Structure and elevator mechanism of the mammalian sodium/proton exchanger NHE9
2020; Springer Nature; Volume: 39; Issue: 24 Linguagem: Inglês
10.15252/embj.2020105908
ISSN1460-2075
AutoresIven Winkelmann, Rei Matsuoka, Pascal F. Meier, Denis Shutin, Chen‐Ou Zhang, Laura Orellana, Ricky Sexton, Michael Landreh, Carol V. Robinson, Oliver Beckstein, David Drew,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoArticle29 October 2020Open Access Transparent process Structure and elevator mechanism of the mammalian sodium/proton exchanger NHE9 Iven Winkelmann Iven Winkelmann Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden Search for more papers by this author Rei Matsuoka Rei Matsuoka Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden Search for more papers by this author Pascal F Meier Pascal F Meier Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden Search for more papers by this author Denis Shutin Denis Shutin Department of Chemistry, University of Oxford, Oxford, UK Search for more papers by this author Chenou Zhang Chenou Zhang Department of Physics, Center for Biological Physics, Arizona State University, Tempe, AZ, USA Search for more papers by this author Laura Orellana Laura Orellana Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden Search for more papers by this author Ricky Sexton Ricky Sexton Department of Physics, Center for Biological Physics, Arizona State University, Tempe, AZ, USA Search for more papers by this author Michael Landreh Michael Landreh orcid.org/0000-0002-7958-4074 Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Carol V Robinson Carol V Robinson Department of Chemistry, University of Oxford, Oxford, UK Search for more papers by this author Oliver Beckstein Oliver Beckstein Department of Physics, Center for Biological Physics, Arizona State University, Tempe, AZ, USA Search for more papers by this author David Drew Corresponding Author David Drew [email protected] orcid.org/0000-0001-8866-6349 Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden Search for more papers by this author Iven Winkelmann Iven Winkelmann Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden Search for more papers by this author Rei Matsuoka Rei Matsuoka Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden Search for more papers by this author Pascal F Meier Pascal F Meier Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden Search for more papers by this author Denis Shutin Denis Shutin Department of Chemistry, University of Oxford, Oxford, UK Search for more papers by this author Chenou Zhang Chenou Zhang Department of Physics, Center for Biological Physics, Arizona State University, Tempe, AZ, USA Search for more papers by this author Laura Orellana Laura Orellana Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden Search for more papers by this author Ricky Sexton Ricky Sexton Department of Physics, Center for Biological Physics, Arizona State University, Tempe, AZ, USA Search for more papers by this author Michael Landreh Michael Landreh orcid.org/0000-0002-7958-4074 Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden Search for more papers by this author Carol V Robinson Carol V Robinson Department of Chemistry, University of Oxford, Oxford, UK Search for more papers by this author Oliver Beckstein Oliver Beckstein Department of Physics, Center for Biological Physics, Arizona State University, Tempe, AZ, USA Search for more papers by this author David Drew Corresponding Author David Drew [email protected] orcid.org/0000-0001-8866-6349 Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden Search for more papers by this author Author Information Iven Winkelmann1,†, Rei Matsuoka1,†, Pascal F Meier1,†, Denis Shutin2, Chenou Zhang3, Laura Orellana1, Ricky Sexton3, Michael Landreh4, Carol V Robinson2, Oliver Beckstein3 and David Drew *,1 1Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden 2Department of Chemistry, University of Oxford, Oxford, UK 3Department of Physics, Center for Biological Physics, Arizona State University, Tempe, AZ, USA 4Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden † These authors contributed equally to this work *Corresponding author. Tel: +46 816 2295; E-mail: [email protected] The EMBO Journal (2020)39:4541-4559https://doi.org/10.15252/embj.2020105908 Correction added on 15 December 2020, after first online publication: In the previous version of Figure 1C, the colour scheme in the magnification had been inverted between NHE9 WT and NHE9 N243A-D244A. Additionally, the label in Figure 1C for NHE9 WT was updated to NHE9 Correction added on 15 December 2020, after first online publication: The PDB codes for NHE9* and NHE9 ΔCTD were interchanged. The numbers 6131 and 6115 were moved from 'Model composition' to 'Non-hydrogen atoms'. The number of protein residues was changed from - to 771 and from - to 769 for NHE9* and NHE9 ΔCTD, respectively. The percentages of poor rotamers for NHE9* and NHE9 ΔCTD were interchanged. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Na+/H+ exchangers (NHEs) are ancient membrane-bound nanomachines that work to regulate intracellular pH, sodium levels and cell volume. NHE activities contribute to the control of the cell cycle, cell proliferation, cell migration and vesicle trafficking. NHE dysfunction has been linked to many diseases, and they are targets of pharmaceutical drugs. Despite their fundamental importance to cell homeostasis and human physiology, structural information for the mammalian NHEs was lacking. Here, we report the cryogenic electron microscopy structure of NHE isoform 9 (SLC9A9) from Equus caballus at 3.2 Å resolution, an endosomal isoform highly expressed in the brain and associated with autism spectrum (ASD) and attention deficit hyperactivity (ADHD) disorders. Despite low sequence identity, the NHE9 architecture and ion-binding site are remarkably most similar to distantly related bacterial Na+/H+ antiporters with 13 transmembrane segments. Collectively, we reveal the conserved architecture of the NHE ion-binding site, their elevator-like structural transitions, the functional implications of autism disease mutations and the role of phosphoinositide lipids to promote homodimerization that, together, have important physiological ramifications. Synopsis Exchange of sodium for protons across cell membranes regulates intracellular pH and cell volume. Here, cryo-EM combined with functional assays provides first insights into the structure and membrane context of a mammalian Na+/H+ exchanger (NHE). The Equus caballus NHE9/SLC9A9 monomer comprises 13 transmembrane segments and forms a homodimer with similar topology as bacterial antiporter homologues. The NHE9 structure at 3.2 Å shows an inward-facing conformation, with a highly conserved Na+-binding site. Functional assays suggest stabilization of the NHE9 homodimer by phosphoinositide lipid binding to an extracellular loop domain. Autism-associated disease mutants map onto highly flexible regions in NHE9, likely impairing endosomal pH regulation by NHE9. Intrinsic NHE9 dynamics support an elevator-like alternating access mechanism in mammalian Na+/H+ exchange. Introduction Na+/H+ exchangers (NHEs) are ion transporters found in all kingdoms of life (Orlowski & Grinstein, 2004; Donowitz et al, 2013; Fuster & Alexander, 2014; Pedersen & Counillon, 2019). They directly couple the transfer of protons across biological membranes to the counter-transport of Na+/Li+/(K+), a mechanism first proposed in bacteria (West & Mitchell, 1974) and later observed in isolated, rat kidney vesicles (Murer et al, 1976). In mammals, there are 13 distinct NHE orthologues that are thought to perform electroneutral (1:1) ion-exchange: NHE1-9 also known as SLC9A1-9, NHA1-2 also known as SLC9B1-2 and sperm-specific NHE also known as SLC9C (Brett et al, 2005; Fuster & Alexander, 2014; Pedersen & Counillon, 2019). NHEs differ in substrate preferences, kinetics and tissue localizations (Pedersen & Counillon, 2019). NHE1, for example, is ubiquitously expressed in the plasma membrane of most tissues, and its major physiological role is the regulation of intracellular pH and cell volume (Slepkov et al, 2007; Pedersen & Counillon, 2019). NHE3, on the other hand, is highly expressed in the intestine and kidneys and is important for Na+ reabsorption and acid–base