump CusCFBA. In this work, we have investigated the structure and function of the N-terminal region of CusB, which includes the metal-binding site and is missing from previously determined crystal structures. Results from mass spectrometry and X-ray absorption spectroscopy show that the isolated N-terminal 61 residues (CusB-NT) bind metal in a 1:1 stoichiometry with a coordination site composed of M21, M36, and M38, consistent with full-length CusB. NMR spectra show that CusB-NT is mostly disordered in the apo state; however, some slight structure is adopted upon metal binding. Much of the intact protein's function is maintained in this fragment as CusB-NT binds metal in vivo and in vitro, and metal is transferred between the metallochaperone CusF and CusB-NT in vitro. Functional analysis in vivo shows that full-length CusB is necessary in an intact polypeptide for full metal resistance, though CusB-NT alone can contribute partial metal resistance. These findings reinforce the theory that the role of CusB is not only to bind metal but also to play an active role in efflux.

Original languageEnglish (US)
Pages (from-to)6767-6775
Number of pages9
JournalBiochemistry
Volume51
Issue number34
DOIs
StatePublished - Aug 28 2012

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Metallochaperones
Metals
Membrane Fusion Proteins
Cell Division
Escherichia coli
Crystal structure
X-Ray Absorption Spectroscopy
Pumps
Periplasm
Functional analysis
X ray absorption spectroscopy
Membrane Transport Proteins
Heavy Metals
Gram-Negative Bacteria
Stoichiometry
Mass spectrometry
Mass Spectrometry
Bacteria
Binding Sites
Cells

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mealman, T. D., Zhou, M., Affandi, T., Chacón, K. N., Aranguren, M. E., Blackburn, N., ... McEvoy, M. M. (2012). N-Terminal region of CusB is sufficient for metal binding and metal transfer with the metallochaperone CusF. Biochemistry, 51(34), 6767-6775. https://doi.org/10.1021/bi300582

N-Terminal region of CusB is sufficient for metal binding and metal transfer with the metallochaperone CusF. / Mealman, Tiffany D.; Zhou, Mowei; Affandi, Trisiani; Chacón, Kelly N.; Aranguren, Mariana E.; Blackburn, Ninian; Wysocki, Vicki H.; McEvoy, Megan M.

In: Biochemistry, Vol. 51, No. 34, 28.08.2012, p. 6767-6775.

Research output: Contribution to journalArticle

Mealman, TD, Zhou, M, Affandi, T, Chacón, KN, Aranguren, ME, Blackburn, N, Wysocki, VH & McEvoy, MM 2012, 'N-Terminal region of CusB is sufficient for metal binding and metal transfer with the metallochaperone CusF', Biochemistry, vol. 51, no. 34, pp. 6767-6775. https://doi.org/10.1021/bi300582
Mealman, Tiffany D. ; Zhou, Mowei ; Affandi, Trisiani ; Chacón, Kelly N. ; Aranguren, Mariana E. ; Blackburn, Ninian ; Wysocki, Vicki H. ; McEvoy, Megan M. / N-Terminal region of CusB is sufficient for metal binding and metal transfer with the metallochaperone CusF. In: Biochemistry. 2012 ; Vol. 51, No. 34. pp. 6767-6775.
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abstract = "Gram-negative bacteria, such as Escherichia coli, utilize efflux resistance systems in order to expel toxins from their cells. Heavy-metal resistance is mediated by resistance nodulation cell division (RND)-based efflux pumps composed of a tripartite complex that includes an RND-transporter, an outer-membrane factor (OMF), and a membrane fusion protein (MFP) that spans the periplasmic space. MFPs are necessary for complex assembly and have been hypothesized to play an active role in substrate efflux. Crystal structures of MFPs are available, however incomplete, as large portions of the apparently disordered N- and C-termini are unresolved. Such is the case for CusB, the MFP of the E. coli Cu(I)/Ag(I) efflux pump CusCFBA. In this work, we have investigated the structure and function of the N-terminal region of CusB, which includes the metal-binding site and is missing from previously determined crystal structures. Results from mass spectrometry and X-ray absorption spectroscopy show that the isolated N-terminal 61 residues (CusB-NT) bind metal in a 1:1 stoichiometry with a coordination site composed of M21, M36, and M38, consistent with full-length CusB. NMR spectra show that CusB-NT is mostly disordered in the apo state; however, some slight structure is adopted upon metal binding. Much of the intact protein's function is maintained in this fragment as CusB-NT binds metal in vivo and in vitro, and metal is transferred between the metallochaperone CusF and CusB-NT in vitro. Functional analysis in vivo shows that full-length CusB is necessary in an intact polypeptide for full metal resistance, though CusB-NT alone can contribute partial metal resistance. These findings reinforce the theory that the role of CusB is not only to bind metal but also to play an active role in efflux.",
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AU - Mealman, Tiffany D.

AU - Zhou, Mowei

AU - Affandi, Trisiani

AU - Chacón, Kelly N.

AU - Aranguren, Mariana E.

AU - Blackburn, Ninian

AU - Wysocki, Vicki H.

AU - McEvoy, Megan M.

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N2 - Gram-negative bacteria, such as Escherichia coli, utilize efflux resistance systems in order to expel toxins from their cells. Heavy-metal resistance is mediated by resistance nodulation cell division (RND)-based efflux pumps composed of a tripartite complex that includes an RND-transporter, an outer-membrane factor (OMF), and a membrane fusion protein (MFP) that spans the periplasmic space. MFPs are necessary for complex assembly and have been hypothesized to play an active role in substrate efflux. Crystal structures of MFPs are available, however incomplete, as large portions of the apparently disordered N- and C-termini are unresolved. Such is the case for CusB, the MFP of the E. coli Cu(I)/Ag(I) efflux pump CusCFBA. In this work, we have investigated the structure and function of the N-terminal region of CusB, which includes the metal-binding site and is missing from previously determined crystal structures. Results from mass spectrometry and X-ray absorption spectroscopy show that the isolated N-terminal 61 residues (CusB-NT) bind metal in a 1:1 stoichiometry with a coordination site composed of M21, M36, and M38, consistent with full-length CusB. NMR spectra show that CusB-NT is mostly disordered in the apo state; however, some slight structure is adopted upon metal binding. Much of the intact protein's function is maintained in this fragment as CusB-NT binds metal in vivo and in vitro, and metal is transferred between the metallochaperone CusF and CusB-NT in vitro. Functional analysis in vivo shows that full-length CusB is necessary in an intact polypeptide for full metal resistance, though CusB-NT alone can contribute partial metal resistance. These findings reinforce the theory that the role of CusB is not only to bind metal but also to play an active role in efflux.

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