ESTABLISHING THE MOLECULAR MECHANISM OF SODIUM/PROTON EXCHANGERS

Povilas Uzdavinys

Establishing the molecular mechanismof sodium/proton exchangers

Povilas Uzdavinys

©Povilas Uzdavinys, Stockholm University 2017 ISBN print 978-91-7649-964-1ISBN PDF 978-91-7649-965-8 Printed in Sweden by Universitetsservice US-AB, Stockholm 2017Distributor: Department of Biochemistry and Biophysics

Abstract

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 pro-liferation, cell migration and vesicle trafficking. Dysfunction of these pro-teins 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.

List of publications

This thesis is based upon the following publications: Paper I:

Lee C, Kang HJ, von Ballmoos C, Newstead S, Uzdavinys P, Dotson DL, Iwata S, Beckstein O, Cameron AD, Drew D. A two-domain elevator mechanism for sodium/proton antiport. Nature. 2013 Sep 26; 501(7468):573–577.

Paper II:

Coincon M*, Uzdavinys P*, Nji E, Dotson DL, Winkelmann I, Abdul-Hussein S, Cameron AD, Beckstein O, Drew D. Crystal structures reveal the molecular basis of ion translocation in sodi-um/proton antiporters. Nature Structural & Molecular Biology. 2016 Mar; 23(3):248–255.

Paper III: Uzdavinys P*, Coincon M*, Nji E, Ndi M, Winkelman I, von Ballmoos C, Drew D. Dissecting the proton-transport pathway in electrogenic Na+/H+ anti-porters. Proc Natl Acad Sci U S A. 2017 Feb 14; 114(7):E1101–E1110.

Paper IV: Landreh M, Marklund EG*, Uzdavinys P*, Degiacomi MT, Coincon M, Gault J, Gupta K, Liko I, Benesch JL, Drew D, Robinson CV. Integrating mass spectrometry with MD simulations reveals the role of li-pids in Na+/H+ antiporters. Nature Communications. 2017 Jan 10; 8:13993.

* These authors contributed equally to this work

Additional publications

Lee C, Yashiro S, Dotson DL, Uzdavinys P, Iwata S, Sansom MS, von Ballmoos C, Beckstein O, Drew D, Cameron AD. Crystal structure of the sodium-proton antiporter NhaA dimer and new mechanistic insights. Journal of General Physiology. 2014 Dec; 144(6):529–544.

Landreh M, Liko I, Uzdavinys P, Coincon M, Hopper JT, Drew D, Rob-inson CV. Controlling release, unfolding and dissociation of membrane protein complexes in the gas phase through collisional cooling. Chemical Communications (Camb). 2015 Nov 4; 51(85):15582–15584.

Gupta K, Donlan JA, Hopper JT, Uzdavinys P, Landreh M, Struwe WB, Drew D, Baldwin AJ, Stansfeld PJ, Robinson CV. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature. 2017 Jan 19; 541(7637):421–424.

Author’s contribution to the publications

Paper I: I produced and purified all NapA variants: wild-type, MVV, D156N and D157N. I performed proteoliposome functional assays of NapA wild-type and all variants.

Paper II: I generated, produced and purified NapA variants. I carried out

cysteine accessibility experiments using maleimide-PEG-5k. I carried out functional assays and established their kinetics.

Paper III: I generated, produced and purified NapA variants. I produced

and purified human NHA2 wild-type and variants. I carried out cysteine accessibility experiment using maleimide-PEG-5k. I performed functional assays and established kinetics (calculation of apparent affinities KM) of NapA and NHA2 variants, including determination of kinetics at different pH, determination of electrogenicity, experiments for membrane potential generation and ion transport driven by membrane potential. All figures were made by me, except Figure 7 and supplementary Figure 2.

Paper IV: I have performed mutagenesis of NapA variants, together with

protein expression and purification of all proteins (NhaA, NapA variants, NHA2).

Contents

Introduction ................................................................................................. 13 Transporters and Channels: the basics ................................................. 14

Secondary active transporters ............................................................... 15 The alternating access model ................................................................. 17

Rocking-Bundle Models ....................................................................... 17 The establishment of the “elevator” alternating-access mechanism ..... 18

Structure and function of sodium/proton exchangers ............................. 20 Mammalian Na+/H+ exchangers ............................................................. 20

NHEs ..................................................................................................... 21 NHA1 and NHA2 ................................................................................. 22 SLC9C .................................................................................................. 23 Pathophysiology of mammalian Na+/H+ exchangers ............................ 23

Bacterial Na+/H+ exchangers .................................................................. 24 The crystal structure of E. coli NhaA ................................................... 24

Summaries of the papers ............................................................................ 27

Concluding remarks and future perspective ............................................ 34

Sammanfattning på svenska ...................................................................... 36

Acknowledgments ....................................................................................... 38

Reference ..................................................................................................... 39

