Sulfonation of glycopeptide antibiotics by sulfotransferase StaL depends on conformational flexibility of aglycone scaffold

Abstract

Although glycopeptide antibiotics (GPAs), including vancomycin and teicoplanin, represent the most important class of anti-infective agents in the treatment of serious gram-positive bacterial infections, their usefulness is threatened by the emergence of resistant strains. GPAs are complex natural products consisting of a heptapeptide skeleton assembled via nonribosomal peptide synthesis and constrained through multiple crosslinks, with diversity resulting from enzymatic modifications by a variety of tailoring enzymes, which can be used to produce GPA analogues that could overcome antibiotic resistance. GPA-modifying sulfotransferases are promising tools for generating the unique derivatives. Despite significant sequence and structural similarities, these sulfotransferases modify distinct side chains on the GPA scaffold. To provide insight into the spatial diversity of modifications, we have determined the crystal structure of the ternary complex of bacterial sulfotransferase StaL with the cofactor product 3'-phosphoadenosine 5'-phosphate and desulfo-A47934 aglycone substrate. Desulfo-A47934 binds with the hydroxyl group on the 4-hydroxyphenylglycine in residue 1 directed toward the 3'-phosphoadenosine 5'-phosphate and hydrogen-bonded to the catalytic His67. Homodimeric StaL can accommodate GPA substrate in only one of the two active sites because of potential steric clashes. Importantly, the aglycone substrate demonstrates a flattened conformation, in contrast to the cup-shaped structures observed previously. Analysis of the conformations of this scaffold showed that despite the apparent rigidity due to crosslinking between the side chains, the aglycone scaffold displays substantial flexibility, important for enzymatic modifications by the GPA-tailoring enzymes. We also discuss the potential of using the current structural information in generating unique GPA derivatives.

Fig. 1.

StaL–PAP–DSA complex. (A) Cartoon representation of the StaL–PAP–DSA ternary complex with DSA (yellow carbon) and PAP (orange carbon) shown as stick mode. Subunit A is multicolored (blue for N-terminal to red for C-terminal), and B is magenta. Disordered regions are marked by dotted lines. (B) Stereoview of DSA (yellow carbons) and PAP (orange carbons) in the StaL-binding pocket. Residues 1–7 of DSA are labeled. The final 2mFo-DFc electron density contoured at 1.0 σ is shown as a blue mesh. Black dashed lines indicate H-bonds. Subunit A is green, and subunit B is magenta. (C) Only one DSA molecule can bind to the StaL dimer. DSA molecules bound simultaneously to the two active site clash with each other.

Fig. 2.

Modeling the binding of teicoplanin and ristocetin A to StaL. (A) Superposition of teicoplanin from PDB 2XAD (cyan carbons) onto the DSA (yellow carbons) indicates that teicoplanin could bind to StaL without steric clashes. (B) The tetrasaccharide at HPG₄ in ristocetin A (green carbons; CCDC 718620) clashes with subunit B.

Fig. 3.

Conformational flexibility of the aglycone scaffold of teicoplanin class molecules. (A) Superpostion of the cup-shaped teicoplanin aglycone 1 (carbon in salmon; PDB ID code 3MG9; chain B) onto the DSA molecule in the StaL–PAP–DSA ternary complex indicating that the cup-shaped scaffold must be flattened to allow deprotonation of the hydroxyl group (indicated by *) of res-1 (indicated by the black dotted line) by catalytic His67 and sulfonation by PAPS. Residues 1–7 of both aglycone molecules are labled. (B) Superposition of the aglycone portions of the teicoplanin class molecules. DSA (yellow, this study), teicoplanin aglycone 1 (salmon; PDB ID code 3MG9; chain B), teicoplanin aglycone 2 (blue; PDB ID code 3MGB; chain C), teicoplanin aglycone 3 (white; PDB ID code 3MGB; chain D), and A40926 aglycone (magenta; CCDC 134958) are superposed using residues 4–7 of the scaffold. Ristocetin A aglycone (CCDC 718620) and dalbavancin aglycone (PDB ID code 3RUL) are highly similar to teicoplanin aglycone 1, and teicoplanin (PDB ID code 2XAD) is similar to DSA. For clarity, these are omitted in this figure.