X-ray crystallographic structure of a bacterial polysialyltransferase provides insight into the biosynthesis of capsular polysialic acid

Abstract

Polysialic acid (polySia) is a homopolymeric saccharide that is associated with some neuroinvasive pathogens and is found on selective cell types in their eukaryotic host. The presence of a polySia capsule on these bacterial pathogens helps with resistance to phagocytosis, cationic microbial peptides and bactericidal antibody production. The biosynthesis of bacterial polySia is catalysed by a single polysialyltransferase (PST) transferring sialic acid from a nucleotide-activated donor to a lipid-linked acceptor oligosaccharide. Here we present the X-ray structure of the bacterial PST from Mannheimia haemolytica serotype A2, thereby defining the architecture of this class of enzymes representing the GT38 family. The structure reveals a prominent electropositive groove between the two Rossmann-like domains forming the GT-B fold that is suitable for binding of polySia chain products. Complex structures of PST with a sugar donor analogue and an acceptor mimetic combined with kinetic studies of PST active site mutants provide insight into the principles of substrate binding and catalysis. Our results are the basis for a molecular understanding of polySia biosynthesis in bacteria and might assist the production of polysialylated therapeutic reagents and the development of novel antibiotics.

Figure 1

PST catalysed biosynthesis of polysialic acid and structure of MhPST. (a) The biosynthesis of capsular polysialic acid (polySia) is initiated at the cytoplasmic side of the plasma membrane by the transfer of a Kdo linker onto the lipid carrier lyso-phosphatidyl glycerol catalysed by KpsS and KpsC. Extension to polySia requires sialic acid priming involving the action of NeuE, before PST can catalyse the assembly of homopolymeric, α-2,8-linked polySia. The completed, lipid-bound structure is translocated to the cell surface in an ATP-dependent manner by an export complex composed of KpsDEMT. (b) In vitro polysialylation activity of MhPST using a soluble BDP-Sia₂Lac acceptor substrate was analysed by TLC. Polysialylation results in suppressed migration of the acceptor (A complete scan of the TLC plate is shown in Supplementary Figure S7). (c) Ribbon diagrams of the Δ20MhPST apo structure showing the GT-B fold. In the N-terminal Rossmann fold, helices are coloured in marine and β-sheets are coloured in purple, whereas in the C-terminal Rossmann fold, helices are coloured in light blue and β-sheets are coloured in pink. The presumed position of the N-terminal membrane anchor is indicated.

Figure 2

Structures of substrate-bound complexes of MhPST. (a) Experimental Fo – Fc omit electron density map for CDP (yellow sticks) bound to MhPST (ribbon diagram of monomer A) is shown as a blue mesh contoured at 3.0 σ. Structure factor calculation was performed prior to CDP docking. (b) Nucleotide donor binding site of MhPST (monomer A) with residue side chains interacting with bound CDP (yellow sticks) shown in sticks and labelled. Potential hydrogen bonds are indicated as black dashes. (c) Superimposition of donor binding sites of the apo-structure (coloured in cyan) and of the CDP-bound structure (coloured in wheat, pink, and light blue). The shift of the side chain of H291 by 1.3 Å is shown. (a–c) The C-terminal domain is omitted for clarity. (d) Experimental Fo – Fc omit electron density map for fondaparinux (green sticks) bound to MhPST (ribbon diagram of monomer A) is shown as a blue mesh contoured at 3.0 σ. Structure factor calculation was performed prior to ligand docking. (e) Acceptor substrate binding site of MhPST (monomer A) with residue side chains interacting with bound fondaparinux (green sticks) shown in sticks and labelled. The five monosaccharide units of fondaparinux are labelled and potential hydrogen bonds are indicated as black dashes. (a,d) Electron density improved for all ligands upon refinement.

Figure 3

The MhPST acceptor substrate binding groove at the interface of the N-terminal and the C-terminal domain. (a) Surface representation of MhPST (monomer A in wheat) with bound acceptor ligand analogue fondaparinux shown in green sticks. The surface of residue K293 is coloured in blue and is labelled. (b) Electrostatic surface potential of fondaparinux bound MhPST structure. (c) Electrostatic surface potential of CMP bound structure of mono-sialyltransferase PmST1 (PDB: 3s44). (b,c) Surface potentials were calculated with the Adaptive Poisson Boltzmann Server^, with a PARSE force field, with linear interpolation of colours at intermediate potentials (blue, 4 kT/e; red, −4 kT/e; white, 0 kT/e; probe radius, 1.4 Å).

Figure 4

MhPST active site and identification of the catalytic base. (a) Modelled ternary complex by superimposition of the CDP-bound structure and the fondaparinux-bound structure. MhPST (monomer A) is shown as a ribbon diagram with the same color-coding as in Fig. 1c, CDP is shown as yellow sticks, fondaparinux is shown as green sticks and the five monosaccharide units are indicated. Residues with a supposed role in catalysis are shown in sticks and are labelled. Potential hydrogen bonds between E153 and Q41 and Q44, respectively are indicated as black dashes. The location of the mutation K69A is indicated. (b) Superimposition of active site of MhPST (wheat ribbon diagram) and mono-sialyltransferase PmST1 in open conformation (PDB: 3s44, cyan ribbon diagram). The catalytic acid and base are shown in sticks and are labelled for both enzymes, and the distance between E153 and H291 is indicated for MhPST. (c) S_N2-like reaction mechanism of MhPST. The catalytic base E153 (red box) abstracts a proton from the C8′ hydroxyl group of the sialic acid acceptor concerted with the nucleophilic attack on the anomeric C2′ carbon (orange circle) of the CMP sialic acid donor substrate, thereby generating an α-2,8 glycosidic linkage. The resulting negatively charged CMP leaving group is stabilized by H291 (blue box) assisted by S339 and T340 (grey boxes). R = α-2,8-linked oligosialyl.