Manganese (Mn) is a chemical element that was first isolated in 1774 by the Swedish chemist Carl Wilhelm Scheele. Within the periodic table, Mn has atomic number of 25 and is present in group 7 along with other silvery-grey, hard brittle transition metals. Chemically and physically, manganese and iron are roughly similar. Like iron, manganese exhibits different oxidation states such as +2, +3, +4, +6 and +7. Of these, Mn2+ is the most stable oxidation state, which is biologically the most relevant and often competes with Mg2+ inside cells (1). Manganese is crucial for life because it represents an essential trace element and plays a key role in many biochemical processes. For example, manganese controls enzymatic activity because it serves as a cofactor (2). The classes of enzymes that utilize manganese cofactors are very broad and include oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. The role of manganese in enzymatic activity is shown in figure 1 (adapted from 2).
Within mitochondria, superoxide dismutase (SOD) is responsible for the inactivation of reactive oxygen species such as superoxide (O2. -). This enzyme contains manganese in its active site and employs its 2+ and 3+ state for the shuttling of electrons. Specifically, in the first step, O2. – is converted into molecular oxygen, thereby reducing Mn3+ into Mn2+. Subsequently, Mn2+ is oxidized into Mn3+ while another molecule of O2. – is converted into hydrogen peroxide (H2O2). In pyruvate carboxylase, manganese directly participates in its function, namely: Mn2+ interacts with water to stabilize oxaloacetate (OAA) and oxygen from GTP, thereby bringing these compounds closely together enabling the conversion of OAA into pyruvate (PYR). Arginase, by contrast, does not utilize the redox properties of manganese to perform catalysis but employs it as a Lewis acid instead to accept electrons from water allowing the formation of a reactive hydroxide intermediate. In addition to its established role in enzymology, manganese is also essential for bacterial virulence (3). In prokaryotes, manganese uptake is mainly facilitated by so-called ATP-binding cassette (ABC) transporters (3). These are membrane proteins found in all domains of life and represent one of the largest protein superfamilies with about 1100 members described in literature (4,5,6). It is therefore not surprising that ABC transporters make up a large fraction of known proteomes. For example, the E. coli proteome contains 79 ABC transporters (7), while 49 of these proteins have been identified in humans (8). ABC transporters typically catalyze transport reactions through coupling ATP hydrolysis to the membrane translocation of a broad spectrum of substrates. Structurally, ABC transporters share a common architecture comprising four domains, namely: two highly conserved nucleotide-binding domains (NBDs) that are located to the cytoplasm and two variable transmembrane domains (TMDs). The NBDs hydrolyze ATP, thereby powering conformational changes in the attached TMDs. These make up the translocation pathway and the NBD-induced conformational changes, therefore, allow the membrane translocation of substrates into or out of the cytoplasm. (4,5) To obtain insight into how bacterial manganese importers work, the detailed structure of PsaBC, an ABC transporter responsible for Mn2+ import in Streptococcus pneumoniae, was determined recently (9). Here, I will discuss the structural features of PsaBC as well as the mechanistic bases of metal ion specificity and membrane translocation.
Overview of ABC transporters
In order to sustain cellular metabolism, the uptake of nutrients and the removal of waste products is essential. This means that molecules that enter or leave a cell must cross one or more membranes. These vital tasks are typically performed by specialized membrane proteins known as transporters. Given the wide variety of potential substrates, it is not surprising that transporters make up a large fraction of a typical membrane proteome. For example, 40% of the E. coli membrane proteome comprises potential transporters, while this is 15% in humans and 32% in S. cerevisiae (10,11,12). The ABC transporter superfamily represents one of the largest classes of transporters with about 1100 members described in literature. These proteins are ubiquitously present in all organisms and based on their evolutionary conservation they probably comprise one of the most ancient protein groups. ABC transporters utilize the power of ATP hydrolysis to drive the membrane translocation of substrates that include vitamins, steroids, lipids, ions, proteins, polysaccharides and xenobiotics (4,5,6). Functionally, ABC transporters can be classified into importers and exporters (Figure 2 left upper panel adopted from 13). Importers are responsible for the uptake of nutrients into cells and are primarily found in prokaryotes, while exporters actively remove toxins, drugs and lipids out of cells. ABC transporters are also medically relevant because in humans, mutations in ABC transporters are associated with a range of disorders such as cystic fibrosis and diabetes (4,5,6). Furthermore, ABC exporters mediate multidrug resistance in both bacteria and human cancer cells, thereby contributing to antibiotic resistance and the failure of anticancer therapies. Structurally, all ABC transporters share a similar architecture (figure 2 left upper panel) comprising two NBDs that are located in the cytoplasm and two TMDs which are firmly embedded in the membrane. The NBDs are responsible for ATP binding and hydrolysis, whereas the TMDs form the translocation pathway (4,5,6).
