Funktionell och strukturell studie av Solute Carrier (SLC) Transporters
Forskningsgrupp: David Drew

I urval från Stockholms universitets publikationsdatabas

• Elucidating the molecular basis of Na⁺/H⁺ exchange

  2022. Pascal F. Meier.

  Avhandling (Dok)

  Solute carrier (SLC) transporters are membrane transport proteins, which catalyse the movement of nutrients, ions, and drugs across cell membranes. Here, I will present our contribution to understanding the mechanism of the sodium/proton exchangers (NHE), belonging to the SLC9 family of membrane transporters. NHEs exchange sodium ions for protons across biological membranes, which is a critical reaction for the fine-tuning of cytoplasmic and organelle pH, sodium levels and volume homeostasis. Dysfunction of NHE members has been linked to a number of diseases and disorders, such as hypertension, heart failure, autism spectrum disorder, epilepsy and the susceptibility of long COVID. Protein structures are important for developing mechanistic models, but due to technical challenges only bacterial homologue structures of NHE proteins were previously available.

  Accumulating many years of effort, we were able to determine the first structure of a mammalian Na⁺/H⁺ exchanger, the endosomal isoform NHE9 by single-particle cryo-EM. The structure of NHE9 demonstrated that NHE proteins are architecturally most similar to bacterial homologues with 13-TM segments and likely operated by a similar elevator mechanism (I). Interestingly, native MS and thermal-shift assays indicted that the NHE9 homodimer is stabilized by the binding of a rare lipid only found in late endosomes, which implies the cell may use this lipid as means to switch-on NHE9 activity once it reaches its correct functional localization. We further provided evidence that the large cytoplasmic tail in NHE proteins likely acts in an auto-inhibitory manner. It is only removed upon the binding of extrinsic proteins (II). Indeed, the first structure of a potassium specific K⁺/H⁺ exchanger KefC reveals how its cytoplasmic tail restricts movement of the ion-transporting domain to directly inhibit transport. The structure of KefC is also the first ion-bound state seen for this family and, unlike to the modeled Na⁺/H⁺ exchanger sites with a hydrated Na⁺ ion, coordinates K⁺ as a dehydrated ion (IV). Lastly, we determining the structure of a bacterial Na⁺/H⁺ exchanger NhaA to high-resolution at an active pH of 6.5. With this structure we demonstrated how a cytoplasmic “pH gate” controlled by the pH activated NhaA (III).

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• Crystal structure of the Na+/H+ antiporter NhaA at activating pH reveals the mechanistic basis for pH sensing

  Iven Winkelmann (et al.).

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• Structure and elevator mechanism of the mammalian sodium/proton exchanger NHE9

  2020. Iven Winkelmann (et al.). EMBO Journal 39 (24)

  Artikel

  Na⁺/H⁺ exchangers (NHEs) are ancient membrane-bound nanoma- chines 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 pharma- ceutical drugs. Despite their fundamental importance to cell home- ostasis and human physiology, structural information for the mammalian NHEs was lacking. Here, we report the cryogenic elec- tron 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 atten- tion 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.

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• The MEMbrane Protein Single ShoT Amplification Recipe

  2017. Abdul Aziz-Qureshi (et al.). A Structure-Function Toolbox for Membrane Transporter and Channels, 123-138

  Kapitel

  Here, we present a simple overexpression condition for high-throughput screening of membrane proteins in Escherichia coli. For the vast majority of bacterial membrane protein targets tested the MEMbrane protein Single shoT Amplification Recipe-MemStarleads to high production yields of target protein. The use of MemStar has facilitated structural studies of several transport proteins.

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