Abbreviations

9.9 MAG – 1-(9Z-octadecenoyl)-rac-glycerol ABC – ATP binding cassette AFM – Atomic force microscopy ATP – Adenosine triphosphate CPA – Cation-proton antiporter Cryo-EM – Cryo electron microscopy EPR – Electron paramagnetic resonance GPCR – G protein-coupled receptor KO – Knock out MD – Molecular dynamics MFS – Major Facilitator Superfamily NHE – Sodium-Proton Exchanger PE – 1-palmitoyl-2-oleoyl-glycerophospho-ethanolamine PEG – Polyethylene glycol PG – 1-palmitoyl-2-oleoyl-phosphatidyl-glycerol PMF – Proton Motive Force RT-PCR – Real time polymerase chain reaction SLC – Solute carrier smFRET – Single molecule fluorescence resonance energy transfer TM – Transmembrane

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Introduction

About 30% of genes in sequenced genomes code for integral membrane proteins (Wallin and von Heijne, 1998; Krogh et al., 2001). Membrane pro-teins are dynamic nanomachines that control the flow of information across cells and between organelles. Their activities are attuned to the composition and the structure of the membrane, and respond to various external stimuli. X-ray crystallography has proven to be an excellent tool for establishing detail atomic structures of membrane proteins and the complexes they form. Biophysical methods such as electron microscopy, scattering and spectro-scopic techniques, and molecular dynamics simulations provide important complementary insights into their molecular mechanisms (Caffrey and Drew, 2017). The importance of understanding the structure and the function of membrane proteins is also highlighted by the fact that 50% of all pharma-ceutical drug targets are membrane proteins (Overington et al., 2006).

Integral membrane proteins can be divided into -barrel and -helical pro-teins. -barrel membrane proteins are made up from an even number of

-sheets that come together to form a barrel (Figure 1A). These proteins are found in the outer membranes of bacteria, mitochondria and chloroplasts. -barrel membrane proteins encode about 2–3% of the genes in gram-negative bacterial genomes (Wimley, 2003). -helical membrane proteins make up about 27% of the human proteome and typically are referred to simply as membrane proteins (Almén et al., 2009) (Figure 1B). Major classes of -helical membrane proteins are receptors, transporters and channels, enzymes and others (Almén et al., 2009).

Receptors are membrane proteins that typically respond to the signals outside the cell causing intracellular signalling cascades e.g., G protein-coupled receptors (GPCRs) and receptor type tyrosine kinases. Enzymes are proteins that catalyse a chemical reaction, such as oxidoreductase, transfer-ases, hydrolases, lyases, isomerases and ligases. Transporters and channels catalyse the movement of solute across the membrane.

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Another structural feature of the secondary active transporters is the pres-ence of discontinuous helices. These helices contain an unwound region that exposes backbone carbonyl oxygen atoms which are often crucial for ion coordination in the membrane (Screpanti and Hunte, 2007). The first exam-ples of transporters with discontinuous helices were the Na+/H+ antiporter NhaA (Hunte et al., 2005) and the sodium-coupled amino acid transporter LeuT (Yamashita et al., 2005). Both proteins contain two discontinuous helices that are arranged in an antiparallel (NhaA) or in a parallel (LeuT) fashion with respect to each other. Discontinuous helices also create a dipo-lar moment that further lowers the energetic barrier for binding charged ions in the hydrophobic membrane.

The alternating access model

Rocking-Bundle Models Regardless of the source of energy, transporters have to undergo multiple

conformational changes to be able to transport the substrate across the mem-brane. The concept of such translocation of solutes across the membrane was suggested by Peter Mitchell (Mitchell, 1957; Mitchell, 1990). It was de-scribed as a mobile barrier mechanism, which involve the opening and clos-ing of a substrate-binding site on either side of the membrane. The large number of crystal structures have been consistent with the alternating-access models proposed in the 1960s by Oleg Jardetzky (Jardesky, 1966). In es-sence, alternating access to the substrate-binding site is achieved when the barrier formed between two domains on the cytoplasmic side of the mem-brane is moved apart and re-formed on the extracellular side (Figure 4) (Drew and Boudker, 2016). For transporters made up of two structurally similar domains this is referred to as the “rocker–switch” mechanism (Figure 4A). The best-known examples of proteins operating by the rocker-switch mechanism is the proton-coupled lactose symporter LacY from Escherichia coli (Abramson et al., 2003), which is the member of the large Major Facili-tator Superfamily (MFS). Transporters with structurally divergent domains function according to the “rocking-bundle” mechanism (Figure 4B). Alt-hough similar to rocker-switch proteins, there are typically larger, gating type conformational changes in one of the structurally distinct bundles. One of the best characterised transporters operating by this mechanism is the bacterial amino acid leucine transporter LeuT (Krishnamurthy and Gouaux, 2012).

