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.

Dynamic elements govern the catalytic activity of CapE, a capsular polysaccharide‐synthesizing enzyme from Staphylococcus aureus

“…The dynamic region known as the latch (residues 287–309) occupies the same position in both structures. Because the latch was not observed in holo‐CapE [8] we conclude that its structuring is coupled to the binding of UDP‐sugar, but not to the binding of coenzyme [8]. We note that, in the crystal, the active site of CapE is accessible to the solvent and does not form part of crystal‐packing contacts.…”

“…2). Overall, the structure of CapE is virtually identical to that of apo‐CapE in complex with a substrate analog (PDB code http://3W1V; rmsd = 0.41 Å) [8]. The dynamic region known as the latch (residues 287–309) occupies the same position in both structures.…”

“…Crystals of holo‐CapE were grown as previously reported [8] and soaked with substrate UDP‐ d ‐GlcNAc. The high concentration of enzyme in the crystal (∼10 mM) ensures a rapid conversion to products.…”

“…We note that, in the crystal, the active site of CapE is accessible to the solvent and does not form part of crystal‐packing contacts. In addition, the structure of substrate‐free CapE have previously demonstrated the possibility of very large conformational changes of the catalytic domain in the crystal form [8], thus enabling enzymatic activity.…”

“…Purified CapE was dialyzed in 10 mM Tris/HCl (pH 9.0), 30 mM NaCl, and 1 mM DTT, and concentrated with a 100 kDa Centriprep filtration unit (Millipore) prior to crystallization. Crystallization was carried out as described for wild‐type protein in the substrate‐free form [8]. Suitable crystals were soaked in mother liquor supplemented with 25% (v/v) glycerol and 500 μM UDP‐ d ‐GlcNAc.…”