Geula Davidov

Self-recognition mechanism of MamA, a magnetosome-associated TPR-containing protein, promotes complex assembly

The magnetosome, a biomineralizing organelle within magnetotactic bacteria, allows their navigation along geomagnetic fields. Magnetosomes are membrane-bound compartments containing magnetic nanoparticles and organized into a chain within the cell, the assembly and biomineralization of magnetosomes are controlled by magnetosome-associated proteins. Here, we describe the crystal structures of the magnetosome-associated protein, MamA, from Magnetospirillum magneticum AMB-1 and Magnetospirillum gryphiswaldense MSR-1. MamA folds as a sequential tetra-trico-peptide repeat (TPR) protein with a unique hook-like shape. Analysis of the MamA structures indicates two distinct domains that can undergo conformational changes. Furthermore, structural analysis of seven crystal forms verified that the core of MamA is not affected by crystallization conditions and identified three protein–protein interaction sites, namely a concave site, a convex site, and a putative TPR repeat. Additionally, relying on transmission electron microscopy and size exclusion chromatography, we show that highly stable complexes form upon MamA homooligomerization. Disruption of the MamA putative TPR motif or N-terminal domain led to protein mislocalization in vivo and prevented MamA oligomerization in vitro. We, therefore, propose that MamA self-assembles through its putative TPR motif and its concave site to create a large homooligomeric scaffold which can interact with other magnetosome-associated proteins via the MamA convex site. We discuss the structural basis for TPR homooligomerization that allows the proper function of a prokaryotic organelle.

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. CDFs 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 domains conformational changes upon ligand binding allows the priming of the CDF into its transport mode.

MamA as a Model Protein for Structure-Based Insight into the Evolutionary Origins of Magnetotactic Bacteria.

MamA is a highly conserved protein found in magnetotactic bacteria (MTB), a diverse group of prokaryotes capable of navigating according to magnetic fields – an ability known as magnetotaxis. Questions surround the acquisition of this magnetic navigation ability; namely, whether it arose through horizontal or vertical gene transfer. Though its exact function is unknown, MamA surrounds the magnetosome, the magnetic organelle embedding a biomineralised nanoparticle and responsible for magnetotaxis. Several structures for MamA from a variety of species have been determined and show a high degree of structural similarity. By determining the structure of MamA from Desulfovibrio magneticus RS-1 using X-ray crystallography, we have opened up the structure-sequence landscape. As such, this allows us to perform structural- and phylogenetic-based analyses using a variety of previously determined MamA from a diverse range of MTB species across various phylogenetic groups. We found that MamA has remained remarkably constant throughout evolution with minimal change between different taxa despite sequence variations. These findings, coupled with the generation of phylogenetic trees using both amino acid sequences and 16S rRNA, indicate that magnetotaxis likely did not spread via horizontal gene transfer and instead has a significantly earlier, primordial origin.

Inter-phylum structural conservation of the magnetosome-associated TPR-containing protein, MamA.

Magnetotactic bacteria enclose the magnetosome, a unique prokaryotic sub-cellular organelle that allows the biomineralization of magnetic nano-crystals. Membrane-coated magnetosomes are arranged into a linear chain that permits magnetotactic bacteria to navigate geomagnetic fields. Magnetosome assembly and biomineralization are controlled by conserved magnetosome-associated proteins, including MamA, a tetra-trico-peptide repeat (TPR)-containing protein that was shown to coat the magnetosome membrane. In this study, two MamA structures from Candidatus Magnetobacterium bavaricum (Mbav) were determined via X-ray crystallography. These structures confirm that Mbav MamA folds as a sequential TPR protein and shares a high degree of structural similarity with homologous MamA proteins from Magnetospirillum species. Furthermore, the two TPR-containing domains of MamA are separated by an interphylum-conserved region containing a flexible hinge that is involved in ligand binding and recognition. Finally, substantial differences were found in the local stabilization of the MamA N-terminal domain as a result of the loss of an evolutionary conserved salt bridge.

Structure-function studies of the magnetite-biomineralizing magnetosome-associated protein MamC.

Magnetotactic bacteria are Gram-negative bacteria that navigate along geomagnetic fields using the magnetosome, an organelle that consists of a membrane-enveloped magnetic nanoparticle. Magnetite formation and its properties are controlled by a specific set of proteins. MamC is a small magnetosome-membrane protein that is known to be active in iron biomineralization but its mechanism has yet to be clarified. Here, we studied the relationship between the MamC magnetite-interaction loop (MIL) structure and its magnetite interaction using an inert biomineralization protein-MamC chimera. Our determined structure shows an alpha-helical fold for MamC-MIL with highly charged surfaces. Additionally, the MamC-MIL induces the formation of larger magnetite crystals compared to protein-free and inert biomineralization protein control experiments. We suggest that the connection between the MamC-MIL structure and the proteins charged surfaces is crucial for magnetite binding and thus for the size control of the magnetite nanoparticles.

