Evidence of Ball-and-chain Transport of Ferric Enterobactin through FepA

Abstract

The Escherichia coli iron transporter, FepA, has a globular N terminus that resides within a transmembrane ␤-barrel formed by its C terminus. We engineered 25 cysteine substitution mutations at different locations in FepA and modified their sulfhydryl side chains with fluorescein maleimide in live cells. The reactivity of the Cys residues changed, sometimes dramatically, during the transport of ferric enterobactin, the natural ligand of FepA. Patterns of Cys susceptibility reflected energy-and TonB-dependent motion in the receptor protein. During transport, a residue on the normally buried surface of the N-domain was labeled by fluorescein maleimide in the periplasm, providing evidence that the transport process involves expulsion of the globular domain from the ␤-barrel. Porin deficiency much reduced the fluoresceination of this site, confirming the periplasmic labeling route. These data support the previously proposed, but never demonstrated, ball-and-chain theory of membrane transport. Functional complementation between a separately expressed N terminus and C-terminal ␤-barrel domain confirmed the feasibility of this mechanism.FepA is a Gram-negative bacterial outer membrane (OM) 6 protein that transports ferric enterobactin (FeEnt) (1-3). The crystal structures of FepA (4) and other bacterial metal transporters (FhuA, BtuB,57)), contain a C-terminal, 22-stranded ␤-barrel, placing them in the porin superfamily (5). Their ϳ150-residue globular N termini (N-domain; see Fig. 1) reside within their ␤-barrels. This architecture is potentially consistent with the "ball-and-chain" mechanism of membrane transport, whereby the globule controls solute (ligand) uptake by moving in and out of the channel. This process was postulated for nervous system channels (6), but no demonstrated examples of ball-and-chain transport are known.FepA and its relatives are unlike other porins (7, 8), because they selectively adsorb metal chelates with high affinity (3, 9 -14). Ligand binding causes small conformational changes that activate them to transport competency (15-17), hence their designation "ligand-gated porin" (LGP). The requirements for metabolic energy (18 -20) and another cell envelope protein, TonB (21-24), in LGP mediated transport are well known but unaccounted for: the OM has no source of energy and cannot sustain an ion gradient because of its open porin channels (7); TonB is a minor cell envelope protein whose functions are not yet understood.In live cells, FepA binds and transports FeEnt via sub-reactions with different dependences on energy and TonB. (i) In the absence of ligand the receptor opens, and its flexible surface loops extend outward (25). (ii) FeEnt binds to FepA in a biphasic reaction (26) that begins with adsorption to aromatic amino acids in the loop extremities (27, 28). Multiple determinants in multiple loops, including L7 (25), converge on the iron complex, creating a closed conformation that associates the negatively charged (Ϫ3), catecholate iron center with basic and aromatic residues in the r...

Studies on colicin B translocation: FepA is gated by TonB

“…Obvious pathways for transport of ligands are not apparent, raising the question of whether the globular domain exits the b-barrel during ligand transport or colicin translocation. It was predicted (Vakharia and Postle, 2002) and subsequently demonstrated that, when expressed as individual peptides, the globular domain and the b-barrel were capable of restoring transport through interprotein complementation Ma et al, 2007). These data suggested that the internal Fig.…”

“…When tethered near the amino-terminal end of the globular domain by disulphides between the amino-terminus and the barrel wall, the TonB-dependent transporters FepA and FhuA were inactive Ma et al, 2007). In the case of FhuA, the addition of reducing agent dithiothreitol restored transport in vivo .…”

“…Previous in vivo and in vitro investigations suggested that exposure of the TonB box in the periplasm occurs in the absence of TonB. (Merianos et al, 2000;Coggshall et al, 2001;Cadieux et al, 2003;Ma et al, 2007). According to this model, exposure of the TonB box signals to TonB that a ligandoccupied transporter is ready and waiting for TonB energy transduction.…”

“…The TonB-dependence of T13C labelling was unexpected. In studies on the BtuB or FepA TonB box, the absence of TonB did not prevent ligand-specific increases in Cys residue labelling, or exposure of TonB box-specific spin labels to the external medium (Merianos et al, 2000;Cadieux et al, 2003;Xu et al, 2006;Ma et al, 2007).…”

“…The transporter/receptors are characterized by the b-barrel structure found in virtually all OM proteins, and in addition, an amino-terminal globular domain of~150 residues that occupies the lumen of the barrel (Ferguson et al, 1998;Locher et al, 1998;Buchanan et al, 1999;van der Helm et al, 2002;Yue et al, 2003;Chimento et al, 2005;Cobessi et al, 2005a,b;Cherezov et al, 2006). The amino-terminal globular domain has been referred to as a hatch, cork or plug -terms implying that it transits out of the barrel, although that has not been demonstrated definitively and is a point of controversy Endriss et al, 2003;Eisenhauer et al, 2005;Chakraborty et al, 2006;Ma et al, 2007).…”