Figure 4
Identification of residues involved in UDP-sugars binding. (A) Sequence alignment of YvcK from B. subtilis and from B. halodurans. The highlighted amino acids are those found to interact with NAD in the crystal structure. (B) Crystal structure of a monomer of B. halodurans YvcK in complex with NAD (PDB ID: 2O2Z). The colored residues of YvcK were found to interact with NAD. The figure was realized with PyMOL software. (C) Identification of YvcK residues susceptible to interact with UDP-sugars. This scheme generated by PoseView software represents the residues of B. halodurans YvcK that interact with NAD. A molecule of UDP-Glc or UDP-GlcNAc was superposed to NAD and residues of YvcK that could interact with UDP-Glc or UDP-GlcNAc are encircled. (D) TSA results for the binding of UDP-GlcNAc to B. subtilis YvcK modified proteins. Four YvcK residues found to interact with NAD in the crystal structure were replaced by Ala to generate YvcK-T14A, YvcK-N218A, YvcK-Y265A and YvcK-R301A. The proteins were purified and TSA was performed in the presence of increasing concentrations of UDP-GlcNAc (0 to 4 mM). The difference of temperature was plotted against the concentration of UDP-GlcNAc for YvcK (grey circle) and each modified protein: YvcK-T14A (cyan diamond), YvcK-N218A (pink square), YvcK-Y265A (yellow triangle) and YvcK-R301A (green square). All the values correspond to the average of data from at least 3 independent experiments and the standard deviations are representated by the error bars. (E) Kinetic parameters for the binding of UDP-GlcNAc to B. subtilis YvcK modified proteins from TSA experiments. The apparent K D, the Δ Tm max and the standard deviations were calculated using Microcal Origin 5.0 software. ND indicates that the binding of UDP-GlcNAc to YvcK and the Δ Tm values are too weak to calculate an apparent K D.
