An overview of Neu5Ac metabolism in H. influenzae.

1) H. influenzae is sialidase negative and relies on environmental sialidases to hydrolyse and release terminal Neu5Ac from human glycoconjugates. 2) Outer membrane porins facilitate diffusion of Neu5Ac into the periplasm. 3) A mutarotase, NanM, catalyses the formation of β-Neu5Ac from α-Neu5Ac to prepare for active transport across the inner membrane. 4) Neu5Ac is captured by the high-affinity substrate-binding protein, SiaP. SiaP delivers Neu5Ac to the SiaQM TRAP transporter, which uses a Na+ electrochemical gradient to drive transport. H. influenzae cannot synthesise Neu5Ac and relies solely on SiaPQM for obtaining environmental Neu5Ac. 5) Cytoplasmic processing of Neu5Ac by an anomerase, YhcH, generates the unfavourable open chain form in preparation for use by the first enzyme of the Neu5Ac degradation pathway, NanA. 6) Neu5Ac is sequentially degraded into cell wall constituents or fructose-6-phosphate, which can enter glycolysis. Five conserved enzymes (an aldolase, NanA; kinase, NanK; epimerase, NanE; deacetylase, NagA; and deaminase, NagB) are involved in this pathway which provides H. influenzae with carbon, nitrogen, and energy. 7) Alternatively, Neu5Ac can be activated by cytidine monophosphate and a sialic acid synthetase and added to lipooligosaccharides by a sialyltransferase. Definitions: Neu5Ac, N-acetylneuraminate; manNAc, N-acetylmannosamine; manNAc-6P, N-acetylmannosamine-6-phosphate; glcNAc, N-acetylglucosamine; glcNAc-6P, N-acetylglucosamine-6-phosphate; glcN-6P, glucosamine-6-phosphate; fru-6P, fructose-6-phosphate; galNAc, N-acetylgalactosamine; CMP, cytidine monophosphate; Omp, outer membrane porin; NanM, Neu5Ac mutarotase; YhcH, Neu5Ac anomerase; NanA, Neu5Ac lyase; NanK, manNAc kinase; NanE, manNAc-6P epimerase; NagA, glcNAc-6P deacetylase; NagB, glcN-6P deaminase.

The structure of HiSiaQM.

a, Coulomb maps for the parallel (3.36 Å) and antiparallel (2.99 Å) HiSiaQM homodimers. The periplasmic surfaces of the monomers are facing the same direction for the parallel dimer (PDB: 8THI), whereas the periplasmic surface of one monomer is rotated 180° for the antiparallel dimer (PDB: 8THJ). The transport domain (orange and gold) is in the ‘elevator down’ conformation in all four monomers. The dimeric interface in both maps is distanced and neither has significant protein-protein interactions. The maps are coloured according to the topology in c. Density consistent with phospholipids is coloured grey and is particularly present in the dimer interface of the higher resolution antiparallel dimer map. b, Structural model of the HiSiaQM monomer. The transport domain is in the ‘elevator down’ conformation with the substrate binding site facing the cytoplasm. c, The topology of HiSiaQM is the same as the non-fused PpSiaQM with the addition of the fusion helix. The M-subunit forms the transport domain (orange and gold) and bracing arm helices (pink) as well as a large portion of the scaffold (purple and blue). The Q-subunit is entirely used as a scaffold for the elevator transport mechanism. The fusion helix (purple) connects the scaffold and adds to its size. It also forms a short horizontal helix, similar to the arm helices of the M-subunit. d, A structural overlay of HiSiaQM (2.9 Å structure, green; 4.7 Å structure, purple) and PpSiaQM (2.9 Å structure, orange) shows that the helices of the structures are well aligned, and all three structures are in the same conformation.

