Cysteine-mediated decyanation of vitamin B12 by the predicted membrane transporter BtuM

Abstract

Uptake of vitamin B12 is essential for many prokaryotes, but in most cases the membrane proteins involved are yet to be identified. We present the biochemical characterization and high-resolution crystal structure of BtuM, a predicted bacterial vitamin B12 uptake system. BtuM binds vitamin B12 in its base-off conformation, with a cysteine residue as axial ligand of the corrin cobalt ion. Spectroscopic analysis indicates that the unusual thiolate coordination allows for decyanation of vitamin B12. Chemical modification of the substrate is a property other characterized vitamin B12-transport proteins do not exhibit.

Fig. 1

Function and structure of BtuM_Td. a Schematic representation of cobalamin (Cbl) showing the corrinoid ring with the central cobalt ion (red). The ligand at the β-axial position is in this case a cyano-group, but differs in various Cbl variants (Supplementary Figure 1a, b). The ligand at the α-axial position (base-on conformation) is the 5,6-dimethylbenzimidazole base, which is covalently linked to the corrinoid ring. When this coordination is lost, Cbl is termed base-off. Cbi lacks the 5,6-dimethylbenzimidazole base (indicated by the zigzagged red line). b Growth assays with E. coli ΔFEC was conducted in the presence of 50 μg ml⁻¹ l-methionine or 1 nM Cbl. Additional experiments in the presence of different Cbl concentration are shown in Supplementary Figure 2f and g. All growth curves are averages of nine experiments (three biological triplicates, each with three technical replicates). Top panel: cells containing the empty expression vector (pBAD24) in the presence of methionine (blue line) or Cbl (grey line) and cells expressing the BtuCDF system (black and red lines, respectively). Bottom panel: cells expressing BtuM_Td (black and red lines) or mutant BtuM_Td_C80S (blue and grey) in the presence of methionine and Cbl, respectively. The inset displays a western blot showing that the mutant is expressed to wild-type levels (the full-length western blot can be found in Supplementary Figure 2h). c The structure of BtuM_Td in cartoon representation, coloured from blue (N terminus) to red (C terminus) and viewed from the membrane plane. α-helices (H1-6) and connecting loops (L1-5) are indicated. Cbl is shown in stick representation with carbon atoms coloured wheat, the oxygen and nitrogen atoms in red and blue, respectively, the cobalt ion in pink. Four n-nonyl-β-d-glucopyranoside detergent molecules are also shown in stick representation (carbons in light grey)

Fig. 2

Binding of vitamin B12 by BtuM_Td. a Transparent surface representation (light grey) of the binding pocket of BtuM_Td with bound Cbl. The protein backbone is shown in blue. The Co-ion is coordinated by Cys80 located in L3 (Co to sulphur distance 2.7 Å) and His207 (Co to nitrogen distance 2.4 Å) from a neighbouring symmetry mate (Supplementary Figure 6). A complete description of the interactions of BtuM_Td with its substrate can be found in Supplementary Figure 12. b The spectrum of BtuM_Td_cHis8-bound Cbl (4.3 μM, black line) compared to unbound cyano-Cbl (2.4 μM, red line). The regions of the spectrum with major changes are indicated with arrows. c Same as b but with Cbi bound to the protein (9.2 μM black line), compared to unbound dicyano-Cbi (9 μM, red line). The regions of the spectrum with major changes are indicated with arrows. For comparison, a scaled spectrum of Cbl bound to BtuM_Td (light grey line) from b is included, showing that the spectrum of both substrates bound to the protein is virtually the same, indicating the same binding mode

Fig. 3

Cobinamide (Cbi) binding to BtuM_Td and BtuM_Td-catalysed decyanation. a Representative ITC-measurements of differently tagged BtuM_Td constructs. BtuM_Td with a C-terminal His-tag binds Cbi with a K_d value of 0.65 ± 0.27 μM (top). EPEA-tagged BtuM_Td binds Cbi with essentially the same affinity of K_d 0.58 ± 0.13 μM (middle). For the EPEA-tagged mutant version BtuM_Td_C80S K_d = 5.6 ± 2.8 μM (bottom). All ITC experiments were performed as technical triplicates, error is s.d. b Decyanation of Cbi catalysed by EPEA-tagged BtuM_Td. Upon addition of an excess of BtuM_Td to Cbi, the substrate is slowly decyanated, which can be followed spectroscopically (left) with the main spectral changes indicated by the arrows. The mutant BtuM_Td_C80S, did not catalyse decyanation (right). c Quantification (error bars are s.d. of technical triplicates) of decyanation reveals that the process is slow. The ratio of the absorption at 369 nm over 330 nm of BtuM_Td (black dots) was plotted as function of time. A mono-exponential decay function was fitted to the data (red line) to extract τ = 12 ± 0.7 min (s.d. of technical triplicates), which is comparable to the decyanation rate of the His-tagged protein and the process follows pseudo-first order kinetics (Supplementary Figure 8b, c). The ratio of absorption obtained with the cysteine mutant (open dots), which does not catalyse decyanation, is shown for comparison