Magnetotactic bacteria align along the Earth’s magnetic field using an organelle called the magnetosome, a biomineralized magnetite (Fe(ii)Fe(iii)2O4) or greigite (Fe(ii)Fe(iii)2S4) crystal embedded in a lipid vesicle. Although the need for both iron(ii) and iron(iii) is clear, little is known about the biological mechanisms controlling their ratio1. Here we present the structure of the magnetosome-associated protein MamP and find that it is built on a unique arrangement of a self-plugged PDZ domain fused to two magnetochrome domains, defining a new class of c-type cytochrome exclusively found in magnetotactic bacteria. Mutational analysis, enzyme kinetics, co-crystallization with iron(ii) and an in vitro MamP-assisted magnetite production assay establish MamP as an iron oxidase that contributes to the formation of iron(iii) ferrihydrite eventually required for magnetite crystal growth in vivo. These results demonstrate the molecular mechanisms of iron management taking place inside the magnetosome and highlight the role of magnetochrome in iron biomineralization.
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This work received institutional support from the Commissariat à l’Energie Atomique et aux Energies Alternatives, the Centre National de la Recherche Scientifique, Aix-Marseille University and the Max Planck Society. We are grateful to BM-30 (ESRF, Grenoble, France) and X06SA (SLS, Villigen, Switzerland) staff for technical assistance in synchrotron data collection. We thank J. Perez (SOLEIL, GIF-sur-Yvette) for help in SAXS data collection, and A. Komeili for the gift of the wild-type and ΔmamP AMB-1 strains. We acknowledge S. Siegel and C. Li for their support at the µSpot beamline of BESSY II, Helmholtz Zentrum Berlin. We thank the AFMB laboratory (Marseille) for circular dichroism measurements. M.I.S. was supported by a grant from the Eurotalent and ToxNuc-E programs. D.F. is supported by the Max Planck Society and a Starting Grant from the ERC (256915-MB2). S.R.J. and M.C.Y.C. thank the Defense Advanced Research Projects Agency (N66001-12-1-4230) for support.
The authors declare no competing financial interests.
Extended data figures and tables
Electron density maps are contoured at 1σ around the open (top) and closed (bottom) dimers. In both cases, one monomer is coloured in gold and the other in white.
Extended Data Figure 2 Sequence alignment of MamP proteins from different MTB and structural annotations discussed in the text.
Black circles, acidic residues creating a hydrogen-bond network at the bottom of the crucible; green circles, acidic residues creating a hydrogen-bond network on the side of the crucible together with the propionate moieties of the haem from the MC1 domains; H, polar residues connecting H93 side chain to the exterior of the protein. Secondary structures are indicated at the bottom of the alignment.
a, Gel filtration of MamP using different buffers at different pH indicating a pH-dependent tetramer/dimer equilibrium. SAXS experiments confirm the presence of this equilibrium (see Methods). b, Circular dichroism measurement of MamP at pH 5 and 9 showing that there is no major structural rearrangement between the two pH values. c, The construction of the two different dimers of MamP (one in green, the other in cyan) starting from the two molecules in the asymmetric unit. These two molecules in the asymmetric unit are related by a non-crystallographic symmetry (NCS represented in magenta) axis. The two dimers (AC and BD) are generated using the twofold symmetry axis of the crystal (represented in black). The two dimers are therefore symmetric but they slightly differ, mainly in the orientation of two side chains of important residues located in the crucible (see Extended Data Fig. 4) supporting the notion of an ‘open’ (AC) and a ‘closed’ (BD) dimer. d, Superimposition of the two symmetric open and closed dimers. The root mean square distance between the Cα positions of 176 superimposed residues is 0.51 Å, showing that there is no major structural difference between the two states.
Extended Data Figure 4 Putative hydrogen-bond network in the crucible of the two MamP dimers and protonation states at pH 9 deduced from pKa calculations of protonable residues.
a, Putative hydrogen-bond network and protonation states of the conserved acidic residues in the crucible of the AC (open) dimer of MamP. b, Putative hydrogen-bond network and protonation states in the BD (closed) dimer of MamP. Note the small reorientation of the side chains of E193 and E123 and the repercussion on the calculated charge and, ultimately, the stabilization of two water molecules at the dimeric interface: in the open dimer, the two side chains could stabilize two water molecules (W) through two putative hydrogen bonds, which is not the case in the closed dimer. In the Fe(ii) soaking experiment, the anomalous electron density extends towards these two water molecules in the open dimer, whereas it is not visible in the closed dimer, indicating that this last conformation is not compatible with iron binding. All the putative hydrogen bonds drawn here are below 3.2 Å distance. c, Calculated pKa values of conserved residues at the bottom of the crucible in the open and closed dimers. These pKa values were calculated using PROPKA32 and the charge was deduced assuming a pH of 9.
Extended Data Figure 5 Detail of a putative hydrogen-bond network of conserved polar residues and water molecules connecting the side chain of H93 at the bottom of the crucible to the exterior of the protein.
One monomer is coloured in a ramp from blue (N-Ter) to red (C-Ter), the other one is coloured in white and rendered transparent for clarity.
Size distribution of crystals determined by transmission electron microscopy. Wild type (420 particles, 28 cells), ΔmamP (425 particles, 38 cells), ΔmamP + mamP (320 particles, 29 cells), ΔmamP + mamPΔacid (528 particles, 46 cells).
Extended Data Figure 7 Western blot of MamP to determine expression of MamP and MamP mutant complements.
The lanes are loaded as follows: whole cell extract of (1) wild type AMB-1, (2) ΔmamP, (3) ΔmamP + mamP, (4) ΔmamP + mamPΔacid. The antibodies were raised to a peptide of MamP of approximately 20 amino acids from strain AMB-1 (QLEGAPMILAGPRPHGYR) in rabbits by ProSci (Poway). Western blot analysis of MamP was done for each of the three biological replicates used to collect Cmag and TEM statistics. These images are representative of those collected for all three replicates.
Extended Data Figure 8 TEM images indicating the presence of electron dense particles when MamP is present.
a, Typical TEM image of the synthesis in presence of the protein. The image shows the presence electron-dense particles, probably the magnetite found by X-ray diffraction together with poorly crystalline particulate matter. b, Typical TEM image of the synthesis in the absence of MamP. Only a gangue of iron ions, probably condensate from the solution while preparing the TEM grids, can be detected. These images are representative of those collected during the experiment.
Extended Data Figure 9 Time-resolved analysis of the mineralization synthesis followed by X-ray diffraction.
Reference peaks of ferrihydrite, magnetite and sodium chloride used as salt during the synthesis and their relative intensity are indicated.
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Siponen, M., Legrand, P., Widdrat, M. et al. Structural insight into magnetochrome-mediated magnetite biomineralization. Nature 502, 681–684 (2013). https://doi.org/10.1038/nature12573
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