Genetic manipulation of iron biomineralization enhances MR relaxivity in a ferritin-M6A chimeric complex

Ferritin has gained significant attention as a potential reporter gene for in vivo imaging by magnetic resonance imaging (MRI). However, due to the ferritin ferrihydrite core, the relaxivity and sensitivity for detection of native ferritin is relatively low. We report here on a novel chimeric magneto-ferritin reporter gene – ferritin-M6A – in which the magnetite binding peptide from the magnetotactic bacteria magnetosome-associated Mms6 protein was fused to the C-terminal of murine h-ferritin. Biophysical experiments showed that purified ferritin-M6A assembled into a stable protein cage with the M6A protruding into the cage core, enabling magnetite biomineralisation. Ferritin-M6A-expressing C6-glioma cells showed enhanced (per iron) r2 relaxivity. MRI in vivo studies of ferritin-M6A-expressing tumour xenografts showed enhanced R2 relaxation rate in the central hypoxic region of the tumours. Such enhanced relaxivity would increase the sensitivity of ferritin as a reporter gene for non-invasive in vivo MRI-monitoring of cell delivery and differentiation in cellular or gene-based therapies.

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

Magnetospirillum gryphiswaldense MSR‐1 synthesizes membrane‐enclosed magnetite (Fe3O4) 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.

Disease-Homologous Mutation in the Cation Diffusion Facilitator Protein MamM Causes Single-Domain Structural Loss and Signifies Its Importance.

Cation diffusion facilitators (CDF) are highly conserved, metal ion efflux transporters that maintain divalent transition metal cation homeostasis. Most CDF proteins contain two domains, the cation transporting transmembrane domain and the regulatory cytoplasmic C-terminal domain (CTD). MamM is a magnetosome-associated CDF protein essential for the biomineralization of magnetic iron-oxide particles in magnetotactic bacteria. To investigate the structure-function relationship of CDF cytoplasmic domains, we characterized a MamM M250P mutation that is synonymous with the disease-related mutation L349P of the human CDF protein ZnT-10. Our results show that the M250P exchange in MamM causes severe structural changes in its CTD resulting in abnormal reduced function. Our in vivo, in vitro and in silico studies indicate that the CTD fold is critical for CDF proteins’ proper function and support the previously suggested role of the CDF cytoplasmic domain as a CDF regulatory element. Based on our results, we also suggest a mechanism for the effects of the ZnT-10 L349P mutation in human.

Characterization of the N-terminal domain of BteA: a Bordetella type III secreted cytotoxic effector.

BteA, a 69-kDa cytotoxic protein, is a type III secretion system (T3SS) effector in the classical Bordetella, the etiological agents of pertussis and related mammalian respiratory diseases. Currently there is limited information regarding the structure of BteA or its subdomains, and no insight as to the identity of its eukaryotic partners(s) and their modes of interaction with BteA. The mechanisms that lead to BteA dependent cell death also remain elusive. The N-terminal domain of BteA is multifunctional, acting as a docking platform for its cognate chaperone (BtcA) in the bacterium, and targeting the protein to lipid raft microdomains within the eukaryotic host cell. In this study we describe the biochemical and biophysical characteristics of this domain (BteA287) and determine its architecture. We characterize BteA287 as being a soluble and highly stable domain which is rich in alpha helical content. Nuclear magnetic resonance (NMR) experiments combined with size exclusion and analytical ultracentrifugation measurements confirm these observations and reveal BteA287 to be monomeric in nature with a tendency to oligomerize at concentrations above 200 µM. Furthermore, diffusion-NMR demonstrated that the first 31 residues of BteA287 are responsible for the apparent aggregation behavior of BteA287. Light scattering analyses and small angle X-ray scattering experiments reveal a prolate ellipsoidal bi-pyramidal dumb-bell shape. Thus, our biophysical characterization is a first step towards structure determination of the BteA N-terminal domain.

BtcA, A Class IA Type III Chaperone, Interacts with the BteA N-Terminal Domain through a Globular/Non-Globular Mechanism

Bordetella pertussis, the etiological agent of “whooping cough” disease, utilizes the type III secretion system (T3SS) to deliver a 69 kDa cytotoxic effector protein, BteA, directly into the host cells. As with other T3SS effectors, prior to its secretion BteA binds BtcA, a 13.9 kDa protein predicted to act as a T3SS class IA chaperone. While this interaction had been characterized for such effector-chaperone pairs in other pathogens, it has yet to be fully investigated in Bordetella. Here we provide the first biochemical proof that BtcA is indeed a class IA chaperone, responsible for the binding of BteAs N-terminal domain. We bring forth extensive evidence that BtcA binds its substrate effector through a dual-interface binding mechanism comprising of non-globular and bi-globular interactions at a moderate micromolar level binding affinity. We demonstrate that the non-globular interactions involve the first 31 N-terminal residues of BteA287 and their removal leads to destabilization of the effector-chaperone complex and lower binding affinities to BtcA. These findings represent an important first step towards a molecular understanding of BteA secretion and cell entry.