Conformational Change of a Tryptophan Residue in BtuF Facilitates Binding and Transport of Cobinamide by the Vitamin B12 Transporter BtuCD-F

Abstract

BtuCD-F is an ABC transporter that mediates cobalamin uptake into Escherichia coli. Early in vivo data suggested that BtuCD-F might also be involved in the uptake of cobinamide, a cobalamin precursor. However, neither was it demonstrated that BtuCD-F indeed transports cobinamide, nor was the structural basis of its recognition known. We synthesized radiolabeled cyano-cobinamide and demonstrated BtuCD-catalyzed in vitro transport, which was ATP- and BtuF-dependent. The crystal structure of cobinamide-bound BtuF revealed a conformational change of a tryptophan residue (W66) in the substrate binding cleft compared to the structure of cobalamin-bound BtuF. High-affinity binding of cobinamide was dependent on W66, because mutation to most other amino acids substantially reduced binding. The structures of three BtuF W66 mutants revealed that tight packing against bound cobinamide was only provided by tryptophan and phenylalanine, in line with the observed binding affinities. In vitro transport rates of cobinamide and cobalamin were not influenced by the substitutions of BtuF W66 under the experimental conditions, indicating that W66 has no critical role in the transport reaction. Our data present the molecular basis of the cobinamide versus cobalamin specificity of BtuCD-F and provide tools for in vitro cobinamide transport and binding assays.

Structure of the human transcobalamin beta domain in four distinct states

“…The structure was solved by MR using the same truncated version of TC that was used for the structure solving of apo TC-beta as search model. The ligand structure and geometry-restraints files were downloaded from PDB ID 5M29 [ 31 ]. In the structure of TC-beta:AntiB12 one calcium molecules was placed, coordinated by one His305 and its backbone amide, which is part of the flexible N-terminus, as well as by two water molecules and the surface exposed His345 of a symmetry mate.…”

Structural basis of nanobody-mediated blocking of BtuF, the cognate substrate-binding protein of the Escherichia coli vitamin B12 transporter BtuCD

“…As a first step to assess the effect of the obtained nanobodies on BtuCD-F function, we determined the affinity of the selected nanobodies to BtuF and their ability to displace Cbl from BtuF. Cbl binding to BtuF was analyzed at pH 7.5 and 23 °C by measuring Cbl-induced fluorescence quenching of fluorescently labeled BtuF (BtuF fluo ), an assay developed and described previously 6 (Fig. 2A ).…”

“…BtuF for the pull-down, crystallization, binding and transport assays, was prepared as described previously 6 , 18 . The Avi-tagged BtuF construct for the panning was cloned by insertion of a C-terminal Avi-tag between the BtuF gene and the 3 C cleavable His-tag.…”

“…The ABC importer BtuCD-F catalyzes vitamin B12 (cyanocobalamin or Cbl) and cobinamide uptake into the cytoplasm of Escherichia coli 5 , 6 . Like other members of the ABC transporter superfamily, BtuCD-F couples the energy of ATP binding and hydrolysis to substrate translocation across lipid bilayers 7 .…”

Does a Conjugation Site Affect Transport of Vitamin B12–Peptide Nucleic Acid Conjugates into Bacterial Cells?

“…In this line, Locher et al. showed that one of the receptors (BtuCD‐F), which mediated Cbl uptake into E. coli , might also be involved in the uptake of cobinamide ( 2 ), which is a Cbl precursor that lacks the nucleotide loop . During the uptake process, vitamin B 12 binds to a series of receptors; therefore, alterations to the vitamin structure might have a profound effect on the extent of its uptake.…”