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Structure and function of sodium/proton exchangers

All living cells depend on maintaining an intracellular pH because most proteins have a certain pH range in which they can function. Moreover, eu-karyotic cells have organelles, which can have a different environment and pH than the cytosol. The difference of pH in organelles can be vast, from 4.5 – 5.0 in lysosomes to pH 8 in mitochondria (Casey et al., 2010). The pH can even be different on different side of the same organelle, like in the Golgi complex, with pH 6.7 on cis side and pH 6.0 on trans side (Casey et al., 2010). Therefore, pH or proton (H+) concentration and its gradients play a critical role for maintaining cell viability. Protons, like other ions, cannot easily cross the membrane. One group of integral membrane proteins, which facilitate H+ transport, are the sodium/proton (Na+/H+) antiporters or ex-changers (NHEs).

Na+/H+ antiporters are secondary active transporters found in all king-doms of life and play an important role not only in maintaining internal pH, but also in sodium concentration, cell volume, and have roles in control of the cell cycle, cell migration, cell proliferation and vesicle trafficking (Brett et al., 2005a; Fuster and Alexander, 2014). The first proposal of a coupled Na+/H+ exchanger was described in E. coli in 1974 by Peter Mitchell and colleagues (West and Mitchell, 1974).

Mammalian Na+/H+ exchangers Na+/H+ antiporters belong to the monovalent cation:proton antiporter

(CPA) superfamily that consists of 4 clades from bacteria to man: CPA1, CPA2, PSE, and NaT-DC (Brett et al., 2005a; Landau et al., 2007; Slepkov et al., 2007; Schushan et al., 2010; Chen et al., 2011). It is a commonly thought that Na+/H+ antiporters from the CPA1 family carry out electroneu-tral (nNa+/nH+) transport, whereas members of CPA2 family perform elec-trogenic nNa+/(n+1)H+ exchange (Brett et al., 2005a). Mammalian Na+/H+ exchangers are ubiquitous ion transporters and are present in all animal spe-cies. These proteins can be found in the cytoplasmic membranes of cells and in the membranes of many organelles (Donowitz et al., 2013). Mammalian Na+/H+ exchangers belong to the Solute Carrier SLC9 family, which is di-

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vided into 3 subgroups: SLC9A (NHE1-9), SLC9B (NHA1, NHA2), and SLC9C (Donowitz et al., 2013).

NHEs In humans, there are 13 different NHE isoforms that localise to various

tissues (Orlowski and Grinstein, 2011). All NHEs have a similar topology, consisting of an N-terminal transport domain that is made up of ~12 trans-membrane helices of ~450 residues, and a C-terminal cytosolic domain that is involved in regulation and varies in length. The NHEs, like all SLC9 members, are thought to assemble physiological homodimers (Fafournoux et al., 1994; Hisamitsu et al., 2004; Hisamitsu et al., 2006; Moncoq et al., 2008), but it is not clear if each promoter functions independently (Fafournoux et al., 1994).

Some NHE members reside on the plasma membrane (NHE1–5) while others in distinct intracellular compartments (NHE6–9) (Fuster and Alexan-der, 2014; Ohgaki et al., 2011). Interestingly, NHE8 can be found in both locations, in plasma membrane and in endomembrane (Nakamura et al., 2005; Xu et al., 2008). Moreover, even though NHE3 and NHE5 are classi-fied as plasma membrane proteins, these isomers can traffic between the plasma membrane and the recycling system (Janecki et al., 1998; Chow et al., 1999; Szaszi et al., 2002). The activity of the plasma membrane NHEs is driven by an Na+ electrochemical gradient. In comparison, the organellar NHEs are driven by an H+ gradient, which is generated by the V-type H+-ATPase. Plasma membrane NHEs transport only Na+ and Li+ ions, whilst intracellular NHEs are also thought to be able to transport K+ ions (Brett et al., 2005b; Ohgaki et al., 2011). Until now it was reported that some NHEs perform electroneutral exchange with stoichiometry of either 1:1 or 2:2 (Grinstein et al., 1984; Fuster at al., 2008). Few of NHEs have been found to be glycosylated, however, glycosylation does not appear to be essential for transport function (Counillon et al., 1994; Tse et al., 1994).

NHE1 is the best-characterised member of the SLC9 family and is ubiqui-tously expressed in nearly all mammalian cells (Orlowski and Grinstein, 2004). NHE2 is mostly located in intestines – in the brush borders of colon (Guan et al., 2006; Donowitz et al., 1998). NHE3 mainly expressed in small intestine (Hoogerwerf et al., 1996) and kidneys (Ambühl et al., 1996), but can be found in heart, brain and lungs too (Fuster and Alexander, 2014). NHE4 has highest expression in the stomach. However, it can be found in the kidney, brain, uterus and skeletal muscles (Orlowski and Grinstein, 2004). NHE5 has the highest expression in brain but was also detected in testis, spleen and skeletal muscle (Klanke et al., 1995; Attaphitaya et al., 1999; Baird et al., 1999). NHE6 can be found in skeletal muscle, brain and heart (Orlowski and Grinstein, 2011). NHE6 was also found to be expressed in mineralizing osteoblasts, where it helps to maintain internal pH (Liu et al.,

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2011). NHE7 can be found in skeletal muscle and brain (Numata and Or-lowski, 2001). NHE8 expressed in skeletal muscle and kidneys (Goyal et al., 2003). NHE9 is ubiquitously expressed and can be found in brain, heart, skeletal muscle and even in placenta (Nakamura et al., 2005).