HiSiaQM self-association in L-MNG and amphipol.

a, SV-AUC analysis of HiSiaQM in L-MNG (left panel). Two well resolved species exist at 7.3 S and 9.9 S, with the larger peak constituting 85% of the signal. The species at 7.3 S (Peak 1, blue shading) is most consistent with HiSiaQM as a monomer with ∼98 molecules of L-MNG bound (middle panel; green = measured mass, black = theoretical mass); Peak 1 existing as a dimer is unlikely, as the dimeric protein would only have ∼14 molecules of L-MNG bound. The calculated f/f0 of a monomer for Peak 1 is 1.2, consistent with a protein in a detergent micelle. The species at 9.9 S (Peak 2, pink shading) is most consistent with HiSiaQM as a dimer with ∼116 molecules of L-MNG bound (right panel; purple = measured mass, black = theoretical mass); Peak 2 existing as a monomer is not possible, as the protein clearly has a smaller species in Peak 1 and cannot be divided further than a monomer, and a trimer is also unlikely as the trimeric protein would only have ∼32 molecules of L-MNG bound. The calculated f/f0 of a dimer for Peak 2 is also 1.2, again consistent with a protein in a detergent micelle. These calculations do not account for bound lipid molecules. b, Left panel, SV-AUC analysis of amphipol solubilised HiSiaQM (initially purified in L-MNG) shows two distinct species present at 5.9 S and 8.3 S. These are monomeric and dimeric species, as L-MNG solubilised protein exists as these oligomeric states at 7.3 S and 9.9 S as in a. Right panel, representative size exclusion chromatogram of amphipol solubilised HiSiaQM favouring the dimeric state. The main peak at ∼10.8 mL contains dimeric HiSiaQM and the shoulder at ∼11.8 mL contains monomeric HiSiaQM. The sample used for structure determination is shaded turquoise.

Phospholipids bound to HiSiaQM.

a, The dimer has well-defined areas of density (grey) that correspond to bound phospholipids. Two mechanistically important areas are the dimer interface and fusion helix pocket. b, Phospholipids are present at the dimer interface, including in close contact with the anchoring tryptophan residues (shown as spheres) of the Q-subunits that provide stability to the scaffold domain. c, A single phospholipid is trapped in a pocket formed by the fusion helix (protein model surface shown in colour, EM density in grey). The lipid is on the periplasmic side of the transporter and the headgroup appears to be coordinated by residues surrounding the top of the pocket, which have a generally positive character.

Transport assays demonstrate that 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,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 KM 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.

Sedimentation velocity AUC analysis of the interaction between HiSiaQM and HiSiaP in L-MNG detergent.

a, Titrating increasing concentrations of HiSiaQM (blue, 40 µM (2.88 mg/mL); pink, 20 µM; green, 10 µM; brown, 5 µM; other concentrations omitted for clarity) against fluorescently labelled FITC-HiSiaP (10 nM) identifies a shift in the signal for HiSiaP from 3 S to ∼7.5 S and ∼10.5 S. This shift demonstrates binding to HiSiaQM and identifies two bound species with different sedimentation coefficients. The two species are annotated with the most likely binding stoichiometries (one or two HiSiaP monomers (red and grey) may be binding the dimeric species). b, Fitting a binding model to the data (Fraction bound = [P]total / [P]total + KD) estimates a KD of 65 µM (95% confidence interval = 62–69 µM, R2 = 0.99).

Subunit substitution transport assays.

a, Transport was measured with subunit substitution of HiSiaPQM with the fused SiaPQM from A. actinomycetemcomitans (Aa) and the non-fused SiaPQM from P. profundum (Pp). Transport activity was measured in the presence of a membrane potential and a Na+ gradient. The mean activity is shown as bars with SEM error from at least three technical replicates (n = 3 or 4). b, Electrostatic surface comparison of the putative SiaQM interaction surfaces of HiSiaP, AaSiaP and PpSiaP. The SiaP proteins of the two fused systems have a greater area of negatively charged residues (red, circled) at the N-terminal lobe than in the non-fused system.