Structural insight into magnetochrome-mediated magnetite biomineralization

This article has been updated

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Overall structure of MamP homodimer.
Figure 2: A conserved dimerization interface mediated by the PDZ domain and structure of a magnetochrome domain.
Figure 3: A crucible on the surface of MamP is built on a conserved acidic pocket surrounded by a conserved acidic crown.
Figure 4: MamP, an iron oxidase with a functionally important crucible, mediates ferrihydrite production in an in vitro mineralization experiment starting with Fe(ii).

Accession codes

Accessions

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 4JJ0 (apo form) and 4JJ3 (in complex with iron).

Change history

  • 30 October 2013

    Minor changes were made to affiliation 1 and text citations of figures.

References

  1. 1

    Blakemore, R. Magnetotactic bacteria. Science 190, 377–379 (1975)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Schuler, D. Genetics and cell biology of magnetosome formation in magnetotactic bacteria. FEMS Microbiol. Rev. 32, 654–672 (2008)

    Article  Google Scholar 

  3. 3

    Komeili, A. Molecular mechanisms of compartmentalization and biomineralization in magnetotactic bacteria. FEMS Microbiol. Rev. 36, 232–255 (2012)

    CAS  Article  Google Scholar 

  4. 4

    Bell, P. E., Mills, A. L. & Herman, J. S. Biogeochemical conditions favoring magnetite formation during anaerobic iron reduction. Appl. Environ. Microbiol. 53, 2610–2616 (1987)

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

    Frankel, R. B. & Blakemore, R. P. Precipitation of Fe3O4 in magnetotactic bacteria. Phil. Trans. R. Soc. Lond. B 304, 567–573 (1984)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Baumgartner, J. et al. Magnetotactic bacteria form magnetite from a phosphate-rich ferric hydroxide via nanometric ferric (hydr)oxide intermediates. Proc. Natl Acad. Sci. USA 110, 14883–14888 (2013)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Fdez-Gubieda, M. L. et al. Magnetite biomineralization in Magnetospirillum gryphiswaldense: time-resolved magnetic and structural studies. ACS Nano 7, 3297–3305 (2013)

    CAS  Article  Google Scholar 

  8. 8

    Zhang, C. et al. Two bifunctional enzymes with ferric reduction ability play complementary roles during magnetosome synthesis in Magnetospirillum gryphiswaldense MSR-1. J. Bacteriol. 195, 876–885 (2012)

    Article  Google Scholar 

  9. 9

    Uebe, R. et al. The cation diffusion facilitator proteins MamB and MamM of Magnetospirillum gryphiswaldense have distinct and complex functions, and are involved in magnetite biomineralization and magnetosome membrane assembly. Mol. Microbiol. 82, 818–835 (2011)

    CAS  Article  Google Scholar 

  10. 10

    Siponen, M. I., Adryanczyk, G., Ginet, N., Arnoux, P. & Pignol, D. Magnetochrome: a c-type cytochrome domain specific to magnetotatic bacteria. Biochem. Soc. Trans. 40, 1319–1323 (2012)

    CAS  Article  Google Scholar 

  11. 11

    Lohsse, A. et al. Functional analysis of the magnetosome island in Magnetospirillum gryphiswaldense: the mamAB operon is sufficient for magnetite biomineralization. PLoS ONE 6, e25561 (2011)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Murat, D., Quinlan, A., Vali, H. & Komeili, A. Comprehensive genetic dissection of the magnetosome gene island reveals the step-wise assembly of a prokaryotic organelle. Proc. Natl Acad. Sci. USA 107, 5593–5598 (2010)

    ADS  CAS  Article  Google Scholar 

  13. 13

    Quinlan, A., Murat, D., Vali, H. & Komeili, A. The HtrA/DegP family protease MamE is a bifunctional protein with roles in magnetosome protein localization and magnetite biomineralization. Mol. Microbiol. 80, 1075–1087 (2011)

    CAS  Article  Google Scholar 

  14. 14

    Holm, L. & Sander, C. Dali: a network tool for protein structure comparison. Trends Biochem. Sci. 20, 478–480 (1995)

    CAS  Article  Google Scholar 

  15. 15

    Clausen, T., Kaiser, M., Huber, R. & Ehrmann, M. HTRA proteases: regulated proteolysis in protein quality control. Nature Rev. Mol. Cell Biol. 12, 152–162 (2011)

