Insight into Metal Removal from Peptides that Sequester Copper for Methane Oxidation

Abstract Methanobactins (Mbns) are modified peptides that sequester copper (Cu) methanotrophs use to oxidize methane. Limited structural information is available for this class of natural products, as is an understanding of how cells are able to utilize Mbn‐bound Cu. The crystal structure of Methylosinus sporium NR3K CuI–Mbn provides further information about the structural diversity of Mbns and the first insight into their Cu‐release mechanism. Nitrogen ligands from oxazolone and pyrazinediol rings chelate CuI along with adjacent coordinating sulfurs from thioamides. In vitro solution data are consistent with a CuI–Mbn monomer as found for previously characterized Mbns. In the crystal structure, the N‐terminal region has undergone a conformational change allowing the formation of a CuI 2–Mbn2 dimer with CuI sites bound by chelating units from adjacent chains. Such a structural alteration will facilitate CuI release from Mbns.


Methanobactin Production and Purification
Methylosinus sporium NR3K (Genbank accession: EF619620) [1] was grown, Mbn recovered from the spent media, purified by reverse phase high performance liquid chromatography (HPLC) and purity verified by analytical HPLC, all using previously described procedures. [2,3] We also tested the well-studied methanotrophic bacteria Methylococcus capsulatus (Bath) and Methylomicrobium album BG8, [4] whose genomes do not possess the Mbn operon, for secretion of an Mbn-like molecule. The crude extracts isolated from the spent media in which these two organisms were grown have UV-vis spectra ( Figure S1a) similar to those reported previously. [5] HPLC purification of these crude extracts does not give rise to any fractions with spectral properties similar to those of characterized Mbns (Figure S1b,c), but are consistent with the presence of flavins, particularly for M. capsulatus (Bath). [6] PCR Amplification of 16S Ribosomal RNA Genes from M. sporium NR3K and M. trichosporium OB3b To confirm the identity of M. sporium NR3K, [1]  amplified with an annealing temperature of 47.8 °C, extracted from a 1% agarose gel and sequenced using the above primers. The sequence obtained is shown in Figure S2.
In summary, electrospray ionization mass spectrometry was used to determine molecular weights.
UV-vis spectra were acquired on a PerkinElmer λ35 spectrophotometer, using sealed anaerobic were frozen in 20% glycerol. Diffraction data were collected (100 K) at the ID29 beamline (ESRF, Grenoble, France). Data were processed and integrated with iMOSFLM [7] and scaled using SCALA. [8] The crystal structure was solved by single-wavelength anomalous dispersion based on Cu sites with the SHELX C/D/E suite. [9] The model was completed by iterative cycles of refinement (Refmac) [10] and model-building in COOT. [11] The model was validated with Molprobity, [12] data collection statistics and refinement details are reported in Table S3 and atomic coordinates have been deposited in the Protein Data Bank with the accession code 4oz7. The UV-vis spectrum (b) and the broad emission peak, with a λmax of 528 nm (c), are characteristic of the isoalloxazine ring found in flavins. [6] AGATCTCCAAGAAGGAAGTGCTCCCGGTTCTCGGCCGCCTCGGCGCGCTGTGCGCCTCGTGCTCGATCTGC GGCCCGAACTGCTGAGTTCGCCGCGCGCCGCCCCGGCGCTTGCGCCGGGGCGGCGCATTTTGCGTGGAATC ATTCTCGGAGGCGTCTTCGAAATGCAGATCGGCTTCAATTTCACGACAAACCCGACGCTCGATATCGTTCA GAGGATGATCCGAGAGCGTCAGATCGATTATTGCGAGCTGCTCATCGATAATTTTCTCCACATCCCTCCGC GGGAGTTGGAGAGCGTTTTCAAATGCCCGCTCGCCTTTCACATCATGTTCTCGAGGTTTCTCGAAAGCGAT GCCGAATTTCTCGCCAAATTTGCGAAGCGATTGCGGGAGCTGATCGACGCGCTGAATCCGCTCTATGTCTC GGACCACGTCCTGCGTTTCACGCACGAGGGCCGGCGTCTCTATCATCTCGGAGAGATCGATTACGAAACGG AATACGACCTCGTCAAGCAACGCGTTTCACGATGGCAGGACATGGTCGGACGTCGCCTGTATCTCGAGAAT TATCCATCGCTCATGGAAGGCGGATGGGAGGCGCCGGCCTTTTTCGAGCGGCTCACGAAGGAAACCGGCGC AGGGGTTCTA Figure S2. The sequence of part of the M. sporium NR3K Mbn operon. Reliable sequencing data was obtained ~37 base pairs upstream of the reverse (the final ten 5' base pairs whose sequence is determined by the forward primer did not give good sequencing data) and ~26 bases downstream of the forward primer. Over 83% of the above sequence has been confirmed by sequencing both strands, including the part of the mbnA gene coding for the core peptide. The mbnA and mbnB genes are coloured blue and green respectively with the stop and start codons in bold and underlined.    trichosporium OB3b Mbn. [2] If the revised value [16]  the result of a competition assay between these, analysed by HPLC. [3] The Cu(II) affinity of M.
sporium NR3K Mbn is ~ 2 × 10 12 M -1 , calculated using the Cu(I) affinity and the reduction potential ( Figure S8). [2] The Cu(I) affinity of M. sporium NR3K Mbn decreases by two orders of magnitude when the disulfide bond is reduced, as is also the case for M. trichosporium OB3b Mbn. [2]  diffraction data [3] shows no indication of protonation at this position in M. hirsuta CSC1 Cu(I)-Mbn (2ygi), with some evidence for a small amount in Methylocystis strain M Cu(I)-Mbn (2ygj).
trichosporium OB3b respectively. [a] Average for the sites between the two molecules in the asymmetric unit and their symmetry-related chains that form dimers.
[b] Average for the two molecules in the asymmetric unit. [2] [c] Average for the four molecules in the asymmetric unit. [3] [d] Single molecule in the asymmetric unit. [3] [e] A pyrazinediol (pyra A ) ring provides this nitrogen ligand in M. sporium NR3K and the Methylocystis Cu(I)-Mbns, whilst both nitrogen ligands are provided by oxazolone rings (oxa) in M. trichosporium OB3b Cu(I)-Mbn.    [12]