Fig 1.
Structural features of TarS217-573.
(A) Ribbon representation of TarS217-573 in two views related by a 90° rotation along the x-axis, where each monomer is indicated by color. The bottom view displays methionine residues that cluster among monomers. (B) Electrostatic surface representation of TarS217-573 in two views related by a 90° rotation along the x-axis, in the same orientation as (A).
Fig 2.
Structural features of TarS1-349.
(A) Ribbon representation of TarS1-349 in complex with UDP-GlcNAc (pink), Mn2+ (purple sphere) and sulfates (yellow). (B) Comparison of the position of the CS loop in the superimposed UDP complexed (blue) and UDP-GlcNAc complexed (gold) ribbon structures. (C) Comparison of the disordered and ordered states of the SA loop in surface representations of the UDP complexed (blue) structure (left) and superimposed UDP complexed and UDP-GlcNAc complexed (50% transparent, gold) structures (right) highlighting the partial occlusion of the active site in the ordered state. Only UDP-GlcNAc is displayed for simplicity. Substrates and residues are displayed in stick form and colored according to heteroatom type.
Fig 3.
Structural features of full-length TarS.
(A) Ribbon representation of TarS in complex with UDP (displayed in ball and stick form and colored according to heteroatom type) in two views related by a 90° rotation along the x-axis. Each monomer is indicated by color, and the catalytic and trimerization domains are indicated in the top view. Arrows in the bottom view demonstrate the 3-fold symmetry in the trimerization domain. (B) Overlay of TarS monomer trimerization domains displaying the relative positions of corresponding catalytic domains. The superimposed trimerization domain is represented by a light gray plane and the catalytic domains by colored planes corresponding to (A). (C) Electrostatic surface representation of TarS in two views related by a 180° rotation along the y-axis, with the left view in the same orientation as the bottom view in (A).
Table 1.
Data Collection, phasing and refinement statistics
Fig 4.
(A) Schematic of the proposed SN2 reaction mechanism, with the glycosyltransferase reaction between UDP-GlcNAc and polyRboP resulting in UDP and β-polyRboP-GlcNAc. (B) Close-up of the catalytic site showing interactions of active site residues (green) with UDP-GlcNAc (pink), Mn2+ (purple) and ordered waters (red). Interactions between atoms are displayed by dotted lines, and the distance between the C1 position of GlcNAc and the hydroxyl of the proposed catalytic base (D178) is indicated. (C) Electrostatic surface representation of the TarS active site in complex with UDP-GlcNAc (green), sulfates (yellow), and glycerol (pink) (superimposed from SpsA (PDB:1qgq)). (D) Docking of a PRboPRboP molecule (green) in the TarS active site. The model places an RboP C4 hydroxyl in close proximity to the catalytic D178, as well as a glycerol (purple) hydroxyl superimposed from the SpsA structure (circled). The terminal phosphates of PRboPRboP furthermore superimpose closely with sulfates bound in the structure (circled). The positions of UDP (blue) and UDP-GlcNAc (pink) are indicated. Ligands are displayed in stick form and colored according to heteroatom type.
Fig 5.
Analysis of TarS catalytic activity.
(A) Comparison of activity in the presence and absence of divalent cations by HPLC based UDP detection. A no protein control was included for reference, and reactions proceeded in the presence of 1mM UDP-GlcNAc and 1mM metal/EDTA where indicated. (B) Relative activities of various TarS catalytic site mutants compared to wild-type by HPLC based UDP detection in the presence of 1mM UDP-GlcNAc. For comparative purposes, relative activity is given as a fraction of wild-type activity whose value was adjusted to 1.0. (C) Thermostability of various TarS catalytic site mutants in the presence and absence of UDP-GlcNAc, analyzed by differential static light scattering as a measure of Tagg upon thermodenaturation. (D) Kinetic parameters of full-length and TarS1-349 constructs. Kinetic parameters were determined using continuous fluorescence-based UDP detection with increasing UDP-GlcNAc concentrations (hydrolysis reaction) or increasing polyRboP concentrations (glycosyltransferase reaction; in presence of 1mM UDP-GlcNAc).