Towards high-resolution structural biology of membrane protein complexes in their native lipid environment

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

Magnesium is the most abundant divalent cation in cells and an essential cofactor for many enzymes and nucleotides. It plays a critical role in DNA/RNA synthesis and in stabilizing protein complexes. Maintaining a stable level of magnesium inside the cell is crucial and it is regulated by channels, transporters and exchanger. Dysregulation of magnesium homeostasis is related to a variety of human diseases such as neurodegenerative diseases, diabetes, osteoporosis, hearing loss, lung, heart and kidney diseases, pregnancy complications, and has recently been shown to have a role in obesity. Magnesium deficiency which is quite common in the US population impacts overall health mostly affecting muscle, bone, immune and nervous system. (1) Structure and function of magnesium channels Magnesium channel MRS2 is located in the inner mitochondrial membrane of eukaryotic cells and facilitates the transport of Mg2+ into the mitochondrial matrix playing a crucial role in magnesium homeostasis and mitochondrial function. CorA is the bacterial homolog of MRS2 and characterized as a homo-pentameric channel which forms a symmetric closed state at normal to high concentrations of magnesium with magnesium-binding sites between protomers as well as near the membrane pore. Under low magnesium concentrations the channel undergoes an asymmetric opening likely caused by the destabilization of protomer interactions when magnesium ions dissociate from their binding site. We have solved high-resolution cryo-EM structures of human MRS2 in different conditions and found that it also forms a homo-pentameric complex but it shows significant differences to its bacterial homolog CorA. A 70-amino acid long mitochondrial transit peptide is cleaved off the N-terminal domain of human MRS2 upon translocation into the mitochondrial membrane. Magnesium-binding sites along the pore of the channel as well as in between subunits were identified, some of which may play a role in regulating channel activity. Two pore gates as well as an important inter-subunit salt bridge were identified, that when mutated lead to increased channel activity. Additional structural and functional studies of wild-type and mutant MRS2 in synthetic as well as native nanodiscs as well as liposomes are underway to further investigate the structure, mechanism and regulation of this eukaryotic channel in its native lipid environment. (2) Structure and function of magnesium transporters Magnesium (Mg2+) uptake systems are present in all domains of life, consistent with the vital role of this ion. Bacteria require magnesium for survival, and when magnesium becomes depleted during an active infection, additional magnesium transporters are made and activated, including MgtA. MgtA belongs to the family of P-type ATPases which use ATP hydrolysis to drive ion translocation against its gradient and are structurally characterized as monomeric transporters. Here we solved the single-particle cryo-EM structure of the Mg2+ transporter MgtA from Escherichia coli. After carefully extracting the transporter out of its lipid membrane, we obtained high-resolution structures of this transporter in its dimeric form. The MgtA dimer structure is formed by multiple contacts between residues in adjacent soluble N and P subdomains. Our structures revealed an ion, assigned as Mg2+, in the transmembrane segment. Moreover, we detected two cytoplasmic ion-binding sites and determined the structure of the previously predicted to be unstructured N-terminal tail. Sequence conservation, mutagenesis and ATPase assays indicate dimerization, the ion-binding sites and the N-terminal tail facilitate cation transport or serve regulatory roles. The findings are the first to show the structure for MgtA and is the first example of a dimer structure for this class of important transport proteins. Besides MgtA being a drug target against pathogenic bacteria, this knowledge is also relevant for human conditions caused by altered P-type ATPases, including neurological conditions and copper-related diseases like Menkes disease. (3) Collaborations Collaborations involving structural and computational studies on a variety of membrane proteins including transporters, channels, and receptors as well as viral spike protein conformations in different cellular compartments, virus-like-particle (VLP), SARS-CoV-2 accessory membrane protein, extracellular vesicles and lipid transport across cells, as well as testing new detergents and polymers to gently extract membrane protein complexes from their native lipid environment for high-resolution structural studies.