homeostasis (Zachos et al, 2005; Donowitz et al, 2009). Other NHE isoforms, such as NHE6, NHE7, NHE8 and NHE9, are critical for the maintenance and regulation of organellar and endosomal pH, which in turn are linked to a multitude of physiological functions (Brett et al, 2002; Orlowski & Grinstein, 2007; Pedersen & Counillon, 2019). Figure 1. Horse NHE9 functional characterization and cryo-EM structureȚ. Phylogenetic tree of canonical human NHE1-9 (SLC9A1-9) cluster into those that localize predominantly to either the plasma membrane (pink) or endomembrane (orange); for completeness, more distantly related non-canonical human NHE members SLC9B1-2 (NHA1-2) (yellow) and SLC9C (blue) are also shown as labelled. SEC trace and SDS–PAGE of purified NHE9* (residues 8 and 574 out of 644) as depicted by schematic. Activity of NHE9* co-reconstituted with ATPase into liposomes to mimic the in vivo situation (schematic). Representative ACMA fluorescence traces of liposome reconstituted NHE9* (blue), NHE9* double-mutant N243A-D244A (red) and rat fructose transporter GLUT5 (black). ATP-driven H+ pumping establishes a ΔpH (0–3 min). H+ efflux is initiated by the addition of 40 mM NaCl, and subsequent addition of NH4Cl (4 min) collapses the proton gradient. Michaelis–Menten kinetics for NHE9* (red), NHE9 ΔCTD (green), N243A-D244A (blue) and rat GLUT5 (black) as detected by ACMA dequenching following substrate addition. In all experiments, error bars, s.e.m.; n = 3 technical repeats. The apparent KM values are an average from n = 3 separate protein reconstitutions. Cryo-EM density map of the NHE9 ΔCTD homodimer with the 6-TM core transport domains (coloured in blue), the dimer domain (coloured in orange) and the linker helix (coloured in grey). Download figure Download PowerPoint Dysfunction of NHEs has been linked to many diseases such as cancer, hypertension, heart failure, diabetes and epilepsy (Fuster & Alexander, 2014; Ueda et al, 2017). In particular, NHEs are prime drug targets for cancer therapies (Stock & Pedersen, 2017; Pedersen & Counillon, 2019), since tumour cells typically upregulate NHE expression to re-alkalinize intracellular pH in response to the "Warburg effect" (Cardone et al, 2005; Parks et al, 2013), i.e. as metabolic preference for oxidative glycolysis leads to intracellular acidification. Consequently, many cancer cells are highly dependent on NHE activity, and their inhibition or knockdown interferes with cancer development (Cardone et al, 2005; Stock & Pedersen, 2017; White et al, 2017). NHE1 has further been targeted in heart disease (Odunewu-Aderibigbe & Fliegel, 2014), since NHE1 inhibition protects the myocardium against ischaemic, reperfusion injury and heart failure. Although clinical trials of an NHE1 inhibitor were discontinued, animal models hold promise that NHE1 inhibition could result in an effective therapeutic (Karmazyn, 2013). Since NHE3 has been linked directly to blood volume and pressure (Alexander & Grinstein, 2006), NHE3 is targeted as an avenue to treat hypertension (Linz et al, 2016); so far, this is yet to bear fruit, although an NHE3 inhibitor was awarded FDA-approval for irritable bowel syndrome (Siddiqui & Cash, 2020). The endosomal NHE6 and NHE9 are the only isoforms known, to date, with disease-associated mutations (Fuster & Alexander, 2014). More specifically, human disease mutations of NHE6 are associated with an Angelman syndrome-like disorder (Gilfillan et al, 2008) and NHE9 to neurological disorders such as familial autism, ADHD and epilepsy (Kondapalli et al, 2013, 2014; Ullman et al, 2018). Despite the clear importance of NHE function to human physiology and drug development, their structure and the molecular details of their ion-exchange mechanism have been lacking. Based on substantial biochemical data, all NHEs are thought to form physiological active homodimers (Brett et al, 2005; Fuster & Alexander, 2014; Pedersen & Counillon, 2019), with the respective monomers consisting of a transporter module that performs ion-exchange, and a C-terminal non-membranous cytosolic domain of varying length ~ 125–440 residues, which regulates ion-exchange activity (Fuster & Alexander, 