In bacterial ABC transporters, the four domains are typically four distinct subunits or they are fused into half- transporters made up of one NBD and one TMD that form homo- or heterodimeric complexes. In contrast to the NBDs, the sequences and overall structure of the TMDs are less well conserved owing to the structural variety of the translocated substrates. The NBDs are characterized by the presence of several highly conserved motifs, such as the Walker A, Walker B and signature motif, as well as various structural loop elements (A, D, P and Q-loop) and the H-switch (figure 2 right upper panel adopted from 5). The Walker A and B motifs along with loop elements and the H-switch make up the core of the ATP-binding site. Nucleotide binding is the mainly facilitated by the P-loop of the Walker A motif that binds the phosphate groups of the nucleotide, while other residues of the Walker A motif associate with the Mg2+ ion that is bound to the nucleotide. The Walker B motif together with the catalytic glutamate, that precedes this motif, are essential for the ATPase activity. The H-switch stabilizes the catalytic transition state and the C-loop contributes to ATP-induced dimerization of the NBDs. This is required for the formation of two ATP-binding sites between the P-loops of one NBD and the signature motif of the other. Two different formations are commonly observed in NBD dimers, namely: an open and a closed conformation (4,5,6). The closed NBD conformation is essential for ATPase activity of the complete transporter, whereas the open conformation is associated with an inactivated transporter. In general, changes in the NBD dimeric state are transmitted to the TMDs through the coupling helices (figure 2 left upper panel) that are part of the NBD-TMD interface. These conformational changes make the TMDs alternate between an inward-facing (IF) and outward-facing (OF) conformation. In the OF, the substrate-binding site is oriented towards the extracytoplasmic (periplasmic) region, while in the IF, the substrate-binding site is directed towards the cytoplasm (figure 2 left bottom panel adapted from 4). The transition between the OF and IF is thus associated with changing access to the substrate translocation pathway from the two opposite sides of the membrane. The IF allows exporters to bind their substrate and importers to release it, while the OF enables exporters to release their substrate and importers to bind theirs. Substrate translocation by bacterial importers also requires a high-affinity substrate binding protein (SBP) that specifically binds the substrate extracellularly and delivers it to the right ABC transporter. In Gram-positive bacteria, SPBs are fused to the TMD of the transporter or attached to the membrane via a lipid anchor or a transmembrane helix. In Gram-negative bacteria, however, SPBs are localized to the periplasm. A mechanistic model of bacterial ABC importers is shown in the right bottom panel of figure 2 (adopted from 14) with TMDs in purple and pink, NBDs in cyan and magenta, SPB in orange, substrate as a blue sphere and ATP and ADP as a red and yellow sphere. Import of substrates is initiated (state 1) through docking of the substrate-loaded SPB to the transporter in the IF. This induces a partial closure of the NBDs (state 2). Full dimerization of the NBDs is triggered by binding of ATP, which, in turn, induces reorientation of the TMDs from the IF to the OF conformation. This closes the translocation pathway from the cytoplasmic side and allows access from the periplasmic side. The conformational changes of the TMDs induce opening of the SPB and release of the substrate into the translocation channel (state 3). After ATP hydrolysis, the release of Pi probably resets the transporter to the IF conformation in which the translocation pathway is accessible from the cytoplasm and the substrate can diffuse into the cell (state 4). It has been established that some importers are inhibited when the cytoplasmic substrate concentration is high enough (state 5). In this case, the substrate binds to a so-called regulatory domain, thereby preventing conformational changes in the transporter and blocking its ATPase activity.
Biological and structural features of PsaBC – a bacterial manganese importer
Within cells, manganese is commonly present as Mn2+, which is, therefore, biologically the most relevant form. As such, it is not surprising that cells are equipped with import systems that are only able to handle Mg2+. Two different classes of bacterial manganese importers have been identified, namely: Nramp Mn2+ transporters and ABC Mn2+ importers. Nramp Mn2+ transporters are typically broad-spectrum H+-coupled symporters for divalent cations such as Fe2+ and Mn2+ but these appear restricted to specific microbes. Therefore, the uptake of Mn2+ by bacterial cells is mainly facilitated by dedicated ABC importers. These include PsaBCA from the Gram-positive pathogen Streptococcus pneumoniae, which is made up of three subunits: PsaA, PsaB and PsaC. PsaC is a membrane-embedded protein of 30 kDa with 9 potential transmembrane helices and forms the TMD of the transporter, while PsaB is a cytoplasmic protein 27 kDa with ATPase activity and represents the NBD of the transporter. Additionally, PsaA is a substrate-binding protein of 34 kDa tethered to the extracellular side of the plasma membrane by virtue of its lipid anchor. To assess the mechanistic and molecular properties that underly the import of Mn2+ by PsaBCA, the crystal structure of the importer in an open-inward conformation without the substrate-binding protein was recently determined. To this end, a PsaBC variant carrying a mutation in the TMD was overexpressed, purified and crystallized. Subsequently, the crystals were subjected to X-ray diffraction analysis and from the collected data the structure without any nucleotide bound was solved at 2.8 Å resolution (9).
