Fig. 1: Co^II binding to CobW is enhanced by guanine nucleotides.

a Homology model of CobW (generated with SWISS-MODEL⁶⁷ using E. coli YjiA PDB entry 1NIJ⁶⁸ as template; image generated using CCP4 Molecular Graphics software) showing sulfur atoms from conserved CxCC motif (in yellow) and nucleotide-binding (GxxGxGKT, hhhExxG, SKxD*) motifs^15,16 (in red). *Ordinarily NKxD but [ST]KxD observed in some COG0523 proteins¹⁵. b Purified CobW analysed by SDS-PAGE (full image in Supplementary Fig. 1; n = 1 under these conditions). c ESI-MS analysis (de-convoluted spectra) of purified CobW. d Representative (n = 2) apo-subtracted spectra of Co^II-titrated CobW (26.1 µM); feature at 315 nm (inset) shows a non-linear increase. e Representative (n = 2) elution profile following gel-filtration of a mixture of CobW (10 µM) and Co^II (30 µM) showing no co-migration of metal (red) with protein (black). Fractions were analysed for protein by Bradford assay and for metal by ICP-MS. f Structures of nucleotides used in this work (generated using ChemDraw software). g As in d for a mixture of CobW (24 µM) and GMPPNP (60 µM); feature at 339 nm (inset) showing a linear increase saturating at 2:1 ratio Co^II:CobW (n = 2). h As in e for a mixture of CobW (10 µM), Co^II (30 µM) and GMPPNP (30 µM) showing co-migration of 1.8 equivalents Co^II per CobW (mean value from peak integration, n = 2 independent experiments). Data replicates are shown in Supplementary Fig. 1.