2014; Pedersen & Counillon, 2019). The transport module shares ~18–25% sequence homology to bacterial Na+/H+ antiporters harbouring the "NhaA-fold", so-named after the first crystal structure obtained from Escherichia coli (Brett et al, 2005; Padan, 2008; Fliegel, 2019). The NhaA crystal structure and more recent structures from other bacterial homologues (Hunte et al, 2005; Lee et al, 2013; Paulino et al, 2014; Wöhlert et al, 2014) have shown that the transporter module consists of two distinct domains, a dimerization domain and an ion-transporting (core) domain, made up of six transmembrane (TM) segments. The 6-TM core domain is thought to undergo global, elevator-like structural transitions to translocate ions across the membrane against the anchored dimerization domain (Lee et al, 2013; Coincon et al, 2016; Drew & Boudker, 2016; Okazaki et al, 2019). Due to their highly dynamic nature, the poor stability of detergent-solubilized NHEs has been a bottleneck for structural studies. Here, we focused our structural efforts on NHE9 since this isoform possesses one of the shortest C-terminal regulatory domains, simplifying mechanistic understanding, and an atomic model would allow us to interpret human disease mutations. Whilst the full physiological role of NHE9 still remains uncertain, NHE9 activity is important for regulating vesicular trafficking and turnover of the synaptic membranes, by the fine-tuning of endosomal pH (Kondapalli et al, 2015). In mouse hippocampal neurons, the absence of NHE9 causes impaired synaptic vesicle exocytosis by reducing presynaptic Ca2+ entry as a consequence of altered luminal pH (Ullman et al, 2018). NHE9 is also highly expressed in glioblastoma multiforme (GBM), the most common brain tumour, as endolysosomal pH is critical for epidermal growth factor (EGFR) sorting and turnover (Kondapalli et al, 2015). Results Out of 13 candidates, horse NHE9, which shares 95% sequence identity with human NHE9, was identified to be the most detergent stable using fluorescence-based screening methods in Saccharomyces cerevisiae (Materials and Methods, Figs 1A and B and EV1, and Appendix Fig S1). Purified horse NHE9* (residues 8–574) was reconstituted into liposomes together with F0F1-ATP synthase to mimic the in vivo co-localization of NHE9 with the endosomal V-ATPase (Kondapalli et al, 2015). Proton efflux was monitored in response to the addition of Na+, performed in the presence of valinomycin, which was included to eliminate efflux against a membrane potential (Ψ) (Materials and Methods and Fig 1C). In this experimental setup, proton efflux by NHE9* was ~ 3-fold higher than a NHE9* variant in which the critical ion-binding aspartic acid (D244) and the preceding asparagine (N243)—the strictly conserved "ND" motif (Brett et al, 2005; Masrati et al, 2018)—were substituted to alanine (Fig 1C and D). We could confirm the dequenching observed by the D244A-N243A variant was artefactual, since the unrelated fructose transporter GLUT5 gives a similar response (Fig 1C and D). Whilst an improved assay is required before one can make detailed comparisons between NHE9* variants, the activity was significant enough for the determination of an apparent KM of NHE9* for Na+ (20.5 ± 2.9 mM), which was similar to estimates of endosomal isoforms NHE6 (10 mM) and NHE8 (23 mM) (Xu et al, 2005; Pedersen & Counillon, 2019; Fig 1D). Click here to expand this figure. Figure EV1. Multiple sequence alignment of horse NHE9 and human NHE1-9 isoformsResidues with over 70% sequence identity are indicated by purple background. Conserved ion-binding site residues are highlighted with a red border. Positions which have been identified to harbour disease mutations in human NHE9 are indicated with an asterisk (*). Residues which are predicted by SignalP-5.0 (likelihood 0.56–0.98) to be part of a single peptide at the N-terminus are encircled with a dashed red box. Helix breakpoints (s-shaped line), connecting loops (lines), non-modelled loops (dashed line), dimer domain TMs (orange), core 6-TM transport domain TMs (blue) and the linking helix TM7 (grey) are indicated. Download figure Download PowerPoint NHE9* sample preparation