Binding and Transport in BtuB

An in vitro method for measuring the affinity of the E. coli outer membrane transporter, BtuB, for cyanocobalamin (CNCbl) using fluorescence quenching was designed and found capable of accurately measuring affinities in the low nanomolar (K D ~ 5nM) to sub-micromolar range (K D ~ 350nM). Single alanine mutations were introduced into the BtuB binding site for CNCbl and the resulting affinities measured. Two mutants, N57A and S65A, showed changes in affinity comparable to the loss of one hydrogen bond as would be expected from examining the wild-type binding site. The residue R497 was identified as a binding hotspot that when mutated to alanine resulted in only non-specific interactions with CNCbl. The R497A mutant was then crystallized and the x-ray structure solved by molecular replacement to a resolution of 2.4 . This structure was virtually superimposable onto wild-type BtuB (backbone rmsd = 0.3). Molecular dynamics simulations showed that when CNCbl is docked in the R497A binding site, the mobility of the CNCbl linker moiety is similar to that of CNCbl in solution. Furthermore, the R497A mutant had wild-type affinity for cobinamide (a truncated analog of CNCbl). This highlights the role of R497 in stabilizing the CNCbl linker moiety. Active transport conformations of BtuB were examined in vivo by measuring the accessibility of a water soluble probe to 91 individual cysteine mutations in BtuB. The results indicate that the interior of the barrel domain (-strands 16 through 22) remained inaccessible to probe during the transport process while core residues of the luminal domain become more accessible. In particular, a stretch of 10 luminal domain residues forming an alpha helix and a connecting loop were all identified as energydependent labelers. This is in contrast to the lack of labeling of adjacent barrel residues. Almost all residues that labeled in an energy-dependent fashion formed core interactions within the luminal domain. The findings are inconsistent with a transport model where the luminal domain acts like a structured "plug" during transport and suggest disruption of luminal domain core interactions are required for transport.

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