This structure is shown in figure 3 in surface (left top panel) and ribbon representation (right top panel) with PsaB in green and magenta and PsaC is in blue and yellow, respectively. Overall, the structure represents a symmetrical dimeric complex with a PsaB2C2 stoichiometry, 18 transmembrane helices, a short amphipathic N-terminal helix, an extracellular gate helix and two coupling helices (in blue). Within the dimeric transporter, the PsaB subunits dimerize through interactions between TM2, TM4, TM7 and TM9. The translocation pathway (left bottom panel) is formed upon dimerization and comprises residues from TM4 (as orange spheres) as well as Ser117 and Thr118 from the extracellular gate helix (as red ball and sticks in the right bottom panel-top view). The two coupling helices are located at the interface of PsaB and PsaC that interact with each PsaB subunit. It is thought that these helices transmit conformational changes within the NBDs upon ATP hydrolysis to the TMDs.
The translocation channel contains a narrow point below the pore entrance (left top panel of figure 4) formed by Leu104 and Ile199 (as magenta spheres) from the loop that connects TM7 and TM8. The side chains of these residues seal off the translocation pathway (right panel of figure 4 – top view). The dimensions of this restriction probably prevent the entry of other cationic solutes such as Mg2+, Ca2+, Fe2+, Zn2+ and Cu2+. Moreover, it also prevents the entry of water and the reflux of Mn2+. This restriction, therefore, maintains the permeability barrier of the membrane within the translocation channel. Furthermore, a negatively charged cavity is present in TM2 at the cytoplasmic side of the Leu/Ile seal (bottom panel of figure 4 (top view) with negatively charged residues in red, positively charged residues in blue and uncharged residues in grey). This cavity contains Asp46 and His50 from each PsaC subunit (as red and blue ball and sticks). Conceivably, these residues interact with Mn2+ during translocation. This cavity, therefore, represents a Mn2+ binding or coordination site. Consistent with this notion, mutagenesis of these residues strongly impaired the uptake of Mn2+ into the cytosol. Possibly, this coordination site functions as selectivity filter for Mn2+ import. Below the coordination site are two leucine residues (shown as green ball and sticks in the bottom panel of figure 4) at position 43 (one of each PsaC subunit) that form a cytoplasmic gate, which probably drives Mn2+ release into the cytoplasm. In sum, the coordination site (in TM2) and the extracellular gate helix of TM4 are both crucial structural features for Mn2+ translocation into the transporter. These structural elements are highly conserved in related transporters from eubacteria and archaea.
Metal ions are essential for life mainly because these are utilized as enzymatic cofactors in many metabolic processes. For example, a bioinformatic analysis of nearly 1400 enzymes of known structure estimated that about 50% required metals for proper functioning with 41% of these containing a metal in their active site (15 and references therein). Metalloenzymes occur in all enzyme classes and often employ magnesium, iron, zinc or manganese as cofactor, while nickel, cobalt, copper and calcium are used to a lesser extent (15 and references therein). In prokaryotes, the uptake of metal ions is typically facilitated by ABC transporters. These represent a highly conserved class of membrane-embedded proteins present in every organism. ABC transporters typically catalyze transport reactions through coupling ATP hydrolysis to the membrane translocation of a broad spectrum of substrates. ABC transporters function either as an importers or exporters. Importers are responsible for the uptake of nutrients into cells and are primarily found in prokaryotes, while exporters actively remove toxins, drugs and lipids out of cells. Substrate translocation by bacterial importers also requires a high-affinity SBP that specifically binds the substrate extracellularly and delivers it to the right ABC transporter. In Gram-positive bacteria, SPBs are fused to the TMD of the transporter or attached to the membrane via a lipid anchor or a transmembrane helix. In Gram-negative bacteria, however, SPBs are localized to the periplasm. Structurally, ABC transporters are dimeric complexes with a common architecture comprising four domains, namely: two highly conserved NBDs that are located to the cytoplasm and two variable TMDs. The NBDs hydrolyze ATP, thereby powering conformational changes in the attached TMDs. These make up the translocation pathway and the NBD-induced conformational changes, therefore, allow the membrane translocation of substrates into or out of the cytoplasm. (4,5). Within NBD dimers two different structural conformations are commonly observed - an open and a closed conformation. The closed conformation is essential for ATPase activity of the complete transporter, whereas the open conformation is associated with an inactivated transporter. It is thought that changes in the NBD dimeric state are transmitted to the TMDs through the coupling helices that are part of the NBD-TMD interface. These conformational changes make the TMDs alternate between the IF and OF conformation. In the OF, the substrate-binding site is oriented towards the extracytoplasmic (periplasmic) region, while in the IF, the substrate-binding site is directed towards the cytoplasm (4,5). Despite the availability of 3D structures from a variety of ABC transporters, it is still not clear how metal ions are imported through these complexes. It is therefore important to note that recently the structure of PsaBC, an ABC-type manganese importer from the Gram-positive pathogen Streptococcus pneumoniae, was reported in an open-inward conformation (9). Overall, the structure represents a symmetrical dimeric complex with a PsaB2C2 stoichiometry. Mechanistically, Mn2+ import is controlled by the closure of the translocation channel in the open inward conformation. This also prevents reflux of Mn2+. Transport towards the cytoplasm is achieved through the coordination site within the translocation channel. The uptake of Mn2+ is strictly controlled by the intracellular requirements that are governed by the tight regulation of importer expression.
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