Ga3+ as a mechanistic probe in Fe3+ transport: characterization of Ga3+ interaction with FbpA.
The obligate human pathogens Haemophilus influenzae, Neisseria gonorrhoeae, and N. meningitidis utilize a highly conserved, three-protein ATP-binding cassette transporter (FbpABC) to shuttle free Fe(3+) from the periplasm and across the cytoplasmic membrane. The periplasmic binding protein, ferric binding protein (FbpA), is capable of transporting other trivalent cations, including Ga(3+), which, unlike Fe(3+), is not redox-active. Because of a similar size and charge as Fe(3+), Ga(3+) is widely used as a non-redox-active Fe(3+) substitute for studying metal complexation in proteins and bacterial populations. The investigations reported here elucidate the similarities and differences in FbpA sequestration of Ga(3+) and Fe(3+), focusing on metal selectivity and the resulting transport function. The thermodynamic binding constant for Ga(3+) complexed with FbpA at pH 6.5, in 50 mM 4-morpholineethanesulfonic acid, 200 mM KCl, 5 mM KH(2)PO(4) was determined by UV-difference spectroscopy as log K'eff=13.7+/-0.6. This represents a 10(5)-fold weaker binding relative to Fe(3+) at identical conditions. The unfolding/refolding behavior of Ga(3+) and Fe(3+) holo-FbpA were also studied using a matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy technique, stability of unpurified proteins from rates of H/D exchange (SUPREX). This analysis indicates significant differences between Fe(3+) and Ga(3+) sequestration with regard to protein folding behavior. A series of kinetic experiments established the lability of the Ga(3+)FbpA-PO(4) assembly, and the similarities/differences of stepwise loading of Fe(3+) into apo- or Ga(3+)-loaded FbpA. These biophysical characterization data are used to interpret FbpA-mediated Ga(3+) transport and toxicity in cell culture studies.
Weaver
Katherine D
KD
Department of Chemistry, Duke University, Durham, NC 27708-0346, USA.
Heymann
Jared J
JJ
Mehta
Arnav
A
Roulhac
Petra L
PL
Anderson
Damon S
DS
Nowalk
Andrew J
AJ
Adhikari
Pratima
P
Mietzner
Timothy A
TA
Fitzgerald
Michael C
MC
Crumbliss
Alvin L
AL
D005290:Ferric Compounds / Q000737:chemistry* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D005708:Gallium / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D006193:Haemophilus influenzae / Q000187:drug effects / Q000254:growth & development; D033862:Iron-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013816:Thermodynamics
D005290:Ferric Compounds; D005456:Fluorescent Dyes; D033862:Iron-Binding Proteins; D033902:Periplasmic Binding Proteins; D005708:Gallium