was optimized for grid preparation, cryo-EM data acquisition and structural determination at an active pH of 7.5 (Materials and Methods). We combined 3D classes from two independent data collections of ~ 1.4 million particles, from which an EM map was reconstructed to 3.5 Å according to the gold-standard Fourier shell correlation (FSC) 0.143 criterion, which used ~ 5% of the total collected particles (Table 1, Appendix Figs S2 and S3A). The EM maps were well resolved for the TMs, but a few of the connecting loop residues and a larger, extracellular loop of 51 residues, located between TM2 and TM3, could not be built (Fig EV2A). Surprisingly, there was no obvious density for any of the 93-residue long C-terminal tail regulatory domain (CTD), likely a result of its predicted dynamics and intrinsic disorder (Fig EV2A; Norholm et al, 2011; Hendus-Altenburger et al, 2014; Pedersen & Counillon, 2019). We repeated cryo-EM structural determination for a horse NHE9 construct lacking the entire CTD domain (NHE9 ΔCTD), which displayed similar kinetics as the close-to-full-length NHE9* construct (Figs 1D and EV3 and Materials and Methods). Consistent with removal of the entire flexible CTD, 36% of the auto-picked particles now contributed to the final 3D class with an improved EM map resolution of 3.2 Å (Figs 1E, and EV2B and EV3, and Appendix Fig S3B). As model building revealed only minor differences between NHE9* and NHE9 ΔCTD structures, the later was used for all subsequent analysis due to its moderately better resolution (Fig EV2C). Table 1. Data collection, processing and refinement statistics of NHE9 structures.§ NHE9* (EMDB-11066) (PDB 6Z3Y) NHE9 ΔCTD (EMDB-11067) (PDB 6Z3Z) Data collection and processing statistics Magnification 165,000 165,000 Voltage (kV) 300 300 Electron exposure (e−/Å2) 80 80 Defocus range (μm) 0.7–2.5 0.7–2.5 Pixel size (Å) 0.83 0.83 Symmetry imposed C1 C1 Initial particle images (no.) 2,781,170 1,629,483 Final particle images (no.) 139,511 595,024 Map resolution (Å) 3.51 3.19 FSC threshold 0.143 0.143 Map resolution range (Å) 3.5–4.9 3.1–4.1 Refinement Initial model used (PDB code) 4cz9 4cz9 Model resolution (Å) 3.69 3.6 FSC threshold 0.50 0.50 Map sharpening B factor (Å2) -77.5 -133.8 Model composition Non-hydrogen atoms 6131 6115 Protein residues 771 769 Ligands – – B factors (Å2) Protein 104.99 116.89 Ligand – – R.m.s. deviations Bond lengths (Å) 0.004 0.006 Bond angles (°) 0.525 0.783 Validation MolProbity score 2.03 1.86 Clashscore 8.76 8.31 Poor rotamers (%) 0.5% 1.5% Ramachandran plot Favoured (%) 94.38 93.94 Allowed (%) 5.62 6.06 Disallowed (%) 0 0 Click here to expand this figure. Figure EV2. Cryo-EM density of horse NHE9* and NHE9 ΔCTD before and after density subtraction NHE9* (left purple) and NHE9 ΔCTD (right purple) structures docked into their respective cryo-EM density maps before micelle density subtraction. Non-modelled cryo-EM density for the 51-residue long TM2–TM3 loop was apparent at the dimer interface (black-dotted circle). The NHE9* construct contains 93 out of 163 residues of the C-terminal regulatory domain (blue-dotted circle), but no corresponding additional density was apparent as compared to the NHE9 ΔCTD density maps. Cryo-EM density map and model are shown for all transmembrane segments for NHE9 ΔCTD in the dimer domain (orange), transport domain (blue) and linker TM7 (grey). Residues D215 and D244 (encircled) on TM5 and TM6, respectively, have been modelled after PaNhaP at pH 8.0 (PDB id: 4cz8). Superimposition of NHE9* (wheat) and NHE9 ΔCTD (core transport domains (blue), dimerization domains (orange), linker helices (grey)) structures, which show only small differences in the most mobile regions. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. The data-processing workflow of horse NHE9 ΔCTDThe dataset contained 4,739 movies that were corrected by MotionCor2 and CTFFind. After reference-based auto-picking, 1,629,483 particles were picked. Several rounds of 2D classification were performed, yielding 714,870 particles, which were subjected to 3D classification. One of the three 3D classes was selected, and it contained 595,024 particles. After several rounds of refinement with global and local search using ctf refine and polishing, a final resolution of 3.19 Å was achieved at gold-standard FSC (0.143), with a local resolution range of 3.1–4.1 Å. Download figure Download PowerPoint The NHE9 monomer consists of 13 TMs with an extracellular N-terminus and intracellular C-terminus (Fig 2A and Appendix Fig S4A). The NHE9 structure is therefore more similar to the bacterial homologue structures with 13 TMs and a 6-TM topology inverted repeat, namely NapA (Lee et al, 2013), MjNhaP (Paulino et al, 2014) and PaNhaP (Wöhlert et al, 2014), rather than the more commonly used NHE models that, like NhaA, have 12 TMs and a 5-TM topology inverted repeat (Hunte et al, 2005; Landau et al, 2007; Kondapalli et al, 2013; Hendus-Altenburger et al, 2014; Pedersen & Counillon, 2019; Li et al, 2020; Appendix Fig S4B). The expansion of the inverted-topology repeats establishes a dimerization interface that in NHE9, and the bacterial antiporters with 13 TMs, is formed predominantly by tight interactions between TM1 on one monomer and TM8 on the other, burying a total surface area of ~ 1,700 to 2,000 Å2 (Fig EV4A). In NhaA, with 12 TMs, the dimerization interface is instead formed by interactions between an antiparallel β-hairpin extension in a loop domain that buries a total surface area of only ~ 700 Å2 (Lee et al, 2014). In NHE9, the substrate-binding cavity is located between the dimer and core ion-transport domains and is open towards the intracellular side (Figs 2B and EV4A). Near the base of the cavity is the strictly conserved aspartate Asp244 (TM6) (Figs 2B–D and EV1), essential for ion-binding and transport (Padan, 2008; Maes et al, 2012; Lee et al, 2013; Wöhlert et al, 2014; Coincon et al, 2016; Pedersen & Counillon, 2019). The ion-binding site and the negatively charged funnel are highly conserved across all NHEs (Figs 2B and C and EV1), enabling the generation of plausible models for all NHEs, e.g. including the clinical drug targets NHE1 and NHE3 (Pedersen & Counillon, 2019; Fig EV4B and Appendix Fig S5). Figure 2. NHE9 architecture and ion-binding site of the inward-facing NHE9 homodimer Cartoon representation of the NHE9 homodimer shown from the extracellular side in the endosomal lumen (left) and along the membrane plane (right). Ion translocation 6-TM core transport domains (blue), dimerization domains (orange) and linker helix TM7 (grey), are coloured as in Fig 1E with the respective helices numbered. Cartoon representation of dimeric NHE9 ΔCTD from the cytosolic side with the electrostatic surface representation through the ion-binding site of one monomer (coloured blue to red, for positive to negative charges, respectively). The strictly conserved ion-binding residue Asp244 is labelled and shown in yellow as sticks. The inward open ion-binding cavity is encircled. NHE9 ΔCTD as in (B), but coloured according to conservation scores from the alignment of 650 mammalian NHE1-9 representative sequences calculated with ConSurf server (Ashkenazy et al, 2016; see Materials and Methods). left: cartoon representation of the NHE9 ion-binding site in the 6-TM core transport domain, which is made up of two broken helices TM5a-b (green) and TM12a-b (purple) accessible to a sodium ion from the cytoplasm (yellow sphere). right: ion-binding site residues are shown as yellow sticks and labelled with the corresponding residues in PaNhaP (PDB id: 4cz8) shown as grey sticks. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Cross-sectional comparison of NHE9 against Na+/H+ antiporters and CitS Slice through electrostatic surface representation of horse NHE9 and bacterial homologues as labelled. The more distantly related inward-facing structure of the citrate transporter CitS is also shown as a further example of cavity depth and hydrophobic gap located between protomers. The approximate buried surface area between monomers shown was calculated with PBDePISA. Electrostatic surface representation of the cytoplasmic view of the inward-facing NHE9 monomer (left) and the NHE1 model based on the NHE9 structure (right). Download figure Download PowerPoint The 6-TM core domain is typified by two