Binding Mode for UDP-GlcNAc and Metal Ion
The structure of the DPAGT1/UDP-GlcNAc complex reveals an overall stabilization of the active site, due to movements of CL1 and CL9, and the N terminus of TMH4 (Figure 2C), without any global changes in conformation. The C-terminal end of TMH9b and the following loop region (Phe286-Ile304) display the largest conformational change with an induced fit around the GlcNAc-PP (Figure 2C).
The uridyl moiety of UDP-GlcNAc lies in a narrow cleft at the back of the active site formed by CL5 and CL7. The uracil ring is sandwiched between Gly189 and Phe249 with additional recognition conferred by hydrogen bonds between the Leu46 backbone amide, the Asn191 sidechain to the uracil carbonyls (Figures 2D and 2E) and an extensive hydrogen bond network involving two waters links the uracil ring to five residues (Figures 2D and 2E). Hydrogen-bonding of the ribosyl hydroxyls to the Gln44 mainchain carbonyl and Glu56 sidechain carboxylate complete the recognition of the uridyl nucleotide.
The pyrophosphate bridge is stabilized by interactions with Arg301 and by the catalytic Mg2+ (Figure 2D). The Arg301 sidechain coordinates one pair of α and β phosphate oxygen atoms, whilst the Mg2+ ion is chelated by the second pair of α and β phosphate oxygens. Each oxygen atom is thus singly coordinated in an Arg-Mg2+-“pyrophosphate pincer.” The octahedral coordination of the Mg2+ ion is completed by the sidechains of conserved residues Asn185 and Asp252, and 2 water molecules. Data from DPAGT1 co-crystallized with UDP-GlcNAc and Mn2+ gave a single anomalous difference peak at the metal ion binding site, confirming the presence of a single Mg2+ ion in the active site (Figure 2D). The position of the Mg2+ ion differs by 4 Å from that observed in MraY unliganded structure and the co-ordination differs (Chung et al., 2013).
The GlcNAc moiety-binding site is formed by the CL9 domain and the CL5 loop, although all the direct hydrogen bond interactions are with the CL9 domain. The OH3 and OH4 hydroxyls of GlcNAc form hydrogen bonds with the sidechain of His302 and the mainchain amide of Arg303, respectively. The mainchain of residues 300–303 and the sidechain of Arg303 define the GlcNAc recognition pocket by specifically recognizing the N-acetyl substituent, forming a wall to the sugar-recognition pocket that appears intolerant of larger substituents, thereby “gating” substrate. This structure is absent in MraY, which has a much smaller CL9 loop.
Surprisingly, this structure does not support prior predictions that the highly conserved “aspartate rich” D115Dxx(D/N/E)119 motif is directly involved in Mg2+ binding and/or catalysis (Lloyd et al., 2004). This sequence is adjacent to the active site, but these residues do not directly coordinate Mg2+ or substrate (Figure 2D). Instead, Asp115 is hydrogen-bonded to Lys125 and Tyr256. Lys125 lies adjacent to the phosphates (Figure 2D) and has been implicated in catalysis (Al-Dabbagh et al., 2008). Asp116 forms hydrogen bonds to Ser57 and Thr253 and N-caps TMH8, thus stabilizing residues that interact with the UDP ribosyl moiety (Glu56) and the Mg2+ (Asp252). DPAGT1 with residues Asp115 and Asp116 mutated to Asn, Glu, or Ala retained at least 10% of WT activity (Figure 3A), suggesting they are not essential for catalysis. The third residue in this motif, Asn119, makes no significant interactions. Thus, 2 of the 3 conserved residues perform structural roles; none are directly involved in Mg2+-binding or catalysis.
Figure 3 Proposed DPAGT1 Catalytic Mechanism
(A) Relative activity of active site mutant residues; (n=9).
(B) UDP-GlcNAc complex active site structure with Dol-P modelled based on tunicamycin complex lipid chain position.
(C) Proposed DPAGT1 catalytic mechanism.
For all panels, data presented are means ± SD.