NHA1 and NHA2 With the use of bioinformatics methods, Rao and co-workers identified a

new family of NHEs, which are most similar to the SLC9A genes (Brett et al., 2005a). This new family was named NHA (SLC9B), because it is similar to fungal NHA1 also, and has two paralogs NHA1 and NHA2. These genes are present in all completely sequenced genomes, including nematodes, fly, puffer fish, mouse and human (Brett et al., 2005a). When the first NHA1 was cloned, RT-PCR method revealed that NHA1 is only expressed in testis (Ye et al., 2006). The physiological role of NHA1 is still unclear and it has not been linked to any human disease. NHA2 was identified in 2008 in microarray screening by Battaglino and co-workers (Battaglino et al., 2008). The protein was expressed in differenti-ated osteoclasts and has been found in mitochondria (Battaglino et al., 2008). However, later it was shown that NHA2 is ubiquitously expressed, including brain, heart, lung, kidney, skeletal muscle, and colon. Moreover, NHA2 was found to be intracellular and plasma membrane Na+/H+ exchang-er but a cell-specific (Fuster et al., 2008). Two independent groups showed that NHA2 is highly expressed in kidney distal convoluted tubule (DCT) (Fuster et al., 2008; Kondapalli et al., 2017). DCT is partly responsible for the salt homeostasis and pH regulation. Consequently, experiments with mice, which were fed with a diet in high sodium, proposed a model that NHA2 can play a dual role in salt reabsorption or secretion, and in pH regu-lation in kidneys (Kondapalli et al., 2012; Kondapalli et al., 2017). NHA2 is also overexpressed in - -cell lines reduced sulfonylurea- and secretagogue-induced insulin secretion. The deficit of insulin secretion could be rescued by NHA2 overexpression, but not by non-functional NHA2 mutant (Deisl et al., 2013). Interestingly, loss of NHA2 didn’t affect pH of endosomes, indicating that it may have a role of controlling Na+ concentration rather than pH regulation (Deisl et al., 2013).

NHE activity is driven by a Na+ gradient. However, no Na+-driven H+ ef-flux could be measured in NHA2 expressing mammalian cells (Fuster et al., 2008). In contrast to NHEs, bacterial Na+/H+ antiporters are driven by an H+ gradient (Padan et al., 2005). Because NHA2 is evolutionary closer to the bacterial Na+/H+ antiporters than the NHEs, Rao and co-workers also tested the ability to drive the activity by a H+ gradient. Indeed, H+-driven antiport activity of NHA2 was measured and this observation was strongly supported by co-localization of NHA2 with V-type H+-ATPase (Kondapalli et al., 2012; Hofstetter et al., 2010). A salt-sensitive growth experiment showed

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that NHA2 can tolerate Na+ and Li+ but not K+ in a pH dependence manner, where cells grew only at low pH (3.0 – 4.5) (Xiang et al., 2007; Fuster et al., 2008; Schushan et al., 2010). Nevertheless, the same group showed that NHA2 can be inactivated by intracellular acidification and it is more active from neutral to alkaline pH (Kondapalli et al., 2012).

SLC9C The newly identified SLC9C subgroup contains sperm specific Na+/H+

exchangers, SLC9C1 and SLC9C2 (Fuster and Alexander, 2014). Sperm- SLC9C1 is similar to NHEs, with an N-terminal transport domain and a cy-tosolic regulatory domain. Experiments with KO mice showed that a loss of sperm-NHE is responsible for mice infertility, sperm motility and pH regula-tion. The addition of NH4Cl or cyclic AMP analogues, which elevates intra-cellular pH, partially or completely rescued the motility and fertility defects, respectively (Wang et al., 2003). However, human sperm-NHE has not been connected to male infertility.

Pathophysiology of mammalian Na+/H+ exchangers As Na+/H+ exchangers are highly expressed and have various physiologi-

cal roles, it is not surprising that the dysfunction of these proteins can be linked to various pathological diseases, such as diabetes (Hannan et al., 1998) or neurological disorders. KO NHE1 murine exhibited epileptic-like seizures, ataxia and growth retardation (Bell et al., 1999; Cox et al., 1997). Patients with mutations in SLC9A6 exhibit a syndrome associated with mi-crocephaly, seizures, and ataxia. Some patients demonstrated symptoms of Angelman syndrome. (Gilfillan et al., 2008). Furthermore, it was noticed that NHEs might play an important role in cardiovascular diseases. Inhibi-tion or overexpression of NHE1 in heart of rabbits or mice lead to heart fail-ure (Aker et al., 2004; Nakamura et al., 2008), while NHE3 gene deficiency increased blood pressure in mice (Noonan et al., 2005). Recently it was shown that NHE1 might also be involved in cancer (Stock and Pedersen, 2016; Ariyoshi et al., 2017; Sanhueza et al., 2017). Therefore, a better un-derstanding of Na+/H+ exchangers role in various diseases is significant and makes these membrane proteins potential targets for therapeutics (Brett et al., 2005a).

