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Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli

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Abstract

ZnuA is the periplasmic Zn2+-binding protein associated with the high-affinity ATP-binding cassette ZnuABC transporter from Escherichia coli. Although several structures of ZnuA and its homologs have been determined, details regarding metal ion stoichiometry, affinity, and specificity as well as the mechanism of metal uptake and transfer remain unclear. The crystal structures of E. coli ZnuA (Eco-ZnuA) in the apo, Zn2+-bound, and Co2+-bound forms have been determined. ZnZnuA binds at least two metal ions. The first, observed previously in other structures, is coordinated tetrahedrally by Glu59, His60, His143, and His207. Replacement of Zn2+ with Co2+ results in almost identical coordination geometry at this site. The second metal binding site involves His224 and several yet to be identified residues from the His-rich loop that is unique to Zn2+ periplasmic metal binding receptors. Electron paramagnetic resonance and X-ray absorption spectroscopic data on CoZnuA provide additional insight into possible residues involved in this second site. The second site is also detected by metal analysis and circular dichroism (CD) titrations. Eco-ZnuA binds Zn2+ (estimated K d < 20 nM), Co2+, Ni2+, Cu2+, Cu+, and Cd2+, but not Mn2+. Finally, conformational changes upon metal binding observed in the crystal structures together with fluorescence and CD data indicate that only Zn2+ substantially stabilizes ZnuA and might facilitate recognition of ZnuB and subsequent metal transfer.

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Abbreviations

ABC:

ATP-binding cassette

ANS:

8-Anilino-1-naphthalenesulfonic acid

apoZnuA:

Apo form of ZnuA protein from Escherichia coli

CD:

Circular dichroism

CoZnuA:

Co2+-loaded form of ZnuA protein from Escherichia coli

Eco-ZnuA:

ZnuA protein from Escherichia coli

EPR:

Electron paramagnetic resonance

EXAFS:

Extended X-ray absorption fine structure

HEPES:

N-(2-Hydroxyethyl)piperazine-N′-ethanesulfonic acid

Hin-Pzp1:

Zn2+-binding protein from Haemophilus influenzae

ICP-AES:

Inductively coupled plasma atomic emission spectroscopy

MBR:

Metal-binding receptor

MF:

Mag-Fura-2

MOPS:

3-(N-Morpholino)propanesulfonic acid

PDB:

Protein Data Bank

PEG:

Poly(ethylene glycol)

PLBP:

Periplasmic ligand-binding proteins

RMSD:

RMS deviation

Spn-PsaA:

Mn2+-binding protein from Streptococcus pneumoniae

Syn-MntC:

Mn2+-binding protein from Synechocystis PCC sp. 6803

Syn-ZnuA:

Zn2+-binding protein from Synechocystis PCC sp. 6803

Tpa-TroA:

Mn2+-binding protein from Treponema pallidum

Tris:

Tris(hydroxymethly)aminomethane

Znu:

Zn2+-specific uptake system

ZnZnuA:

Zn2+-loaded form of ZnuA protein from Escherichia coli

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Acknowledgements

This work was supported by National Institutes of Health Grants GM079411 (to M.W.C.), AI056231 and EB001980 (to B. B.), P20RR-16480 (to D.L.T.), and GM58518 (to A.C.R.). Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-06CH11357. We thank the staff at the APS DND-CAT, SBC-CAT, and GM/CA-CAT beamlines for assistance with data collection, P. Focia for preliminary data collection, the Northwestern Keck facility for use of the fluorimeter and UV/vis spectrometer, and J. Argüello, E. Eren, B. Mitra, and M. Golynskiy for valuable discussions.

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Authors and Affiliations

  1. Departments of Biochemistry, Molecular Biology, and Cell Biology and of Chemistry, Northwestern University, Evanston, IL, 60208, USA

    Liliya A. Yatsunyk, Lydia R. Kim & Amy C. Rosenzweig

  2. Department of Chemistry and Biochemistry, Miami University, Oxford, OH, 45056, USA

    J. Allen Easton, Stacy A. Sugarbaker & Michael W. Crowder

  3. National Biomedical EPR Center, Department of Biophysics, Medical College of Wisconsin, Milwaukee, WI, 53226, USA

    Brian Bennett

  4. Department of Chemistry and Chemical Biology, University of New Mexico, Albuquerque, NM, 87131, USA

    Robert M. Breece & David L. Tierney

  5. Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611, USA

    Ivan I. Vorontsov

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  1. Liliya A. Yatsunyk
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  2. J. Allen Easton
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  3. Lydia R. Kim
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Corresponding authors

Correspondence to Michael W. Crowder or Amy C. Rosenzweig.

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Yatsunyk, L.A., Easton, J.A., Kim, L.R. et al. Structure and metal binding properties of ZnuA, a periplasmic zinc transporter from Escherichia coli . J Biol Inorg Chem 13, 271–288 (2008). https://doi.org/10.1007/s00775-007-0320-0

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  • DOI: https://doi.org/10.1007/s00775-007-0320-0

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