Cysteine-Mediated Decyanation of Vitamin B12 by the Predicted Membrane Transporter Btum
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ARTICLE DOI: 10.1038/s41467-018-05441-9 OPEN Cysteine-mediated decyanation of vitamin B12 by the predicted membrane transporter BtuM S. Rempel 1, E. Colucci 1, J.W. de Gier 2, A. Guskov 1 & D.J. Slotboom 1,3 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. 1234567890():,; 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. 1 Groningen Biomolecular and Biotechnology Institute (GBB), University of Groningen, Nijenborgh 4, 9474 AG Groningen, The Netherlands. 2 Department of Biochemistry and Biophysics, Stockholm University, 10691 Stockholm, Sweden. 3 Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands. Correspondence and requests for materials should be addressed to D.J.S. (email: [email protected]) NATURE COMMUNICATIONS | (2018) 9:3038 | DOI: 10.1038/s41467-018-05441-9 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05441-9 fi obalamin (Cbl) is one of the most complex cofactors well as re nement statistics are summarized in Table 1. BtuMTd C(Supplementary Figure 1a) known, and used by enzymes consists of six transmembrane helices with both termini located catalyzing for instance methyl-group transfer and ribo- on the predicted cytosolic side (Fig. 1c). The amino acid nucleotide reduction reactions1,2. For example, in the methionine sequences of BtuM proteins are not related to any other protein5 synthase MetH, the cofactor is used to transfer a methyl moiety but, surprisingly, BtuMTd resembles the structure of S- onto L-homocysteine to produce L-methionine1,3. Many bacteria components from energy-coupling factor (ECF)-type ABC require Cbl for survival1,2,4,5, but only a small subset of prokar- transporters10 (Supplementary Figure 4 and Supplementary yotic species can produce this molecule de novo, via either an Table 1). In contrast to BtuM proteins, ECF-type ABC trans- aerobic or anaerobic pathway4,5. Roughly two thirds of archaea porters are predominantly found in Gram-positive bacteria. They and eubacteria are Cbl-auxotrophs that rely on uptake of either are multi-subunit complexes consisting of two peripheral Cbl or its precursor cobinamide2,5,6 (Cbi, Fig. 1a, Supplementary ATPases and two transmembrane components (EcfT and S- Figure 1b). Dependence on uptake has probably evolved, because component)10,11. EcfT and the ATPases together form the so- synthesis of Cbl involves roughly 30 different enzymes and is called ECF-module. S-components bind the transported sub- energetically costly. Gram-negative bacteria require the TonB- strate, and dynamically associate with the ECF-module to allow dependent transporter BtuB1 to transport Cbl across the outer substrate translocation10,12–14. Intriguingly, no homologues of membrane (Supplementary Figure 1c). For subsequent transport EcfT could be found in T. denitrificans. In addition, all ABC-type of vitamin B12 across the cytoplasmic membrane, the only ATPases encoded by the organism are predicted to be part of characterized bacterial uptake system is the ABC transporter classical ABC transporters, and not ECF transporters. Therefore, BtuCDF, which is predicted to be present in approximately 50% we conclude that the organism does not encode an ECF-module, 5,7 of Cbl-auxotrophic bacteria . Many Cbl-auxotrophic Gram- and hypothesize that solitary BtuMTd may be responsible for Cbl negative bacteria do not encode BtuCDF, whereas they do contain uptake. This hypothesis is supported by the ability of BtuMTd to BtuB. Metabolic reconstruction and chromosomal context ana- transport vitamin B12 when expressed heterologously in E. coli lyses, e.g. co-localization with the gene for BtuB, have identified ΔFEC. Importantly, E.coli also does not encode an ECF- 11 potential alternative inner membrane vitamin B12 transporters, module , hence BtuMTd cannot interact with a module from 5 one of which is BtuM . BtuM homologues are small membrane the host, and BtuMTd must be able to support Cbl uptake using a proteins of ~22 kDa, and found predominantly in Gram-negative different mechanism than that of ECF transporters10,11. In a few species, distributed mostly over α-, β-, and γ-proteobacteria cases, the biotin-specific S-component BioY15 has also been (Supplementary Data 1). found in organisms lacking an ECF-module and was shown to Here, we sought to characterize the predicted vitamin B12 mediate transport without the need for an ECF-module15. fi transporter BtuM from Thiobacillus denitri cans (BtuMTd). We However, organisms encoding only BioY without an ECF-module 15 show that BtuMTd is involved in transport of Cbl in vivo