Investigation of the structure and function of a Shewanella oneidensis arsenical-resistance family transporter.
The toxic metalloid arsenic is an abundant element and most organisms possess transport systems involved in its detoxification. One such family of arsenite transporters, the ACR3 family, is widespread in fungi and bacteria. To gain a better understanding of the molecular mechanism of arsenic transport, we report here the expression and characterization of a family member, So_ACR3, from the bacterium Shewanella oneidensis MR-1. Surprisingly, expression of this transporter in the arsenic-hypersensitive Escherichia coli strain AW3110 conferred resistance to arsenate, but not to arsenite. Purification of a C-terminally His-tagged form of the protein allowed the binding of putative permeants to be directly tested: arsenate but not arsenite quenched its intrinsic fluorescence in a concentration-dependent fashion. Fourier transform infrared spectroscopy showed that the purified protein was predominantly alpha-helical. A mutant bearing a single cysteine residue at position 3 retained the ability to confer arsenate resistance, and was accessible to membrane impermeant thiol reagents in intact cells. In conjunction with successful C-terminal tagging with oligohistidine, this finding is consistent with the experimentally-determined topology of the homologous human apical sodium-dependent bile acid transporter, namely 7 transmembrane helices and a periplasmic N-terminus, although the presence of additional transmembrane segments cannot be excluded. Mutation to alanine of the conserved residue proline 190, in the fourth putative transmembrane region, abrogated the ability of the transporter to confer arsenic resistance, but did not prevent arsenate binding. An apparently increased thermal stability is consistent with the mutant being unable to undergo the conformational transitions required for permeant translocation.
Xia
Xiaobing
X
Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, UK.
Postis
Vincent L G
VL
Rahman
Moazur
M
Wright
Gareth S A
GS
Roach
Peter C J
PC
Deacon
Sarah E
SE
Ingram
Jean C
JC
Henderson
Peter J F
PJ
Findlay
John B C
JB
Phillips
Simon E V
SE
McPherson
Michael J
MJ
Baldwin
Stephen A
SA
D001152:Arsenicals / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D001692:Biological Transport; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D011489:Protein Denaturation; D055550:Protein Stability; D020592:Shewanella / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship
D001152:Arsenicals; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins