Cysteine scanning mutagenesis of TM5 reveals conformational changes in OxlT, the oxalate transporter of Oxalobacter formigenes.
We constructed a single-cysteine panel encompassing TM5 of the oxalate transporter, OxlT. The 25 positions encompassed by TM5 were largely tolerant of mutagenesis, and functional product was recovered for 21 of the derived variants. For these derivatives, thiol-directed MTS-linked agents (MTSEA, MTSCE, and MTSES) were used as probes of transporter function, yielding 11 mutants that responded to probe treatment, as indicated by effects on oxalate transport. Further study identified three biochemical phenotypes among these responders. Group 1 included seven mutants, exemplified by G151C, displaying substrate protection against probe inhibition. Group 2 was comprised of a single mutant, P156C, which had unexpected behavior. In this case, we observed increased activity if weak acid/base or neutral probes were used, while exposure to probes introducing a fixed charge led to decreased function. In both instances, the presence of substrate prevented the observed response. Group 3 contained three mutants (e.g., S143C) in which probe sensitivity was increased by the presence of substrate. The finding of substrate-protectable probe modification in groups 1 and 2 suggests that TM5 lies on the permeation pathway, as do its structural counterparts, TM2, TM8, and TM11. In addition, we speculate that substrate binding facilitates TM5 conformational changes that allow new regions to become accessible to MTS-linked probes (group 3). These biochemical data are consistent with the recently developed OxlT homology model.
Wang
Xicheng
X
Department of Physiology, Johns Hopkins Medical School, Baltimore, Maryland 21205, USA.
Ye
Liwen
L
McKinney
Caleb C
CC
Feng
Mingye
M
Maloney
Peter C
PC
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D001692:Biological Transport; D003545:Cysteine / Q000737:chemistry*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D008954:Models, Biological; D008968:Molecular Conformation; D016296:Mutagenesis; D009154:Mutation; D015345:Oligonucleotide Probes / Q000737:chemistry; D020624:Oxalobacter formigenes / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D001426:Bacterial Proteins; C488962:GlpT protein, E coli; D026901:Membrane Transport Proteins; D015345:Oligonucleotide Probes; C099549:OxlT protein, bacteria; D003545:Cysteine