and we are rare , and in the large majority of organisms BioY is asso- solved its structure to 2 Å resolution. A cobalt–cysteine interac- ciated with an ECF-module11. In contrast, BtuM homologues tion allows for chemical modification of the substrate prior to (apart from one exception) are found exclusively in organisms translocation, which is a rare feature among uptake systems. lacking an ECF-module (Supplementary Data 1). Further experiments, for instance using purified protein Results reconstituted in proteoliposomes, are required to test whether BtuM also catalyses transport in vitro without any additional BtuM supports vitamin B12-dependent growth.Totest Td Td component involved. However, the in vivo assay gives a very experimentally whether BtuM is a potential Cbl-transporter, we Td strong indication that BtuM is a transporter itself, as the protein constructed an Escherichia coli triple knockout strain, E. coli ΔFEC, Td was expressed in a heterologous host that does not contain any based on Cadieux et al.8. In this strain, the gene encoding the Cbl- ECF-module or S-component. Similar in vivo experiments have independent methionine synthase, MetE9, is deleted. The metE been used extensively in the past to identify other transporters (for deletion makes it impossible for E. coli ΔFEC to synthesize instance ref.16) and have the advantage over in vitro assays that methionine, unless it can import Cbl8,9.Inthatcase,L-methionine physiologically relevant conditions are used. can be synthesized using the Cbl-dependent methionine synthase, MetH3,8. E. coli ΔFEC has additional deletions in btuF and btuC, encoding subunits of the endogenous Cbl-transporter BtuCDF7,8. Δ BtuMTd binds cobalamin using cysteine ligation. Close to the Therefore, E. coli FECcannotimportCbl,prohibitingMetH- fi predicted periplasmic surface of BtuMTd, we found well-de ned mediated L-methionine synthesis. Consequently, E. coli ΔFEC can electron density (Supplementary Figure 5) representing a bound grow only if L-methionine is present or if vitamin B12 import is Cbl molecule. The binding mode of Cbl in the crystal structure restored by (heterologous) expression of a Cbl-transport system8. (Fig. 2a) is striking for two reasons. First, the essential Cys80 is The phenotype of E. coli ΔFEC was confirmed in growth assays the α-axial ligand of the cobalt ion. To our knowledge, cobalt (Supplementary Figure 2a). Cells that are not expressing any Cbl- coordination by cysteine has not been observed in any other Cbl- transporter did not exhibit substantial growth in methionine-free binding protein of known structure. Binding of cysteine to cobalt medium, whereas cells complemented with an expression plasmid in a corrinoid has been hypothesized for the mercury methylating for BtuCDF grew readily (Fig. 1b). Cells expressing BtuM had a Td enzyme HgcA17 and observed in a synthetic cyclo-decapeptide, similar growth phenotype, indicating that BtuM is a potential Td but in the latter case the residue replaced the β-ligand18. transporter for vitamin B12 (Fig. 1b). Second, Cbl is bound to BtuMTd in the base-off conformation in which the 5,6-dimethylbenzimidazole moiety does not bind to Crystal structure of BtuMTd bound to vitamin B12. The BtuM the cobalt ion (Fig. 2a). In contrast, at physiological pH the family contains an invariably conserved cysteine residue (Sup- conformation of free Cbl in aqueous solution is base-on with the plementary Figure 3a). In BtuMTd, this cysteine is located at 5,6-dimethylbenzimidazole moiety coordinated to the cobalt ion position 80, and mutation to serine abolishes the ability of the in the α-axial position1 (Fig. 1a). The base-off conformation has protein to complement the E. coli ΔFEC strain (Fig. 1b). To been found only in a subset of Cbl-containing enzymes, but not in investigate the role of the cysteine, we solved a crystal structure at Cbl-binding proteins without enzymatic activity1, such as the 19 2 Å resolution of BtuMTd in complex with Cbl. Data collection as periplasmic substrate-binding protein BtuF , the outer 2 NATURE COMMUNICATIONS | (2018) 9:3038 | DOI: 10.1038/s41467-018-05441-9 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-018-05441-9 ARTICLE a b 0.4 Base-off (AU) 0.3 600 CH N 3 OD 0.2 HO 0.1 P Base-on N CH 0400200 600 800 1000 O 3 O α-axial position Time (min) N + 0.4 N _C80S Td Td N BtuM BtuM N (AU) 0.3 25 kDa 600 OD 0.2 β-axial position 15 kDa CN 0.1 0 200 400 600 800 1000 Time (min) c L5 L5 L3 L3 Out L1 L1 H6 H3 90° H4 H2 H5 H6 H1 L2 H4 In L4 L4 Fig. 1 Function and structure of BtuMTd. a Schematic representation of cobalamin (Cbl) showing the corrinoid ring with the central cobalt ion (red).