Crystal structure of the capsular polysaccharide synthesizing protein CapE of Staphylococcus aureus

Abstract

Enzymes synthesizing the bacterial CP (capsular polysaccharide) are attractive antimicrobial targets. However, we lack critical information about the structure and mechanism of many of them. In an effort to reduce that gap, we have determined three different crystal structures of the enzyme CapE of the human pathogen Staphylococcus aureus. The structure reveals that CapE is a member of the SDR (short-chain dehydrogenase/reductase) super-family of proteins. CapE assembles in a hexameric complex stabilized by three major contact surfaces between protein subunits. Turnover of substrate and/or coenzyme induces major conformational changes at the contact interface between protein subunits, and a displacement of the substrate-binding domain with respect to the Rossmann domain. A novel dynamic element that we called the latch is essential for remodelling of the protein-protein interface. Structural and primary sequence alignment identifies a group of SDR proteins involved in polysaccharide synthesis that share the two salient features of CapE: the mobile loop (latch) and a distinctive catalytic site (MxxxK). The relevance of these structural elements was evaluated by site-directed mutagenesis.

Figure 1. Biosynthetic pathway of UDP-L-FucNAc in S. aureus

(a) Synthesis of UDP-L-FucNAc requires the sequential activity of enzymes CapE, CapF and CapG. The enzyme CapE is a bifunctional enzyme catalysing the 4,6-dehydration of UDP-D-GlcNAc and a subsequently C5-epimerization. CapF catalyses the C3-epimerization of the previous intermediate, followed by the reduction of the keto-sugar at position C4. CapG catalyses a C2-epimerization to yield the final product UDP-L-FucNAc. The asterisks indicate the chemical groups subjected to enzymatic modification. This pathway is adapted from previous mechanistic studies [12,14]. (b) Structure of the substrate analogue UDP-6N₃-GlcNAc, where the hydroxyl group at C6 of the substrate is replaced by an azide substituent [20].

Figure 2. Crystal structure of CapE

Structure of (a) CapE with substrate analogue UDP-6N₃-GlcNAc bound, and (b) K126E mutein with coenzyme NADPH bound. The coenzyme-binding domain, substrate-binding domain, and latch are coloured in blue, yellow and orange, respectively. The ligands are depicted as spheres with CPK colours. The figure was prepared with CHIMERA [50]. (c) Primary and secondary structure of CapE. The underlined sequence (red line) corresponds to the disordered region in K126E.

Figure 3. Conformation of substrate analogue and coenzyme

(a) Stereoview of the substrate analogue UDP-6N₃-GlcNAc. The electron density correspond the sigma-A weighted 2Fo–Fc map contoured at 1.0 σ. The substrate analogue is depicted with CPK colours. (b) Conformation of various nucleotide-sugars bound to representative SDR enzymes. The panel depicts CapE (this study, red); CDP-β-D-xylose bound to CDP-D-glucose 4,6-dehydratase (1wvg, yellow); GDP-rhamnose bound to GDP-mannose 4,6-dehydratase (1n7g, cyan); dTDP-D-glucose bound to dTDP-glucose 4,6-dehydratase (D128N/E129Q mutein, 1r6d, pink); and UDP-GlcNAc bound to FlaA1 (UDP-GlcNAc inverting 4,6-dehydratase, 2gn4, green). (c) Residue environment around the substrate analogue in wild-type CapE and (d) in the binding pocket of the coenzyme in K126E. Panels (c) and (d) were generated with the program LIGPLOT [51].

Figure 4. Hexameric organization of CapE

(a) CapE forms a hexamer organized as a trimer of dimers (3×2). The structure corresponds to wild-type CapE in complex with substrate analogue (ligand is not shown). The protein subunits of one of the pseudo-dimers are depicted in dark and light blue (chains A and B). The other subunits are depicted in black and grey. (b) SEC profile of CapE. The arrows indicate the elution of the calibration standards. CapE elutes with an apparent molecular weight of 210 kDa. A minor fraction of monomer (8%) with an apparent molecular weight of 40 kDa is also observed. The predicted molecular weight of one subunit of CapE is 39 kDa, and that of the hexamer is 230 kDa. The peak corresponding to the dimer was not observed. (c) Interaction surfaces between protein subunits. The three main contact interfaces correspond to subunits A–B, A–F and A–C. (d) Same interaction surfaces in mutein K126E.

Figure 5. Conformational changes in the substrate-binding domain

(a) Topology of the hexamer. The coenzyme-binding domain (blue) and the substrate-binding domain (red) occupy the central and peripheral regions of the complex, respectively. The latch (orange) is located in an intermediate position. (b) Conformational change. Binding of the substrate analogue, and loss of the coenzyme, induces a rotation of 11° and a displacement of the substrate domain towards the Rossmann domain. Dark and light colours correspond to wild-type protein and K126E, respectively. The rotation angle was calculated with the program DYNDOM [43].

Figure 6. Analysis of the latch

(a) Structure of the latch at the A–B dimer interface. The latch (orange) of subunit A (grey ribbons) associates with the substrate-binding domain of molecule B (grey surface). The substrate analogue is depicted only in subunit B (CPK representation). (b) Same view of K126E. The latch is not modelled because of disorder. The dotted line represents the hypothetical position of the latch in the same position as shown in panel (a). (c) Close view of the latch of subunit A interacting with the substrate-binding pocket of subunit B. The hydrophobic residues Leu²⁸⁸, Tyr²⁹⁰, Tyr²⁹³ and Ile³⁰¹ fit on the groves of subunit B with high surface complementarity. (d) Sequence alignment of the residues belonging to the latch. The panel shows the top-ten solutions found by BLAST [52]. The sequences belong to various genus of Gram-positive and Gram-negative pathogenic bacteria (from top to bottom: Staphylococcus, Pseudomonas, Acidovorax, Hyphomicrobium, Vibrio, Pasteurella, Fusobacterium, Listeria, Bacillus and Enterococcus). Each sequence is identified by their accession code in the UNIPROT database.

Figure 7. Mutational analysis

(a) Wild-type CapE or muteins (2 μM) were incubated with UDP-D-GlcNAc (200 μM) for 2 h at 37°C. Consumption of substrate UDP-D-GlcNAc was monitored by HPLC as described in experimental procedures. The bars correspond to the average of three independent assays ± S.D. (b) Representative HPLC profiles. The substrate UDP-D-GlcNAc was incubated with no CapE (top), with wild-type CapE (middle) and with mutein Y293A (bottom).