Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site

Wahlgren, Weixiao Yuan; Dunevall, Elin; North, Rachel A.; Paz, Adrian; Scalise, Mariafrancesca; Bisignano, Paola; Bengtsson‐Palme, Johan; Goyal, Parveen; Claesson, Elin; Caing-Carlsson, Rhawnie; Andersson, Rebecka; Beis, Konstantinos; Nilsson, Ulf J.; Farewell, Anne; Pochini, Lorena; Indiveri, Cesare; Grabe, Michael; Dobson, Renwick C. J.; Abramson, Jeff; Ramaswamy, S.; Friemann, Rosmarie

doi:10.1038/s41467-018-04045-7

Cited by 68 publications

(138 citation statements)

References 80 publications

(96 reference statements)

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“…How does substrate binding in the 1:1 K + :Cl − cotransporter KCC4 differ from the 1:1:2 Na + :K + :Cl − cotransporter NKCC1 30, 37 ? Overlays of the relevant sites are shown in Figure 4B,C,E.…”

Section: Resultsmentioning

confidence: 99%

Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs

Reid

Kern

Brohawn

2019

Preprint

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AbstractCation-chloride-cotransporters (CCCs) catalyze transport of Cl− with K+ and/or Na+ across cellular membranes. CCCs play roles in volume regulation, neural development and function, audition, blood pressure regulation, and renal function. CCCs are targets of drugs including loop diuretics and their disruption is implicated in pathophysiologies including epilepsy, hearing loss, and the genetic disorders Andermann, Gitelman, and Bartter syndromes. Here we present the cryo-EM structure of a CCC, the Mus musculus K+-Cl− cotransporter KCC4, in lipid nanodiscs. The structure, captured in an inside-open conformation, reveals the architecture of KCCs including an extracellular domain poised to regulate transport activity through an outer gate. We further identify substrate K+ and Cl− binding sites and provide a structural explanation for differences in substrate specificity and transport ratio between CCCs. These results provide mechanistic insight into the function and regulation of a physiologically important transporter family.

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Section: Resultsmentioning

confidence: 99%

Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs

Reid

Kern

Brohawn

2019

Preprint

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“…Multiple protein sequence alignment was performed between Sa SiaT and additional SiaT from various bacterial species, as described elsewhere (Wahlgren et al, 2018 ). This was used to compare conservation of the Neu5Ac binding site between organisms.…”

Section: Methodsmentioning

confidence: 99%

“…Purified Sa SiaT was reconstituted into proteoliposomes using a protocol previously optimized for Pm SiaT, with some modifications (Wahlgren et al, 2018 ). Briefly, 5 μg of protein was mixed with 120 μL 10% C 12 E 8 , 100 μL of 10% egg yolk phospholipids (w/v, sonicated as previously described to form liposomes, Scalise et al, 2014 ).…”

Section: Methodsmentioning

confidence: 99%

“…Bacteria that import sialic acids have evolved multiple mechanisms of transport across the cytoplasmic membrane. To date, four unique transporter families have been recognized, including those from the ATP binding cassette (ABC) (Post et al, 2005 ), tripartite ATP-independent periplasmic (TRAP) (Allen et al, 2005 ), major facilitator superfamily (MFS) (Vimr and Troy, 1985 ), and sodium solute symporter (SSS) (Severi et al, 2010 ; Wahlgren et al, 2018 ) transporter families (North et al, 2017 ). Whilst most bacteria possess only one type of sialic acid transporter, there are a few exceptions that are predicted to express two family types (Severi et al, 2010 ).…”

Section: Introductionmentioning

confidence: 99%

“…Recently, we determined the high-resolution outward-facing, and open, substrate-bound structure of the SiaT sialic acid transporter from Proteus mirabilis ( Pm SiaT) (Wahlgren et al, 2018 ). SiaT transporters belong to the SSS family.…”

Section: Introductionmentioning

confidence: 99%

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The Sodium Sialic Acid Symporter From Staphylococcus aureus Has Altered Substrate Specificity

et al. 2018

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Mammalian cell surfaces are decorated with complex glycoconjugates that terminate with negatively charged sialic acids. Commensal and pathogenic bacteria can use host-derived sialic acids for a competitive advantage, but require a functional sialic acid transporter to import the sugar into the cell. This work investigates the sodium sialic acid symporter (SiaT) from Staphylococcus aureus (SaSiaT). We demonstrate that SaSiaT rescues an Escherichia coli strain lacking its endogenous sialic acid transporter when grown on the sialic acids N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). We then develop an expression, purification and detergent solubilization system for SaSiaT and demonstrate that the protein is largely monodisperse in solution with a stable monomeric oligomeric state. Binding studies reveal that SaSiaT has a higher affinity for Neu5Gc over Neu5Ac, which was unexpected and is not seen in another SiaT homolog. We develop a homology model and use comparative sequence analyses to identify substitutions in the substrate-binding site of SaSiaT that may explain the altered specificity. SaSiaT is shown to be electrogenic, and transport is dependent upon more than one Na+ ion for every sialic acid molecule. A functional sialic acid transporter is essential for the uptake and utilization of sialic acid in a range of pathogenic bacteria, and developing new inhibitors that target these transporters is a valid mechanism for inhibiting bacterial growth. By demonstrating a route to functional recombinant SaSiaT, and developing the in vivo and in vitro assay systems, our work underpins the design of inhibitors to this transporter.

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Functional and solution structure studies of amino sugar deacetylase and deaminase enzymes from Staphylococcus aureus

et al. 2018

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N‐Acetylglucosamine‐6‐phosphate deacetylase (NagA) and glucosamine‐6‐phosphate deaminase (NagB) are branch point enzymes that direct amino sugars into different pathways. For Staphylococcus aureus NagA, analytical ultracentrifugation and small‐angle X‐ray scattering data demonstrate that it is an asymmetric dimer in solution. Initial rate experiments show hysteresis, which may be related to pathway regulation, and kinetic parameters similar to other bacterial isozymes. The enzyme binds two Zn2+ ions and is not substrate inhibited, unlike the Escherichia coli isozyme. S. aureus NagB adopts a novel dimeric structure in solution and shows kinetic parameters comparable to other Gram‐positive isozymes. In summary, these functional data and solution structures are of use for understanding amino sugar metabolism in S. aureus, and will inform the design of inhibitory molecules.

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Substrate-bound outward-open structure of a Na+-coupled sialic acid symporter reveals a new Na+ site

Cited by 68 publications

References 80 publications

Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs

Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs

The Sodium Sialic Acid Symporter From Staphylococcus aureus Has Altered Substrate Specificity

Functional and solution structure studies of amino sugar deacetylase and deaminase enzymes from Staphylococcus aureus

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