Cation Diffusion Facilitators Transport Initiation and Regulation Is Mediated by Cation Induced Conformational Changes of the Cytoplasmic Domain

Cation diffusion facilitators (CDF) are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all domains of life. CDF's were shown to be involved in several human diseases, such as Type-II diabetes and neurodegenerative diseases. In this work, we employed a multi-disciplinary approach to study the activation mechanism of the CDF protein family. For this we used MamM, one of the main ion transporters of magnetosomes - bacterial organelles that enable magnetotactic bacteria to orientate along geomagnetic fields. Our results reveal that the cytosolic domain of MamM forms a stable dimer that undergoes distinct conformational changes upon divalent cation binding. MamM conformational change is associated with three metal binding sites that were identified and characterized. Altogether, our results provide a novel auto-regulation mode of action model in which the cytosolic domain's conformational changes upon ligand binding allows the priming of the CDF into its transport mode

Bacterial Magnetosome Biomineralization - A Novel Platform to Study Molecular Mechanisms of Human CDF-Related Type-II Diabetes

<div><p>Cation diffusion facilitators (CDF) are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all organisms. CDFs were found to be involved in numerous human health conditions, such as Type-II diabetes and neurodegenerative diseases. In this work, we established the magnetite biomineralizing alphaproteobacterium <i>Magnetospirillum gryphiswaldense</i> as an effective model system to study CDF-related Type-II diabetes. Here, we introduced two ZnT-8 Type-II diabetes-related mutations into the <i>M. gryphiswaldense</i> MamM protein, a magnetosome-associated CDF transporter essential for magnetite biomineralization within magnetosome vesicles. The mutations' effects on magnetite biomineralization and iron transport within magnetosome vesicles were tested <i>in vivo</i>. Additionally, by combining several <i>in vitro</i> and <i>in silico</i> methodologies we provide new mechanistic insights for ZnT-8 polymorphism at position 325, located at a crucial dimerization site important for CDF regulation and activation. Overall, by following differentiated, easily measurable, magnetism-related phenotypes we can utilize magnetotactic bacteria for future research of CDF-related human diseases.</p></div

Metal binding sites of MamM-CTD and V260R mutant.

<p>MamM-CTD presents a central binding site formed by symmetrical Asp249-His285 and two symmetrical peripheral binding sites formed by His264-Glu289. The twisted dimeric fold of V260R allows the formation of the central putative binding site and two alternative peripheral symmetrical binding sites between Glu268-His264, each from a different monomer.</p

Thermodynamics of zinc binding to MamM-CTD dimer and mutants, as measured by Isothermal Titration Calorimetry.

<p>Thermodynamics of zinc binding to MamM-CTD dimer and mutants, as measured by Isothermal Titration Calorimetry.</p

Structural overlay of the wild type MamM-CTD in green and V260R mutant in purple.

<p>(A) Monomers overlay present a similar metallochaperone-like fold. In contrast to wild type MamM-CTD structure that presents a flexible C-terminal tail, one of V260R mutant monomers presents an additional beta-sheet (b4). A 2.05 Å 2F_o – F_c electron density omit map was calculated and is presented around b4. The map is countered at 1.0 σ (light brown). (B) Dimer packing overlay reveals altered and twisted dimer packing for V260R mutant.</p

Altered dimerization interface of V260R mutant.

<p><i>(Top)</i> The interactions at the top of the V-shaped dimer are maintained by a hydrophobic core of the two symmetrical Trp247 surrounded by two polar interaction patches. The first polar patch includes Arg238-Glu282 salt bridge with additional polar interactions to Asp245 side chain and the backbone carbonyl of Val242. The second patch includes a double Arg238-Glu282-Arg240 salt bridge. <i>(Bottom)</i> The interactions at the bottom of the V-shaped dimer include two interaction patches, the first is stabilized by an Arg260-Glu265 salt bridge with additional polar interactions to the side chains of Glu268 and His264. The second, lower, interaction patch is stabilized by a single Arg260-Glu268 salt bridge.</p

ZnT-8 homology model construction.

<p>(A) Multiple sequences and structural alignments reveal that the variable ZnT-8 position 325 overlaps Valine 260 of MamM protein. Valine 260 is located at the bottom of the V-shaped dimer and serves as the stand-alone hydrophobic interaction stabilizing the CTD dimerization interface. (B) ZnT-8-CTD homology model shares great structural similarity to MamM-CTD. Valine 260 of MamM and the representative arginine allele at position 325 of ZnT-8 are presented as red sticks.</p

MamM-CTD and Type-II diabetes-related mutants are dimers that bind zinc.

<p>(A) MamM-CTD and Type-II diabetes-related mutants elute form size-exclusion chromatography (Superdex200) in a volume appropriate for dimers. (B) Zn²⁺ pH-dependent binding of the MamM-CTD mutants. ZnCl₂ (5 mM) was titrated into MamM-CTD V260W (92 µM) and MamM-CTD V260R (91 µM) in 1.8 µl aliquots every 150 s. Measurements were performed at 25°C in 10 mM Tris·HCl pH 8.0, 150 mM NaCl. Top panels show the heat change during injection and bottom panels represent the data after peak integration. Data were fit using the Origin software. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097154#pone-0097154-t001" target="_blank">Table 1</a> for the binding parameters.</p

Thermodynamics of zinc binding to MamM-CTD dimer and mutants as measured by Isothermal Titration Calorimetry.

<p>Data was fitted to the single-site binding isotherm using ORIGIN 7.0 software.</p