From conservation to structure, studies of magnetosome associated cation diffusion facilitators (CDF) proteins in Proteobacteria

Abstract

Magnetotactic bacteria (MTB) are prokaryotes that sense the geomagnetic field lines to geolocate and navigate in aquatic sediments. They are polyphyletically distributed in several bacterial divisions but are mainly represented in the Proteobacteria. In this phylum, magnetotactic Deltaproteobacteria represent the most ancestral class of MTB. Like all MTB, they synthesize membrane-enclosed magnetic nanoparticles, called magnetosomes, for magnetic sensing. Magnetosome biogenesis is a complex process involving a specific set of genes that are conserved across MTB. Two of the most conserved genes are mamB and mamM, that encode for the magnetosome-associated proteins and are homologous to the cation diffusion facilitator (CDF) protein family. In magnetotactic Alphaproteobacteria MTB species, MamB and MamM proteins have been well characterized and play a central role in iron-transport required for biomineralization. However, their structural conservation and their role in more ancestral groups of MTB like the Deltaproteobacteria have not been established. Here we studied magnetite cluster MamB and MamM cytosolic C-terminal domain (CTD) structures from a phylogenetically distant magnetotactic Deltaproteobacteria species represented by BW-1 strain, which has the unique ability to biomineralize magnetite and greigite. We characterized them in solution, analyzed their crystal structures and compared them to those characterized in Alphaproteobacteria MTB species. We showed that despite the high phylogenetic distance, MamBBW-1 and MamMBW-1 CTDs share high structural similarity with known CDF-CTDs and will probably share a common function with the Alphaproteobacteria MamB and MamM.

Fig 1. Cation diffusion facilitators comparison.

(A) Multiple sequence alignment of MamB and MamM CTDs from the Alphaproteobacteria (MSR-1 and QH-2) and Deltaproteobacteria BW-1. Secondary structure representative, base on MamM_MSR-1 structure (PDB code 3W5Y). The blue and red frames highlight conserved sequences. (B) Structural overlay of MamB_BW-1, MamM_BW-1 and MamB_QH-2 CTD apo-form structures (PDB codes: 6QFJ, 6QEK, and 5HO5, respectively). Residues that are suggested to participate in the central metal ion-binding site represented as sticks. (C) Structural overlay of MamB_BW-1 and MamM_MSR-1 CTD structures (PDB codes: 6QFJ and 3W5Y, respectively) Residues that hypothetically participate in central and peripheral metal ion-binding sites represented as sticks.

Fig 2. Comparison of MamB-MamM crystal structures.

Structural overlay of MamB and MamM CTD monomer apo-form structures from Alphaproteobacteria and Deltaproteobacteria species (PDB codes: 6QFJ (blue), 6QEK (green), 5HO5 (purple) and 3W5Y (yellow)). Root-mean-square deviation (RMSD) values calculated for monomers and dimers respectively, using Swiss-PDB-Viewer [35]. Overall, MamM_BW-1 and MamM_MSR-1 share the highest structural similarities with RMSD of 0.66 Å over 77 common backbone atoms.

Fig 3. MamB_BW-1 and MamM_BW-1 CTD structures and dimerization interface.

(A) MamB_BW-1 folded to a metallochaperone-like fold to create a V-shaped dimer. Overlap of the MamM_BW-1 monomers on MamB_BW-1 V-dimer structure (PDB codes: 6QFJ (Green), 6QEK (blue). (B) MamB_BW-1 may create a stable V-shape dimer while the parallel S-shaped typical backbone structure, revealed in the dimerization interface after overlapping the monomers on MamM_BW-1 dimer. Hydrophobic interactions between Val235-Pro231 and Ile233-Thr234 from each monomer may hold the dimer. (C) MamM_BW-1 presents a stable dimerization interface located at the bottom of the V-shaped dimer. Dimerization interface stability rests on symmetrical backbone interaction between Pro278-Val283 and Ser280-Ile282.

Fig 4. Proteins CTDs electrostatic potential map.

Alphaproteobacteria and Deltaproteobacteria MamB and MamM CTDs crystal structures, electrostatic potential maps based on the PDB codes: 3HO5, 3W5Y, 6QFJ, and 6QEK. Top of the structures has hydrophobic and positive patches that may fit the interaction model between the CTD-TMD and CTD-magnetosome membrane. Hydrophobic and negative electrostatic charge distribution located at the bottom of the structures. The Central V-shaped dimer cavity showed negative-charge patches found in correlation with the central metal-binding site. MamM structures present an additional negative-charge patch in the periphery above the V-bottom that correlated with the peripheral metal-binding site.

Fig 5. Phylogenetic tree showing the evolutionary relationships between magnetotactic bacteria and other non-magnetotactic Proteobacteria species.

The tree was built using the Maximum-Likelihood method implemented in IQ-TREE and the concatenation of 53 ribosomal proteins. 500 replicates of a non-parametric bootstrap approach were conducted to test the robustness of the tree topology. Internal branches with support superior to 95% are annotated with a circle. Support values superior to 70% are associated with a grey circle while those below that value are not shown. Magnetotactic species names are in bold. The branch length represents the number of substitutions per site.

Fig 6. Maximum-likelihood tree showing the evolutionary relationships between MamM and MamB proteins involved in magnetite biomineralization within magnetotactic Proteobacteria strains.

The tree was built using the Maximum-Likelihood method implemented in IQ-TREE and the trimmed alignment of FieF, MamB and MamM sequences detected in the 6 strains. Color of the names of the strains correspond to their affiliation given in the species tree (Fig 5): Alphaproteobacteria (blue), Ca. Etaproteobacteria (brown) and Deltaproteobacteria (Purple). The branch length represents the number of substitutions per site. The robustness of the tree topology was tested with 500 replicates of a non-parametric bootstrap approach (black values). The posterior probability of each clade was also inferred with a Bayesian approach implemented in MrBayes (red values).