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

Abstract

Many pathogenic bacteria utilise sialic acids as an energy source or use them as an external coating to evade immune detection. As such, bacteria that colonise sialylated environments deploy specific transporters to mediate import of scavenged sialic acids. Here, we report a substrate-bound 1.95 Å resolution structure and subsequent characterisation of SiaT, a sialic acid transporter from Proteus mirabilis. SiaT is a secondary active transporter of the sodium solute symporter (SSS) family, which use Na+ gradients to drive the uptake of extracellular substrates. SiaT adopts the LeuT-fold and is in an outward-open conformation in complex with the sialic acid N-acetylneuraminic acid and two Na+ ions. One Na+ binds to the conserved Na2 site, while the second Na+ binds to a new position, termed Na3, which is conserved in many SSS family members. Functional and molecular dynamics studies validate the substrate-binding site and demonstrate that both Na+ sites regulate N-acetylneuraminic acid transport.

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

“…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.…”

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

“…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.…”

“…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 ).…”

“…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 ).…”

“…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.…”