The dual role of MamB in magnetosome membrane assembly and magnetite biomineralization

Abstract

Magnetospirillum gryphiswaldense MSR-1 synthesizes membrane-enclosed magnetite (Fe O ) nanoparticles, magnetosomes, for magnetotaxis. Formation of these organelles involves a complex process comprising key steps which are governed by specific magnetosome-associated proteins. MamB, a cation diffusion facilitator (CDF) family member has been implicated in magnetosome-directed iron transport. However, deletion mutagenesis studies revealed that MamB is essential for the formation of magnetosome membrane vesicles, but its precise role remains elusive. In this study, we employed a multi-disciplinary approach to define the role of MamB during magnetosome formation. Using site-directed mutagenesis complemented by structural analyses, fluorescence microscopy and cryo-electron tomography, we show that MamB is most likely an active magnetosome-directed transporter serving two distinct, yet essential functions. First, MamB initiates magnetosome vesicle formation in a transport-independent process, probably by serving as a landmark protein. Second, MamB transport activity is required for magnetite nucleation. Furthermore, by determining the crystal structure of the MamB cytosolic C-terminal domain, we also provide mechanistic insight into transport regulation. Additionally, we present evidence that magnetosome vesicle growth and chain formation are independent of magnetite nucleation and magnetic interactions respectively. Together, our data provide novel insight into the role of the key bifunctional magnetosome protein MamB, and the early steps of magnetosome formation.

Metal binding to the dynamic cytoplasmic domain of the cation diffusion facilitator (CDF) protein MamM induces a ‘locked‐in’ configuration

“…A). The evolutionary‐closest CDF CTD structure that was solved in a Zn 2+ ‐bound state is of MamB, another iron transporter CDF protein from MTB (Fig. B).…”

“…Namely, only at high levels of metals (concentrations that approach cytotoxicity) will they be bound to the CTD to activate the actual transport. In contrast to the transmembrane binding site, the metal‐binding sites in the CTD are not fully conserved in terms of properties, location and number (although in most proteins more than one binding site has been proposed) . The exact binding mechanism and its energetics, the kinetic pathway by which metal binding induces these proposed structural changes, and how the ions bind and interact with the CTD, remain poorly understood.…”

The cation diffusion facilitator protein MamM’s cytoplasmic domain exhibits metal-type dependent binding modes and discriminates against Mn2+

“…This is due not only to a tighter closed state, but also to a slightly more open apo form, compared with those of MamM CTD. MamB, another CDF protein from MTB, exhibits very little conformational change between the apo and Zn 2+ -bound states (Uebe et al, 2018). However, the degree-of-closure in both MamB CTD structures is larger than that of MamM CTD apo structures, and is similar to that of the MamM Cu 2+ -bound structure (pdb codes: 5HO1 & 5HO5; Figure 2C, Table 1 and Table 2).…”

“…Previously, a number of CDF protein CTD structures were solved either in their apo state, their Zn 2+ -bound state (YiiP from Escherichia coli and YiiP homolog from Shewanella oneidensis, EcYiiP and SoYiiP respectively), or in both apo and Zn 2+ -bound states (MamB and CzrB) (Cherezov et al, 2008;Coudray et al, 2013;Lopez-Redondo et al, 2018;Lu et al, 2009;Lu and Fu, 2007;Uebe et al, 2018) (Figure 2A). According to these solved structures, the only protein to exhibit a distinct closure of the V-shape upon metal binding compared with its apo state is CzrB (pdb codes: 3BYP & 3BYR) (Cherezov et al, 2008).…”

“…Nonetheless, since these Cd 2+ ions possess only half occupancy in the electron density map, on average just one ion is bound in each monomer, as is the case with MamB. When considering all CDF metal-bound structures, a central site exists only in MamM and MamB, and mutations of these CS residues in both proteins were shown to lead to lower functionality both in vivo and in vitro (Uebe et al, 2018;Zeytuni et al, 2014b). Noticeably, copper binding in a unique coordination in the CS facilitates subsequent binding to the PS (as was shown for Zn 2+ (Barber-Zucker et al, 2019)), or vice versa.…”