Experiments with KO mice showed that NHA2 deficiency increases obe-sity- and aging-induced glucose intolerance, however not because of altered insulin sensitivity as both KO and WT mice had similar levels of serum insu-lin (Deisl et al., 2016). Also, NHA2 was implied to play a role in hyperten-sion (Xiang et al., 2007). Nevertheless, more studies are required to better understand the mechanism and the role of NHA2.

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Bacterial Na+/H+ exchangers Due to difficulties in isolating and purifying mammalian Na+/H+ ex-

changers, bacterial homologues (often referred to as antiporters) have proven to be excellent model systems for understanding their molecular mechanism. Unsurprisingly, the bacterial Na+/H+ antiporters are important in their own right, as they establish a sodium-motive-force and are thus critical for cell homeostasis and pathogenicity (Krulwich et al., 2011).

Bacterial Na+/H+ antiporters can be found in both CPA1 and in CPA2 clades. CPA1 clade uses the Na+ gradient to catalyse electroneutral reaction:

Na+/Li+ (out) + H+ (in) Na+ (in) + H+ (out);

Proteins in the CPA2 clade use the H+ gradient to catalyse electrogenic

reaction:

Na+/Li+ (in) + nH+ (out) Na+ (out) + nH+ (in).

The best-characterized Na+/H+ antiporter is NhaA from Escherichia coli.

It belongs to CPA2 clade and it has been showed to catalyse electrogenic reaction where it exchanges Na+ ion for 2H+ ions (Taglicht et al., 1993). NhaA is one of the fastest transporters characterized to date, which reaches a maximum turnover up to 1500 ions per second and its activity is pH depend-ent for Na+ and Li+ ions. The protein becomes active only above pH 6.5 and reaches its maximum activity at pH 8.5 (Taglicht et al., 1991). NhaA was the first Na+/H+ antiporter to obtain an atomic crystal structure (Hunte et al., 2005). It was known from different studies that NhaA is a dimer (Screpanti et at., 2006; Hilger et al., 2007). However, the structure of NhaA was initial-ly determined as a monomer at a low pH where the protein is inactive (Hunte et al., 2005).

The crystal structure of E. coli NhaA The crystal structure of E. coli NhaA (Figure 6) revealed its structural archi-tecture consisting of 12 TMs with both the N- and C-terminal tails located in the cytoplasm. NhaA is made up of two 5 TM structural-inverted repeats. Each monomer forms two distinct domains, a core (ion translocation) do-main and a dimer (scaffold) domain. The core domain is responsible for ion binding and translocation, and contains two discontinuous helices that cross over near the centre of the protein (Figure 6). Near the cross-over helices are two conserved aspartates, D163 and D164. The dimer interface is composed

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regulation (Kozachkov et al., 2007), as mutations of Lys300 increase appar-ent KM and shift of pH dependence profile (Maes et al., 2012).

NhaA has been studied extensively for the past 25 years. Despite a vast wealth of functional data, a transport mechanism is difficult to generate from a single crystal structure (Hunte et al., 2005). No bound ion was observed in the NhaA structure (Hunte et al., 2005). Based on functional data and MD simulations it was proposed that both Asp163 and Asp164 bind sodium, whereas Asp133 with Thr132 are helping to coordinate it, and Lys300 plays an important role in pH regulation (Maes et al., 2012). By solid support elec-trophysiology it was shown that ion exchange in NhaA could be kinetically explained as a simple competition of Na+ and H+ to the same ion-binding site (Mager et al., 2011). In essence, at low pH NhaA is inactive because both aspartates are protonated and unable to bind Na+. As the pH increases to neutral values, Na+ can now bind and the binding of substrate somehow causes conformational changes in the protein.

Despite an overall working hypothesis of the transport cycle in NhaA fur-ther structures were required to solidify a transport mechanism. Of particular importance, was the determination of a Na+/H+ antiporter structure in the opposite facing conformation and a structure at a pH where the antiporter was active. Such structures would enable us to better understand ion recogni-tion and how ion-binding and conformational changers are coupled.

As outlined in my thesis summary we have been able to shed light into the transport mechanism by solving crystal structures of an NhaA homo-logue at active pH in two different conformations, namely NapA from Thermus thermophilus (Paper I and Paper II). It is worth mentioning that during this period, crystal structures of archaeal Na+/H+ antiporters from Pyrococcus abyssi (PaNhaP) and Methanocaldoccocus jannaschii (MjNhaP1) were also determined (Wöhlert et al., 2014; Paulino et al., 2014). These crystal structures, and the comparison to NapA in Paper II, proposed new questions regarding the role of lipids (Paper IV) and the ion-coupling mechanism (Paper III). Overall, the structural and functional data outlined in this thesis has pro-vided a wealth of new mechanistic information, not only important to Na+/H+ antiporters, but to all types of secondary active transporters.

