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Establishing the molecular mechanism of sodium/proton exchangers
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.ORCID iD: 0000-0001-8222-7660
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2017. , 47 p.
Keyword [en]
membrane protein, secondary active transporters, sodium/proton exchangers, proton transport, structure, energetics
National Category
Biochemistry and Molecular Biology Structural Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-147333ISBN: 978-91-7649-964-1 (print)ISBN: 978-91-7649-965-8 (electronic)OAI: oai:DiVA.org:su-147333DiVA: diva2:1143860
Public defence
2017-11-14, William-Olssonsalen, Geovetenskapens hus, Svante Arrhenius väg 14, Stockholm, 10:00 (English)
Opponent
Supervisors
Available from: 2017-10-20 Created: 2017-09-22 Last updated: 2017-10-20Bibliographically approved
List of papers
1. A two-domain elevator mechanism for sodium/proton antiport
Open this publication in new window or tab >>A two-domain elevator mechanism for sodium/proton antiport
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2013 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 501, no 7468, 573-577 p.Article in journal (Refereed) Published
Abstract [en]

Sodium/proton (Na+/H+) antiporters, located at the plasma membrane in every cell, are vital for cell homeostasis1. In humans, their dysfunction has been linked to diseases, such as hypertension, heart failure and epilepsy, and they are well-established drug targets(2). The best understood model system for Na+/H+ antiport is NhaA from Escherichia coli(1,3), for which both electron microscopy and crystal structures are available(4-6). NhaA is made up of two distinct domains: a core domain and a dimerization domain. In the NhaA crystal structure a cavity is located between the two domains, providing access to the ion-binding site from the inward-facing surface of the protein(1,4). Likemany Na+/H+ antiporters, the activity of NhaA is regulated by pH, only becoming active above pH 6.5, at which point a conformational change is thought to occur(7). The only reported NhaA crystal structure so far is of the low pH inactivated form(4). Here we describe the active-state structure of a Na+/H+ antiporter, NapA from Thermus thermophilus, at 3 angstrom resolution, solved from crystals grown at pH7.8. In the NapA structure, the core and dimerization domains are in different positions to those seen in NhaA, and a negatively charged cavity has now opened to the outside. The extracellular cavity allows access to a strictly conserved aspartate residue thought to coordinate ion binding(1,8,9) directly, a role supported hereby molecular dynamics simulations. To alternate access to this ion-binding site, however, requires a surprisingly large rotation of the core domain, some 20 degrees against the dimerization interface. We conclude that despite their fast transport rates of up to 1,500 ions per second(3), Na+/H+ antiporters operate by a two-domain rocking bundle model, revealing themes relevant to secondary-active transporters in general.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-95768 (URN)10.1038/nature12484 (DOI)000324826300064 ()
Note

AuthorCount:10;

Available from: 2013-11-04 Created: 2013-11-04 Last updated: 2017-09-22Bibliographically approved
2. Crystal structures reveal the molecular basis of ion translocation in sodium/proton antiporters
Open this publication in new window or tab >>Crystal structures reveal the molecular basis of ion translocation in sodium/proton antiporters
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2016 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 23, no 3, 248-255 p.Article in journal (Refereed) Published
Abstract [en]

To fully understand the transport mechanism of Na+/H+ exchangers, it is necessary to clearly establish the global rearrangements required to facilitate ion translocation. Currently, two different transport models have been proposed. Some reports have suggested that structural isomerization is achieved through large elevator-like rearrangements similar to those seen in the structurally unrelated sodium-coupled glutamate-transporter homolog Glt(ph). Others have proposed that only small domain movements are required for ion exchange, and a conventional rocking-bundle model has been proposed instead. Here, to resolve these differences, we report atomic-resolution structures of the same Na+/H+ antiporter (NapA from Thermus thermophilus) in both outward- and inward-facing conformations. These data combined with cross-linking, molecular dynamics simulations and isothermal calorimetry suggest that Na+/H+ antiporters provide alternating access to the ion-binding site by using elevator-like structural transitions.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-128517 (URN)10.1038/nsmb.3164 (DOI)000371452500012 ()26828964 (PubMedID)
Available from: 2016-04-06 Created: 2016-03-30 Last updated: 2017-09-22Bibliographically approved
3. Dissecting the proton transport pathway in electrogenic Na+/H+ antiporters
Open this publication in new window or tab >>Dissecting the proton transport pathway in electrogenic Na+/H+ antiporters
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2017 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 7, E1101-E1110 p.Article in journal (Refereed) Published
Abstract [en]

Sodium/proton exchangers of the SLC9 family mediate the transport of protons in exchange for sodium to help regulate intracellular pH, sodium levels, and cell volume. In electrogenic Na+/H+ antiporters, it has been assumed that two ion-binding aspartate residues transport the two protons that are later exchanged for one sodium ion. However, here we show that we can switch the antiport activity of the bacterial Na+/H+ antiporter NapA from being electrogenic to electroneutral by the mutation of a single lysine residue (K305). Electroneutral lysine mutants show similar ion affinities when driven by Delta pH, but no longer respond to either an electrochemical potential (psi) or could generate one when driven by ion gradients. We further show that the exchange activity of the human Na+/H+ exchanger NHA2 (SLC9B2) is electroneutral, despite harboring the two conserved aspartic acid residues found in NapA and other bacterial homologues. Consistently, the equivalent residue to K305 in human NHA2 has been replaced with arginine, which is a mutation that makes NapA electroneutral. We conclude that a transmembrane embedded lysine residue is essential for electrogenic transport in Na+/H+ antiporters.

Keyword
secondary active transporters, proton transport, membrane protein, Na+/H+ exchangers, energetics
National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-141418 (URN)10.1073/pnas.1614521114 (DOI)000393989300010 ()28154142 (PubMedID)
Available from: 2017-04-18 Created: 2017-04-18 Last updated: 2017-09-22Bibliographically approved
4. Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters
Open this publication in new window or tab >>Integrating mass spectrometry with MD simulations reveals the role of lipids in Na+/H+ antiporters
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2017 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, 13993Article in journal (Refereed) Published
Abstract [en]

Na+/H+ antiporters are found in all kingdoms of life and exhibit catalysis rates that are among the fastest of all known secondary- active transporters. Here we combine ion mobility mass spectrometry and molecular dynamics simulations to study the conformational stability and lipid- binding properties of the Na+/H+ exchanger NapA from Thermus thermophilus and compare this to the prototypical antiporter NhaA from Escherichia coli and the human homologue NHA2. We find that NapA and NHA2, but not NhaA, form stable dimers and do not selectively retain membrane lipids. By comparing wild- type NapA with engineered variants, we show that the unfolding of the protein in the gas phase involves the disruption of inter- domain contacts. Lipids around the domain interface protect the native fold in the gas phase by mediating contacts between the mobile protein segments. We speculate that elevator- type antiporters such as NapA, and likely NHA2, use a subset of annular lipids as structural support to facilitate large- scale conformational changes within the membrane.

National Category
Biological Sciences
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-139365 (URN)10.1038/ncomms13993 (DOI)000391641800001 ()
Available from: 2017-02-08 Created: 2017-02-06 Last updated: 2017-09-22Bibliographically approved

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