    CAS  Article  Google Scholar 

  16. 16

    Krojer, T., Garrido-Franco, M., Huber, R., Ehrmann, M. & Clausen, T. Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine. Nature 416, 455–459 (2002)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Lee, H. J. & Zheng, J. J. PDZ domains and their binding partners: structure, specificity, and modification. Cell Commun. Signal. 8, 8 (2010)

    Article  Google Scholar 

  18. 18

    Smith, L. J., Kahraman, A. & Thornton, J. M. Heme proteins–diversity in structural characteristics, function, and folding. Proteins 78, 2349–2368 (2010)

    CAS  Article  Google Scholar 

  19. 19

    Liu, J. et al. Identification and characterization of MtoA: a decaheme c-type cytochrome of the neutrophilic Fe(ii)-oxidizing bacterium Sideroxydans lithotrophicus ES-1. Front. Microbiol 3, 37 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Baumgartner, J., Bertinetti, L., Widdrat, M., Hirt, A. M. & Faivre, D. Formation of magnetite nanoparticles at low temperature: from superparamagnetic to stable single domain particles. PLoS ONE 8, 3 (2013)

    Article  Google Scholar 

  21. 21

    Jolivet, J. P., Chaneac, C. & Tronc, E. Iron oxide chemistry. From molecular clusters to extended solid networks. Chem. Commun. (Camb.) 5, 481–487 (2004)

    Google Scholar 

  22. 22

    Baumgartner, J. et al. Nucleation and growth of magnetite from solution. Nature Mater. 12, 310–314 (2013)

    ADS  CAS  Article  Google Scholar 

  23. 23

    Fischer, H., Neto, M., Napolitano, H. B., Craievich, A. F. & Polikarpov, I. The molecular weight of proteins in solution can be determined from a single SAXS measurement on a relative scale. J. Appl. Cryst. 43, 101–109 (2010)

    CAS  Article  Google Scholar 

  24. 24

    Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D 66, 133–144 (2010)

    CAS  Article  Google Scholar 

  25. 25

    Evans, P. R. Scaling and assessment of data quality. Acta Crystallogr. D D62, 72–82 (2006)

    CAS  Article  Google Scholar 

  26. 26

    Leslie, A. G. W. & Powell, H. R. in Evolving Methods for Macromolecular Crystallography Vol. 245 (eds Read, R. J. & Sussman, J. L. ) Ch. 4 41–51 (Springer, 2007)

    Google Scholar 

  27. 27

    Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Murshudov, G. N., Vagin, A. A., Lebedev, A., Wilson, K. S. & Dodson, E. J. Efficient anisotropic refinement of macromolecular structures using FFT. Acta Crystallogr. D 55, 247–255 (1999)

    CAS  Article  Google Scholar 

  29. 29

    Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 68, 352–367 (2012)

    CAS  Article  Google Scholar 

  30. 30

    Fischer, A., Schmitz, M., Aichmayer, B., Fratzl, P. & Faivre, D. Structural purity of magnetite nanoparticles in magnetotactic bacteria. J. R. Soc. Interface 8, 1011–1018 (2011)

    CAS  Article  Google Scholar 

  31. 31

    Paris, O. et al. A new experimental station for simultaneous X-ray microbeam scanning for small- and wide-angle scattering and fluorescence at BESSY II. J. Appl. Cryst. 40, s466–s470 (2007)

    CAS  Article  Google Scholar 

  32. 32

    Li, H., Robertson, A. D. & Jensen, J. H. Very fast empirical prediction and interpretation of protein pKa values. Proteins 61, 704–721 (2005)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

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.

Author information

Affiliations

Authors

Contributions

M.I.S., M.W., S.R.J. and P.A. performed experiments. M.I.S., P.L. and P.A. performed structure determination. W.-J.Z. prepared genomic DNA. M.I.S., M.W., S.R.J., M.C.Y.C, D.F., P.A. and D.P. analysed the data. M.I.S., D.F., P.A. and D.P. prepared the manuscript. D.F, M.C.Y.C., P.A. and D.P. supervised the work.

Corresponding authors

Correspondence to Pascal Arnoux or David Pignol.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Example of the quality of the 2mFobs − DFcalc electron density map.

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.

Extended Data Figure 3 pH-dependent oligomeric assembly of MamP.

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.

Extended Data Figure 6

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.

Extended Data Table 1 Data collection, phasing and refinement statistics

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing