Conformational Flexibility in the Allosteric Regulation of Human UDP-α-d-Glucose 6-Dehydrogenase

Abstract

UDP-α-D-xylose (UDX) acts as a feedback inhibitor of human UDP-α-D-glucose 6-dehydrogenase (hUGDH) by activating an unusual allosteric switch, the Thr131 loop. UDX binding induces the Thr131 loop to translate ~5 Å through the protein core, changing packing interactions and rotating a helix (α6(136-144)) to favor the formation of an inactive hexameric complex. But how does to conformational change occur given the steric packing constraints of the protein core? To answer this question, we deleted Val132 from the Thr131 loop to approximate an intermediate state in the allosteric transition. The 2.3 Å resolution crystal structure of the deletion construct (Δ132) reveals an open conformation that relaxes steric constraints and facilitates repacking of the protein core. Sedimentation velocity studies show that the open conformation stabilizes the Δ132 construct as a hexamer with point group symmetry 32, similar to that of the active complex. In contrast, the UDX-inhibited enzyme forms a lower-symmetry, horseshoe-shaped hexameric complex. We show that the Δ132 and UDX-inhibited structures have similar hexamer-building interfaces, suggesting that the hinge-bending motion represents a path for the allosteric transition between the different hexameric states. On the basis of (i) main chain flexibility and (ii) a model of the conformational change, we propose that hinge bending can occur as a concerted motion between adjacent subunits in the high-symmetry hexamer. We combine these results in a structurally detailed model for allosteric feedback inhibition and substrate--product exchange during the catalytic cycle.

UDP-glucose Dehydrogenase Activity and Optimal Downstream Cellular Function Require Dynamic Reorganization at the Dimer-Dimer Subunit Interfaces

“…4). As a control, we generated an additional point mutant, ⌬132, in which valine 132 was deleted and which had been reported to be exclusively hexameric (25). Consistent with the prior results, this mutant had undetectable activity (Table 1) and eluted at the molecular mass of the hexamer irrespective of substrate or cofactor addition.…”

“…Several reports have provided structural and enzymologic insights into UGDH function in multiple species (6,7,10,11,(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). In all cases the enzyme oxidizes UDP-glucose through two NAD ϩ -dependent electron transfers.…”

“…Of particular interest to studies relating quaternary structure to cellular function is that bacterial UGDH adopts a strictly dimeric conformation in the presence or absence of its substrate and cofactor (27), whereas higher eukaryotic UGDH orthologues purify in a hexamer-dimer equilibrium that is stabilized to hexameric association under conditions representative of the cellular environment (4,11,16,28). Kinetic data comparing wild-type UGDH to an obligate (inactive) hexamer mutant in which residue 132 was deleted (25) together with the interesting observation of an "open" conformation of the wild-type enzyme observed in cocrystals with the potent UGDH inhibitor UDP-xylose suggest that the transient capacity to dissociate and reorganize the hydrogen bond network at the interface between dimeric units is an important element of the normal catalytic cycle.…”

Allostery and Hysteresis Are Coupled in Human UDP-Glucose Dehydrogenase

“…UDP-Xyl binding stabilizes the E Ω conformation of hUGDH (Figure 1) 16–18 . Competition with UDP-Glc induces the low substrate affinity E Ω conformation of hUGDH to undergo a cooperative conformational change to the high affinity E state 16, 39 .…”

Inhibiting Hexamer Disassembly of Human UDP-Glucose Dehydrogenase by Photoactivated Amino Acid Cross-Linking

“…We previously generated an obligatory homodimeric point mutant of hUGDH, T325D, in which interactions at the dimer-dimer interface were disrupted to show that the enzyme retained only ≈20% of wild-type activity in this state and lacked the ability to contribute significant UDP-glucuronate to downstream products 13 . However, an obligate hexamer caused by a single amino acid deletion, Δ132, also showed no detectable activity 13, 14 . Moreover, crystal structure determination and sedimentation velocity characterization have shown that an endogenous inhibitor of UGDH, UDP-xylose, functions by binding within the UDP-glucose active site and altering subunit association to yield an inactivated hexamer 15, 16 .…”