Functional Characterization of SLC Transporters Using Solid Supported Membranes

Bazzone, Andre; Barthmes, Maria

doi:10.1007/978-1-0716-0724-4_4

Cited by 15 publications

(20 citation statements)

References 67 publications

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“…SSM electrophysiology was conducted using a SURFE 2 R N1 instrument (Nanion Technologies, Munich, Germany) according to published protocols ( Bazzone and Barthmes, 2020 ; Bazzone et al, 2017 ). The sensor was alkylated and painted with lipid solution (7.5 µg/µl 1,2-diphytanoyl-sn-glycero-3-phosphocholine in n-decane), followed immediately by addition of recording buffer (100 mM KCl, 100 mM KPO 4 , pH 7.5).…”

Section: Methodsmentioning

confidence: 99%

Crystal structures of bacterial small multidrug resistance transporter EmrE in complex with structurally diverse substrates

Kermani

Burata

Koff

et al. 2022

eLife

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Proteins from the bacterial small multidrug resistance (SMR) family are proton-coupled exporters of diverse antiseptics and antimicrobials, including polyaromatic cations and quaternary ammonium compounds. The transport mechanism of the Escherichia coli transporter, EmrE, has been studied extensively, but a lack of high-resolution structural information has impeded a structural description of its molecular mechanism. Here we apply a novel approach, multipurpose crystallization chaperones, to solve several structures of EmrE, including a 2.9 Å structure at low pH without substrate. We report five additional structures in complex with structurally diverse transported substrates, including quaternary phosphonium, quaternary ammonium, and planar polyaromatic compounds. These structures show that binding site tryptophan and glutamate residues adopt different rotamers to conform to disparate structures without requiring major rearrangements of the backbone structure. Structural and functional comparison to Gdx-Clo, an SMR protein that transports a much narrower spectrum of substrates, suggests that in EmrE, a relatively sparse hydrogen bond network among binding site residues permits increased sidechain flexibility.

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Section: Methodsmentioning

confidence: 99%

Crystal structures of bacterial small multidrug resistance transporter EmrE in complex with structurally diverse substrates

Kermani

Burata

Koff

et al. 2022

eLife

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“…SSME was performed using the SURFE 2 R N1 device (Nanion Technologies GmbH) and sensor preparation followed the standard protocols as described in detail previously ( Bazzone et al, 2017a ; Bazzone and Barthmes, 2020 ). A summary is provided in Supplementary Methods S1.1 .…”

Section: Methodsmentioning

confidence: 99%

“…In SSME, transporter activity is triggered by substrate concentration jumps at 0 mV with the possibility to apply co-substrate gradients as an additional driving force. In SSME all recorded currents are transient and decay to zero, usually within a second, due to the capacitive read-out ( Bazzone et al, 2017a ; Bazzone and Barthmes, 2020 ). Sugar concentration jumps on SGLT1 generate biphasic transient currents comprised of a fast decaying PSS component and a slowly decaying transport component ( Bazzone et al, 2022a ).…”

Section: Introductionmentioning

confidence: 99%

Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation

et al. 2023

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Beside the ongoing efforts to determine structural information, detailed functional studies on transporters are essential to entirely understand the underlying transport mechanisms. We recently found that solid supported membrane-based electrophysiology (SSME) enables the measurement of both sugar binding and transport in the Na+/sugar cotransporter SGLT1 (Bazzone et al, 2022a). Here, we continued with a detailed kinetic characterization of SGLT1 using SSME, determining KM and KDapp for different sugars, kobs values for sugar-induced conformational transitions and the effects of Na+, Li+, H+ and Cl− on sugar binding and transport. We found that the sugar-induced pre-steady-state (PSS) charge translocation varies with the bound ion (Na+, Li+, H+ or Cl−), but not with the sugar species, indicating that the conformational state upon sugar binding depends on the ion. Rate constants for the sugar-induced conformational transitions upon binding to the Na+-bound carrier range from 208 s−1 for D-glucose to 95 s−1 for 3-OMG. In the absence of Na+, rate constants are decreased, but all sugars bind to the empty carrier. From the steady-state transport current, we found a sequence for sugar specificity (Vmax/KM): D-glucose > MDG > D-galactose > 3-OMG > D-xylose. While KM differs 160-fold across tested substrates and plays a major role in substrate specificity, Vmax only varies by a factor of 1.9. Interestingly, D-glucose has the lowest Vmax across all tested substrates, indicating a rate limiting step in the sugar translocation pathway following the fast sugar-induced electrogenic conformational transition. SGLT1 specificity for D-glucose is achieved by optimizing two ratios: the sugar affinity of the empty carrier for D-glucose is similarly low as for all tested sugars (KD,Kapp = 210 mM). Affinity for D-glucose increases 14-fold (KD,Naapp = 15 mM) in the presence of sodium as a result of cooperativity. Apparent affinity for D-glucose during transport increases 8-fold (KM = 1.9 mM) compared to KD,Naapp due to optimized kinetics. In contrast, KM and KDapp values for 3-OMG and D-xylose are of similar magnitude. Based on our findings we propose an 11-state kinetic model, introducing a random binding order and intermediate states corresponding to the electrogenic transitions detected via SSME upon substrate binding.

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“…SSM-based electrophysiology was conducted using a SURFE2R N1 instrument (Nanion Technologies) following published protocols( 48, 84 ). 3 mm SURFE2R N1 single sensors (Nanion Technologies) were alkylated by adding 100 µl of 0.5 mM thiol solution (1-octadecanethiol in isopropanol), subsequently incubating for 1 hour at room temperature in a closed petri dish.…”

Section: Methodsmentioning

confidence: 99%

Mechanistic basis of choline import involved in teichoic acids and lipopolysaccharide modification

Bärland

Rueff

Cebrero

et al. 2021

Preprint

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Phosphocholine molecules decorating bacterial cell wall teichoic acids and outer-membrane lipopolysaccharide have significant roles in adhesion to host cells, immune evasion, and persistence. Bacteria carrying the operon that performs phosphocholine decoration, synthesize phosphocholine after uptake of the choline precursor by LicB, a conserved transporter among divergent species. Streptococcus pneumoniae is a prominent pathogen where phosphocholine decoration plays a fundamental role in virulence. Here we present cryo-electron microscopy and crystal structures of S. pneumoniae LicB, revealing distinct conformational states and describing architectural and mechanistic elements essential to choline import. Together with in vitro and in vivo functional characterization, we found that LicB displays proton-coupled import activity and promiscuous selectivity involved in adaptation to choline deprivation conditions, and describe LicB inhibition by synthetic nanobodies (sybodies) and hemicholinium-3. Our results provide novel insights into the molecular mechanism of a key transporter involved in bacterial pathogenesis and establish a basis for inhibition of the phosphocholine modification pathway across bacterial phyla.

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Functional Characterization of SLC Transporters Using Solid Supported Membranes

Cited by 15 publications

References 67 publications

Crystal structures of bacterial small multidrug resistance transporter EmrE in complex with structurally diverse substrates

Crystal structures of bacterial small multidrug resistance transporter EmrE in complex with structurally diverse substrates

Functional characterization of SGLT1 using SSM-based electrophysiology: Kinetics of sugar binding and translocation

Mechanistic basis of choline import involved in teichoic acids and lipopolysaccharide modification

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