A Molecular Explanation for Differences in TUN-X,X Analogue Binding to DPAGT1 and MraY
Comparison of the structures of the complexes of tunicamycin with DPAGT1 and MraY (Hakulinen et al., 2017, Yoo et al., 2018; this work) gave an explanation for selectivity of analogues on MraY over DPAGT1. The MraY tunicamycin binding site has a more open, shallow surface than in DPAGT1; in the latter the lipid tail is completely enclosed by Trp122 adjacent to the active site (Figure 6G). The MraY binding site has a disordered loop CL1, a longer TMH9 and a relatively short CL9 region, with only one short α-helix (Figure 1G). In contrast, DPAGT1 has an ordered CL1 which folds over the UDP-binding site. It has a shorter TMH9, followed by a loop and extended strand (residues Gln292 to Arg306) (Figure 1F), which fold over tunicamycin, forming numerous interactions, (e.g., with Arg301, His302, Arg303, Figures 2G and 2H). This extended structure is stabilized by its interactions with the rest of the CL9 domain, a feature found only in eukaryotes.
The N2′′ in GlcNAc is the attachment site of the second lipid chain in TUN-X,X analogues—it occupies distinct environments in the two proteins. In DPAGT1 it is enclosed by the loop at the end of TMH9, and by a tight “gating” cluster of side chains from Trp122, Ile186, Leu293, Cys299, and Arg303 (Figure 6G). By contrast, in MraY, there is a 10 Å gap between Pro108 on TMH4a and Val272 on TMH9, providing ample space for more than one lipid chain to be attached to the amines in TUN-X,X analogues (Figures 6G and 6H).