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Summaries of the papers

Paper I: A two-domain elevator mechanism for sodi-um/proton antiport.

The crystal structure of E. coli NhaA had previously been determined in an inward-facing state (Hunte et al., 2005). Consistent with its fast transport kinetics a mechanism was proposed that hinged upon on small-rearrangements of the discontinuous helices in the core domain.

In this paper, we described a crystal structure of a Na+/H+ antiporter, Na-pA from Thermus thermophilus at 3-Å resolution, in an outward facing state, where a negatively charged funnel is now opened to the outside. NapA was crystallised at 20 °C using the hanging drop vapour diffusion method at pH 7.8. Apart from the conformational differences between NhaA and NapA, the main structural differences observed was that NapA is built from 13 transmembrane (TM) helices, compared to NhaA that is made up of 12 TM helices. NapA has an additional helix at the N-terminus that is involved sta-bilising the NapA dimer. In contrast, we could show that the contacts linking the NhaA protomers together are much weaker and held together by interac-

-hairpin strands (Lee et al., 2014). In the outward-facing NapA structure the conserved ion-binding residues

Asp156 and Asp157 are located near the base of the cavity and next to the point where two antiparallel discontinuous helices cross over in the core domain. We could show that the replacement of either residue to alanine or asparagine completely abolished antiport activity. NapA activity was meas-ured in an isolated system, where purified NapA and E. coli F1F0 ATP syn-thase were co-reconstituted into liposomes (Figure 7A). pH gradient was established by the addition of ATP, proton efflux was observed in response to different concentrations of Na+ or Li+ (Figure 7B). The apparent Michaelis constant (KM) values were determined by plotting ACMA dye dequenching against ion concentration (Figure 7C). The electrogenic nature of NapA was observed by water-soluble pH sensitive dye pyranine (Figure 7D).

Interestingly, another conserved residue Lys305 formed a salt bridge with Asp156, which hadn’t been observed in the initial NhaA crystal structure (Hunte et al., 2005). In a parallel study, we have since shown that the equiv-alent lysine in NhaA also forms a salt-bridge to the ion-binding aspartate (Lee et al., 2014); the TM10 segment harbouring the lysine residue was built out-of-register in the initial NhaA crystal structure (Hunte et al., 2005).

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Paper III: Dissecting the proton transport pathway in elec-

trogenic Na+/H+ antiporters Sodium/proton exchangers belong to the large monovalent cation:proton

antiporter (CPA) superfamily, which includes CPA1 and CPA2 clades and others. It is commonly thought that members of CPA1 clade perform elec-troneutral Na+/H+ exchange, where some members were reported perform the transport with stoichiometry 1H+:1Na+ or 2H+:2Na+. Members of CPA2 clade are thought to be electrogenic, as some members were reported per-form the exchange with stoichiometry of 2H+:1Na+ and 3H+:2Na+.

Bacterial Na+/H+ antiporter NhaA from Escherichia coli is the most thor-oughly studied antiporter, which belongs to CPA2 clade and harbours two conserved aspartate residues. It has been assumed that those two aspartate residues carry two protons across membrane, as both aspartate residues are critical for function, but only one aspartate residue is conserved in the elec-troneutral transporters. However, our structures indicated that another mech-anism could be possible, as a salt-bridge between Asp156 and a neighbour-ing Lys305 has been seen in all NapA crystal structures (Paper I and Paper II). This indicated that rather than Asp156 that lysine residue might be the key proton carrier.

In this paper, we have substituted Lys305 to neutral, positive and nega-tively charged residues. Results showed that all tested variants, except histi-dine (that shows wildtype activity), lost their ability to be (i) stimulated by the dissipation of the membrane potential (Figure 11A), (ii) to be driven by an electrical gradient (Figure 11B), and (iii) to generate an electrical poten-tial while driven by ion gradients (Figure 11C). These results show that the lysine residue is essential for electrogenic activity. Proteoliposomes were prepared as previously mentioned. Moreover, we demonstrated the first-ever rescue phenotype for a key ion-binding site aspartate mutant. We could show that activity of an Asp156 to aspargine mutant could be rescued by the muta-tion of the salt-bridge K305 residue to glutamine.

Human Na+/H+ exchanger NHA2 (SLC9B2) has two ion-binding aspar-tate residues equivalent to bacterial electrogenic transporters NhaA and Na-pA. However, unlike NhaA and NapA, the residue equivalent to K305 has been replaced by arginine. In NapA a K305R mutant shows poor transport activity and is electroneutral i.e., which is consistent for the requirement of a residue that can bind and release protons at neutral pH. If the lysine is essen-tial as a proton carrier then NHA2 activity should also be electroneutral (Figure 11D). Our experiments showed that NHA2 activity is likely electro-neutral, which is consistent with in vivo data.