discontinuous helices TM5a-b and TM12a-b that contain highly conserved unwound regions that cross over each other near the centre of the membrane (Figs 2A, and EV1 and EV5A). These extended helix break points harbour the strictly conserved residues Asp215 and Arg441, which are well-orientated to neutralize the ends of the oppositely charged half-helical TM5a and TM12b dipoles (Fig 2D). Asp215 is positioned at the cytoplasmic funnel and located opposite to Asp244 to create a negatively charged binding pocket readily accessible for a monovalent cation (Fig 2D). In MD simulations, Na+ spontaneously bound to Asp215 and Asp244 residues, but no binding was observed when Asp244 was protonated (Appendix Table S1, Fig 3A and B, and Appendix Fig S6). Although the 6-TM core domain structure is highly conserved (Appendix Fig S4B), the ion-binding site residues themselves are most similar to PaNhaP and MjNhaP which, like NHE9, also catalyse electroneutral exchange (Figs 2D and EV5B). Specifically, in the electrogenic NhaA and NapA antiporters, the residue corresponding to Asn243 in TM6 of NHE9 is replaced by an aspartic acid that is further salt-bridged to a lysine residue, an interaction critical for electrogenic transport (Fig EV5C; Lee et al, 2014; Uzdavinys et al, 2017; Masrati et al, 2018). However, in NHE9 and other electroneutral exchangers, a parallel salt bridge is instead formed between Glu239 in TM6 and Arg408 in TM11 (Figs 2D and EV5C); as yet, the functional role for this highly conserved electroneutral salt bridge is unclear (Masrati et al, 2018). Click here to expand this figure. Figure EV5. NHE9 ion-binding site and location of disease mutations Cartoon representation of the NHE9 6-TM core transport domain. The crossover of broken helices TM5a and TM5b (green) and TM12a and TM12b (purple) is unique to the NhaA-fold and the half-helical dipoles that they create are highlighted. Between the half-helical dipoles are oppositely charged and strictly conserved residues located in the peptide break region of all NHE isoforms (see ED Fig 1) shown here in stick form. Comparison between the ion-binding site of NHE9 (yellow, numbered) and PaNhaP (light grey, PDB id: 4cz9) is indicated. The Tl+ ion in PaNhaP (purple sphere) is further coordinated by the glutamic acid E73 residue (grey sticks; labelled with *) not conserved in NHEs. Further glutamic acid residue D93 in PaNhaP and MjNhaP (light grey and dark grey sticks, respectively; labelled with *) in TM4 is also not conserved in the NHEs, but extends towards the conserved Asn243 in NHE9 reflecting the ion-binding site differences between bacterial electroneutral Na+/H+ antiporters and mammalian NHEs. In electroneutral Na+/H+ antiporters, a salt bridge is formed between residues corresponding to Glu239 and Arg408 in NHE9 (yellow sticks). In the electrogenic Na+/H+ antiporters, a salt bridge is instead formed between an aspartic acid (Asn243 in NHE9) and a lysine residue (Arg408 in NHE9) as shown here for NapA (grey sticks). Cartoon representation of the NHE9 monomer with the 6-TM core transport domain (blue), dimerization domain (orange) and linker helix TM7 (grey) shown from the side (left) and top (right). Conserved proline and glycine residues are shown as yellow spheres. Residues which have been identified to harbour autism disease mutations in patients are shown as red spheres and labelled. Download figure Download PowerPoint Figure 3. MD simulations of NHE9 Na+ density from MD simulation (m2-00-f-3), measured in mol/l. The bulk density is ˜ 150 mM. Only one binding site is visible with this cut through the density. The membrane is omitted for clarity. Top view of the binding site; Na+ positions are shown as small spheres, drawn at 10-ns intervals. Yellow/cyan/solid grey: simulation m2-00-p-2, which started with a Na+ modelled near D244. Red/transparent red/transparent grey: simulation m2-00-f-3 during which a Na+ ion spontaneously entered the binding site and bound in almost the exact same binding pose as in the simulation where the ion was modelled. Cut through the density (same view as (B)) for simulation m2-00
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