Figure 5. Transport assays demonstrate that lauryl maltose neopentyl glycol (L-MNG)-solubilised HiSiaQM is functional.

(a) [³H]-Neu5Ac uptake was measured at multiple time intervals under each condition and used to calculate transport rates. HiSiaQM had the highest activity in the presence of HiSiaP, a membrane potential and a Na⁺ gradient (green circles). Approximately one-third of this rate was present without a membrane potential (orange circles). Transport was low in the absence of Na⁺ (blue circles) and negligible without HiSiaP (pink circles). Error bars represent the standard error of the mean (SEM) for three technical replicates, except without HiSiaP, which has two replicates. The assay diagram contains the parallel HiSiaQM structure, coloured by topology as in Figure 2a and c. This is for visual presentation; it is not known if the transporter exists as a dimeric species in the assay. (b) The rate of transport is dependent on the concentration of Na⁺, showing a sigmoidal response (Hill coefficient = 2.9 [95% C.I. 2.2–3.9]). The K_M for Na⁺ is 12 mM (95% C.I. 10–14 mM). The displayed response shows that HiSiaPQM operates close to its maximum measured rate at a reasonably low external Na⁺ concentration (25 mM). Error bars represent the SEM of five technical replicates. (c) Two Na⁺-binding sites (Na1 and Na2) were identified in HiSiaQM. These sites share highly similar coordinating residues with PpSiaQM. At the Na1 site, a Na⁺ ion (grey) is coordinated by the carbonyl groups of S298, G337, V340 and P342 (orange sticks, coordination shown as black dashes). S295 is also shown but its carbonyl is positioned just outside the coordination distance in our structure. At the Na2 site, a Na⁺ ion (grey) is coordinated by the carbonyl groups of G517, G558, and M564, and the side-chain hydroxyl of T561 (gold sticks, coordination shown as black dashes). (d) A putative substrate-binding site (SBS, outlined) is located in the transport domain of HiSiaQM (orange and gold). The mostly hydrophobic binding site (shown as sticks and electrostatic surface) exists between the two Na⁺-binding sites and is large enough to bind Neu5Ac.

Figure 5—figure supplement 1. The Na⁺ and substrate-binding sites of HiSiaQM.

(a) Closeup views of the cryo-electron microscopy (cryo-EM) density (blue) at the Na1 and Na2 sites of the antiparallel HiSiaQM dimer. Density is present for both Na⁺ ions (green spheres). Na⁺-binding residues are shown as sticks and Na⁺ coordination as black dashes. The density map was automatically sharpened using Phenix (Terwilliger et al., 2018) and the radii of the Na⁺ ions have been reduced to show the density more clearly. (b) A comparison of the Na⁺-binding sites of HiSiaQM from the 2.9 Å structure (green) and the 4.7 Å structure (purple). The Na⁺ ions of the 2.9 Å structure are shown as green spheres and are well positioned to coordinate the identified binding residues in the ‘clamshell’ loops. The positions of these loops are different between the two structures. (c) A comparison of the putative Neu5Ac-binding sites of HiSiaQM from the 2.9 Å structure (green) and the 4.7 Å structure (purple). The higher resolution structure shows that a defined binding pocket exists at the centre of the transport domain.