Sodium/proton exchangers are ubiquitous secondary active transporters that can be found in all kingdoms of life. These proteins facilitate the transport of protons in exchange for sodium ions to help regulate internal pH, sodium levels, and cell volume. Na⁺/H⁺ exchangers belong to the SLC9 family and are involved in many physiological processes including cell proliferation, cell migration and vesicle trafficking. Dysfunction of these proteins has been linked to physiological disorders, such as hypertension, heart failure, epilepsy and diabetes.

The goal of my thesis is to establish the molecular basis of ion exchange in Na⁺/H⁺ exchangers. By establishing how they bind and catalyse the movement of ions across the membrane, we hope we can better understand their role in human physiology.

In my thesis, I will first present an overview of Na⁺/H⁺ exchangers and their molecular mechanism of ion translocation as was currently understood by structural and functional studies when I started my PhD studies. I will outline our important contributions to this field, which were to (i) obtain the first atomic structures of the same Na⁺/H⁺ exchanger (NapA) in two major alternating conformations, (ii) show how a transmembrane embedded lysine residue is essential for carrying out electrogenic transport, and (iii) isolate and recorde the first kinetic data of a mammalian Na⁺/H⁺ exchanger (NHA2) in an isolated liposome reconstitution system.

Membrane proteins make up about one-third of the cellular proteome. The diverse roles that membrane proteins have in cells include major life-sustaining processes, making them major drug targets. The respiratory chain comprises a series of complexes of membrane proteins residing in the inner mitochondrial membrane, which serve as major drivers of ATP synthesis. Assembly of the respiratory chain complexes (RCC) requires coordinated synthesis of nuclear and mitochondrial subunits. Cbp3-Cbp6 complex binds to the mitoribosome as translational activator for cytochrome b synthesis and binds the nascent polypeptide to facilitate its hemylation. Cbp3 consists of an N-terminal domain specific to mitochondrial homologues and a conserved C-terminal ubiquinol-cytochrome c chaperone domain. In this thesis I present the first crystal structure of the C-terminal domain from a bacterial homologue that has enabled us to identify the interaction sites of yeast Cbp3 with Cbp6 and cytochrome b using site-specific photo-crosslinking. Our finding suggests that Cbp3 contacts the mitoribosome via the N-terminal domain in a manner that positions the substrate binding site close to the tunnel exit. In the second project, we have analyzed the effects of disease causing cytochrome b mutations, on bc₁ complex assembly. We found that complex III assembly is blocked at either intermediate 0 or I due to impaired insertion of b_L or b_H heme respectively, which indicates that assembly processes are involved in disease development. We then focused on NADH; a product of alpha-ketoglutarate dehydrogenase complex (KGDH) catalyzed citric acid cycle reaction and one of the substrates that supply electron to the respiratory chain. Kgd4 is a novel subunit of this enzyme complex and two functional variants (Kgd4S and Kgd4L) of unknown origins exist in yeast. We report in our work that Kgd4L originates from a UUG alternative start site, 90 nucleotides upstream and in frame of the annotated start codon. The sequence context upstream of UUG determines the efficiency of recognition of this alternative start codon. Finally, Na⁺/H⁺ antiporters are present in all species and are involved in regulation of intracellular pH, cell volume and sodium concentration. ATP formed during oxidative phosphorylation serves as energy source for Na⁺/K⁺ ATPase to generate Na⁺ gradient across the inner mitochondrial membrane, which drives local Na⁺/H⁺ antiporters. We show that K305 is involved in proton transport and responsible for the electrogenicity of NapA, while human NHA2 shows electroneutral antiporter activity.

Sodium/proton exchangers (NHEs) are secondary active transporters that are ubiquitously found in all kingdoms of life. They facilitate the exchange of protons for sodium ions or other inorganic ions across biological membranes, regulating pH, sodium levels, and osmotic pressure. They are therefore involved in many fundamental cellular processes such as cell migration and proliferation, and trafficking and turnover of vesicles. Their dysfunction consequently implicates them in a number of diseases and disorders, including hypertension, heart failure, epilepsy, autism spectrum disorders, and brain cancer, making them potential targets for drug development. It is therefore crucial to clearly establish their structure, the molecular basis of ion translocation and their regulation.

In this thesis I discuss the key findings of four publications I contributed to with my research. As part of these findings, we could confirm that sodium/proton exchangers operate according to the now broadly accepted elevator transport mechanism. We identified the residues of the ion-binding site that enable electrogenic ion transport in bacterial sodium/proton antiporters, and how they are potentially linked to adaption to different temperature environments. We provide a structural link between two models of regulation by pH of bacterial NhaA; suggesting a channel-like activation of a secondary active transporter. We determined the structure of endosomal NHE9, the first structure of a mammalian sodium/proton exchanger. The structure shows that these transporters, exemplified here by NHE9, share the same topology, fold, and transport mechanism as was observed in bacterial antiporters. In addition, we showed that NHE9 preferentially binds phosphatidylinositol phosphates, a class of lipids enriched in endosomes and involved in a number of signaling and regulation pathways, suggesting a potential regulatory mechanism for NHE9. Taken together, this research contributes to the growing understanding of sodium/proton exchangers and provides direction for future research.