In summary, we could show that the lysine residue Lys305 is essential for electrogenic transport and acts as a proton carrier.

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detergent micelles. Mass spectroscopy (MS) enables to study the stability of Na+/H+ antiporters and the effect of addition of individual lipids.

MS showed that NapA from Thermus thermophilus and NHA2 from Ho-mo sapiens are more stable dimers than NhaA from Escherichia coli. More-over, it showed that NhaA sample contained a bound lipid, which was cardi-olipin. Whereas, NapA sample contained a small amount of the most com-mon phospholipids in E. coli, which was co-purified together, and NHA2 sample did not retain any a significant amount of bound lipids.

Interestingly NapA crossed-linked variants, V71C:L141C and I55C, were restricted to unfolding by trapping the protein in the intermediate unfolding state compared to NapA wild-type.

The stability of NapA wild-type was tested with addition of different li-pids, such as 1-palmitoyl-2-oleoyl-glycerophospho-ethanolamine (PE), 1-palmitoyl-2-oleoyl-phosphatidyl-glycerol (PG), and 1-(9Z-octadecenoyl)-rac-glycerol (9.9 MAG). Results showed that protein interaction with nega-tively charged PG lipids display higher stability than with zwitterionic PE, while uncharged 9.9 MAG lipids have no effect. Moreover, lipid bound (PE) protein resisted to unfolding compared to wild-type, but, the addition of PE had no effect to unfolding profile to NapA cysteine variants.

Molecular dynamics (MD) simulations of NapA in a PE bilayer revealed that the lipids cluster around the dimer domains and the flexible dimer-core domain connections. The highest impact was found to be in the flexible hinges between the core and the dimer domains, where the main concentra-tion of lipids were observed. The unfolding of lipid-free NapA was rapid, where lipid-bound sample was prevented from the collapse because of the lipids.

In conclusion, MS combined with MD simulations showed what is the re-lationship between structural features and lipid interactions in the Na+/H+ antiporter NapA.

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Concluding remarks and future perspective

Na+/H+ exchangers have been extensively studied for several decades. We are just beginning to understand how they function at the atomic level. From previous studies, it was known that mammalian and bacterial Na+/H+ ex-changers are physiological dimers (Fafournoux et al., 1994; Hisamitsu et al., 2004; Hisamitsu et al., 2006; Moncoq et al., 2008; Screpanti et at., 2006; Hilger et al., 2007; Wöhlert et al., 2014; Paulino et al., 2014). These struc-tures are not enough to fully understand their ion-exchange mechanism, alt-hough some computational studies have been used to extrapolate one (Arkin at al., 2007).

Here, we have analysed the function and structure of the electrogenic Na+/H+ antiporter NapA from Thermus thermophilus. For the first time, we managed to crystallise the same Na+/H+ antiporter in two major confor-mations, an outward-facing state (Paper I) and an inward-facing state (Pa-per II). We trapped the inward-facing state by introducing a disulfide bond between two domains. Comparison of two structures showed that the protein has to undergo relatively large structural transitions to alternate-access to the substrate-binding site. In addition, functional studies confirm that these rear-rangements are accessible in a membrane environment (Paper II).

In both crystal structures, a salt-bridge, between Asp156 and Lys305, was observed in the ion-binding site. By testing different Lys305 variants we concluded that this residue is essential for electrogenic transport and is thus the second proton carrier under physiological conditions. To support this hypothesis, we managed to show that activity of the human NHA2 protein, which harbours two aspartates, also appears to be electroneutral (Paper III).

Finally, mass spectroscopy and MD simulations showed that lipids can stabilise NapA by packing closely around the flexible hinge regions, the interface between the dimer domain and the core domain (Paper IV).

Our findings shed new light into how Na+/H+ antiporters translocate ions across the membrane. However, further mechanistic understanding will re-quire an ion-bound structure. We hypothesise that the ion binding is coupled to large-scale structural transitions through breakage of the salt-bridge. In-deed, MD simulations showed that breakage of the NhaA salt-bridge is trigged by sodium binding (Lee et al., 2014). Ideally, we would like to fur-ther monitor conformational changes of a Na+/H+ antiporter in a membrane environment directly, either by smFRET and/or by high-speed AFM. Lastly, the binding of various lipids and globular proteins to a large, cytosolic C-

35

terminal tail continuously regulates the activity of mammalian Na+/H+ ex-changers. This regulatory tail is not present in the bacterial homologues. Thus, to understand their mechanism of action and physiological roles com-pletely, we need to establish how they are allosterically regulated.

