Descripción de los procesos y procedimiento del área de facturación de la

Escherichia coli BtuF Cobalamin Binding Protein. BtuF is a cobalamin ABC transport

protein produced in Salmonella typhimurium and E. coli (Van Bibber et al., 1999; Locher, 2004; Locher & Borths, 2004). Mutagenesis studies conducted by Van Bibber et al. found that S. typhimurium BtuF is involved in cobalamin transport from the periplasm to the cytoplasm via its association with BtuC2 (homodimer membrane permease) (Van Bibber et al., 1999; Borths et al.,

2005). BtuC2 is associated with BtuD2 (homodimer ATPase). Cadieux et al. characterized BtuF

in E. coli and determined a cobalamin binding constant of approximately 15 nM (Cadieux et al., 2002). BtuF, BtuC, and BtuD deletion mutations resulted in cobalamin concentrating in the periplasm, and that all three proteins were required for cobalamin uptake into the cytoplasm.

The crystal structure of BtuC2D2 from E. coli was resolved to 3.2 Å by Locher and Rees

and co-workers (Locher et al., 2002; Locher & Borths, 2004). They proposed a mechanism in which BtuF interacts with BtuCD during cobalamin transport. In their model, BtuF associates with BtuC2, and the two BtuD subunits come together by pivoting up toward the periplasm.

BtuD2 is closely associated with BtuC2 via a hinged, clothes-pin type juncture, and its pivoting

results in the closing of the two BtuC subunits around cobalamin. A stable complex between the BtuF and BtuCD units was verified via SDS-PAGE.

Rees and co-workers determined the crystal structure of BtuF to 2.0 Å (Borths et al., 2002), which showed that BtuF was a bilobal protein connected by one α-helix, with a binding cleft between the two lobes, which identified it as a Group III heme binding protein (Quiocho & Ledvina, 1996; Borths et al., 2002). Rees and co-workers also proposed a docking mechanism in which two glutamate residues on BtuF associate electrostatically with arginine residues on BtuC. Crystallographic evidence showed that the glutamate residues are approximately 46 Å apart, with the binding cleft centered between them. The two positively charged pockets on BtuC are

approximately 48 Å apart, and contain two arginine residues in one pocket and one in the second pocket. Computer simulations showed that aligning the negative and positively charged areas resulted in the cobalamin cleft being located above the entrance of the permease. Hvorup et al. have also seen evidence for this association using crystallography (Hvorup et al., 2007). Sequence alignment has shown that the glutamate and arginine residues are conserved among

numerous iron and cobalamin transporters, and Borths et al. have suggested that their presence may create BtuF-BtuC salt bridges, which could be crucial for the docking of BtuF to BtuC (Borths et al., 2002).

Karpowich et al. have determined the crystal structure of E. coli BtuF at 3.0 Å resolution in the apo form, and 2.0 Å in the holo form (Karpowich et al., 2003). Karpowich et al. suggested that a conformational change caused by the unwinding of the C-terminal α-helix upon BtuF binding to BtuC may trigger the transport of cobalamin from BtuF to BtuC.

Oloo and Tieleman have conducted computer simulations that indicate the binding of one ATP to one BtuD subunit may cause both BtuD subunits to come together (Oloo & Tieleman, 2004). This in turn may trigger a conformational change in the BtuC transmembrane domain, which could create a passage for cobalamin to enter the cell. Their computer simulations also indicate that while ATP is tightly bound to one BtuD subunit, the binding site of the second BtuD subunit may be open and possibly empty. Oloo and Tieleman have suggested that ATP hydrolysis alternates between the two BtuD binding sites.

Ivetac et al. have performed molecular simulations indicating that a change occurs when BtuF docks with BtuCD. Their results have indicated that the opening of the BtuF binding site is coupled with the closing of both BtuD moieties mediated through the BtuC moieties (Ivetac et al., 2007). They proposed that BtuF may indirectly stabilize the transition state of ATP

hydrolysis while at the same time releasing cobalamin to BtuC. BtuD2 closure via ATP binding

could bring the catalytic regions of each BtuD together and thus create conformational changes in BtuC, in agreement with the findings of Oloo and Tieleman (Oloo & Tieleman, 2004).

Molecular simulations performed by Kandt et al. have indicated that the holoBtuF binding pocket cycles through open and closed conformations, with the closed conformation

being predominant (Kandt et al., 2006). In the absence of ligand the pocket also fluctuates, but tends more towards the open conformation, and has a larger range of motion than holoBtuF.

Escherichia coli FhuD Ferrichrome Binding Protein. FhuD (ferric hydroximate uptake D) is

a 29.7 kDa periplasmic binding protein that binds ferrichrome and other Fe(III) hydroximates (Rohrbach et al., 1995; Braun & Braun, 2002; Garcia-Herrero et al., 2007). It is associated with FhuCB, where FhuC and FhuB are the ATPase and membrane permease, respectively (Braun et al., 2004; Carter et al., 2006).

The crystal structure of FhuD with gallichrome (1.9 Å) revealed two domains connected by a single helix, indicating that FhuD is a member of the Group III binding proteins (Quiocho & Ledvina, 1996; Clarke et al., 2000; Braun & Braun, 2002; Braun et al., 2004). Arginine and tyrosine are in direct contact with the siderophore hydroximate moieties via hydrogen bonding (Clarke et al., 2000; Clarke et al., 2001).

Dissociation constants were determined to be 0.3, 0.4, 1, and 5.4 µM for ferric coprogen, ferric aerobactin, ferrichrome, and albomycin, respectively (Rohrbach et al., 1995). Mutations of tryptophan, alanine, and phenylalanine (W68L, A150S, and P175L, respectively) increased dissociation constants.

Campylobacter jejuni CeuE Enterobactin Binding Protein. CeuE is an α-helical periplasmic

ferric enterobactin-binding protein (Muller et al., 2006; Garcia-Herrero et al., 2007). The crystal structure at 2.4 Å resolution revealed a dimer with two iron mecams [1,3,5-N,N’,N’’-tris(2,3- dihydroxybenzoyl)triaminomethylbenzene] bridging the individual binding pockets of the protein, a structure similar to that of FhuD of E. coli (Muller et al., 2006).