In Vitro Functional Characterization of BtuCD-F, the Escherichia coli ABC Transporter for Vitamin B₁₂ Uptake

Abstract

BtuCD is an ATP binding cassette (ABC) transporter that facilitates uptake of vitamin B(12) into the cytoplasm of Escherichia coli. The crystal structures of BtuCD and its cognate periplasmic binding protein BtuF have been recently determined. We have now explored BtuCD-F function in vitro, both in proteoliposomes and in various detergents. BtuCD reconstituted into proteoliposomes has a significant basal ATP hydrolysis rate that is stimulated by addition of BtuF and inhibited by sodium ortho-vanadate. When using different detergents to solubilize BtuCD, the basal ATP hydrolysis rate, the ability of BtuF to stimulate hydrolysis, and the extent to which sodium ortho-vanadate inhibits ATP hydrolysis all vary significantly. Reconstituted BtuCD can mediate transport of vitamin B(12) against a concentration gradient when coupled to ATP hydrolysis by BtuD in the liposome lumen and BtuF outside the liposomes. These in vitro studies establish the functional competence of the BtuCD and BtuF preparations used in the crystallographic analyses for both ATPase and transport activities. Furthermore, the tight binding of BtuF to BtuCD under the conditions studied suggests that the binding protein may not dissociate from the transporter during the catalytic cycle, which may be relevant to the mechanisms of other ABC transporter systems.

A single intact ATPase site of the ABC transporter BtuCD drives 5% transport activity yet supports full in vivo vitamin B 12 utilization

“…S4). This modest stimulation is very similar to what was observed in the past with the nontandem BtuC 2 D 2 (32). From the initial rates of ATP hydrolysis in liposomes and initial transport rates in spheroplasts, we estimated the ATP/substrate ratio of the transport reaction (Tables S1-S3).…”

“…These values are 40-50% lower (i.e., better coupling efficiency) than those determined using transport data from a reconstituted system (32). These differences may indicate that in vivo the coupling is more efficient, or are merely a result of the technical differences between the two experimental systems.…”

“…BtuC 2 D 2 , BtuC 2 -tandem BtuD, double-site, and single-site mutants were reconstituted essentially as previously described (29,32) only that 0.1% (wt/vol) n-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was used throughout the purification and reconstitution process. The lipid: protein ratio used was 30:1 (wt/wt).…”

“…Unlike the maltose transporter, BtuC 2 D 2 has high levels of basal ATP hydrolysis rates. These rates are only mildly stimulated by the substrate-free and substrate-loaded binding protein alike (32). BtuC 2 D 2 (the transporter) and BtuF (the vitamin B 12 binding protein) form an extremely high-affinity, stable complex (K D ∼ 10 −12 M), whereas in the maltose system the complex is transient and is of very low affinity (K D = 50-100 10 −6 M).…”

Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

“…In proteoliposome-based assays, the first reported for a type III BP (38), the addition of the BtuF protein does stimulate the ATPase activity of the transporter, though only ϳ2-fold, as the transporter has a high basal activity. BtuF displays a high affinity for the transporter, suggesting that it may not cycle on and off the transporter as depicted for maltose-BP.…”