36

Sammanfattning på svenska

En cell är den minsta formen av liv. Cellen omges av ett membran, som omsluter cellen och ger skydd mot omgivningen. Innanför membranet är cellen indelad i mindre enheter, kallade organeller, som var och en bidrar med skilda funktioner. Även organellerna omges av egna membraner, vilka i sin tur avskiljer dessa distinkta miljöer från varandra och resten av cellen. Cellens olika membran består av upp till 50% av äggviteämnen, även kallade proteiner, som när de förekommer i membranen kallas membranproteiner. Membranproteinerna fyller viktiga funktioner för cellens överlevnad, då de bland annat kontrollerar och tillåter selektiv passage av små partiklar, som molekyler och joner, genom de annars blockerande membranerna. En intres-sant och viktig typ av membranproteiner är natrium/protonutbytare, vilka släpper in natriumjoner och samtidigt exporterar protoner ut ur cellen i ut-byte. Dessa utbytare är viktiga för att reglera cellens storlek, natriumkon-centration och surhetsgraden (pH). Natrium/protonutbytare återfinns i samt-liga levande celler, vilket motiverar vidare forskning om dessa proteiner. Dysfunktion hos dessa membranproteiner är kopplade till bland annat kardi-ovaskulära och neurologiska sjukdomar. Därför är dessa natrium/protonutbytare viktiga läkemedelsmål. Vissa sjukdomar uppstår till exempel som en följd av mutationer, vilka påverkar regleringen av mem-branproteinet. Utifrån proteinstrukturer och hur dessa proteiner verkar kan läkemedel utvecklas som kan reglera proteinernas aktivitet.

Målet med min avhandling är att presentera vår nuvarande utökade kun-skap om hur dessa natrium/protonutbytare fungerar, samt vad som tidigare var känt innan jag påbörjade mitt doktorandprojekt. Vidare kommer jag pre-sentera mitt bidrag och hur de resultaten relaterar till tidigare publicerade resultat. I samarbete med kollegor lyckades jag lösa två strukturer av samma protein i två olika konformationer, för första gången. Jämförelsen mellan dessa strukturer belyste att den ena proteindomänen behöver genomgå en stor förflyttning för att transportera joner genom membranet. Denna förflytt-ning påvisade jag även i en miljö motsvarande det naturliga membranet. Utifrån mina egna observationer antar jag att det bakteriella NapA proteinet transporterar en natriumjon i utbyte för två protoner. Proteiner som utbyter olika antal laddningar över membranet kallas elektrogena. Transporten möj-liggörs de aminosyrarester som utgör proteinet. För att förstå transportpro-cessen mer i detalj ville jag få klarhet i vilka aminosyror som är ansvariga för transporten. Genom att mutera en specifik lysinaminosyra i protein-sekvensen kunde jag få till stånd en transport av en jon i utbyte för en pro-

37

ton. Därmed skapades ett elektroneutralt protein, där ett jämnt antal ladd-ningar utbyts per transportcykel (en natriumjon mot en proton). Dessa resul-tat är ett stort bidrag till forskningsfältet för natrium/protonutbytare, och de tillät oss att se hur dessa proteiner fungerar i detalj. Dock är inte den kom-pletta mekanismen för dessa proteiner klarlagd och vidare experiment be-hövs.

38

Acknowledgments

My journey towards this accomplishment, my thesis, had some ups and downs, but, in overall, I am happy about my time here in Stockholm and in DBB. First and foremost, I would like to show my gratitude to my supervisor David Drew for accepting me as a PhD student and giving me an opportuni-ty to work on this exciting project and for all your help during my time in the lab.

Secondly, I would like to thank to my dearest friend Mathieu Coinçon. You were like my big brother and I am grateful to have such a teammate not only in the lab but also in the basketball court, even though you suck at it. It was a pleasure to work alongside you in lab and share all our talks and laughs. I will never forget how you welcomed me into your family and being a true friend outside the work.

Christoph von Ballmoos – thank you for teaching me all those transport assays, which were a real headache when I started. However, now I see the power of these experiments and I learn to appreciate it. Also, I am grateful for a warm welcome for my short stay in Bern and in your laboratory.

Aziz, you are a true friend and a great companion in lab. You are my most trustful person at work. You are the only one who I trusted 100%, when I needed help – borrowing solutions or asking for favours, I knew I can count on you.

The Drew lab is the best place I have ever work so far and it is only be-cause of all the people are working/worked here. Saba, it was great to have you as my bench neighbour and share all the laughs. I still remember our “Fact of the day” topic. Yurie and Iven for being such good friends inside and outside the lab. It was nice to work next to others: Emmanuel, Pascal, Magnus, Qie, Robin.

When I first came to Stockholm, it was not easy to be alone in the lab, therefore I am grateful to Sie Germans (Ott’s lab) for welcoming me warm-ly and being patient with my constant questions (especially thanks to Markus). Over the years I have made German friends which made the life in the corridor and the office much nicer. Special thanks to Kirsten, Braulio, Hannah, Tamara, Jacob, Kati, Andy and Caro for all activities outside the lab and my office mates for all the gossips and chitchats. It was lovely to know the rest of the “Germans”: Lorena, Jens, Alex, Mama, Roger, Abeer, Magda and the remaining.

Good luck to everyone!

39

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