The role of intrinsic disorder in the allosteric regulation of human UGDH

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PROJECT SUMMARY (UNCHANGED) Glucuronidation is often the source of unfavorable pharmacokinetics or pharmacodynamics that lead to the failure of drugs during clinical trials, and as such, there is a critical need in drug development for a tool to control glucuronidation. Our long-term goal is to develop strategies to control glucuronidation by limiting its substrate availability. To do this, we will determine the allosteric mechanism that controls human UDP-glucose dehydrogenase (hUGDH), the enzyme that produces the essential substrate for glucuronidation. In our previous grant, we discovered how the 30-residue intrinsically disordered C-terminus (the ID-tail) modifies the structure of the enzyme to favor binding of the feedback inhibitor UDP-Xyl, a downstream metabolite. We also discovered a cryptic allosteric site for inhibiting the enzyme. Briefly, the feedback inhibitor UDP-Xylose competes with substrate for the active site; upon binding, UDP-Xyl induces the enzyme to slowly isomerize into an inactive complex called E. The allosteric transition converts the active site into two novel allosteric sites called SBS and NBS. The SBS site is specific for the UDP-Xyl inhibitor, but the NBS site can bind either UDP-Xyl or the substrate UDP-Glc. We hypothesize that the NBS and SBS allosteric sites cooperatively stabilize E, and the dual-specificity of the NBS is an important feature that allows the abundant substrate UDP-Glc to enhance the binding affinity of the less abundant inhibitor UDP-Xyl in the SBS. This hypothesis is based on our preliminary data that (i) the substrate UDP-Glc can bind to the NBS site and inhibit hUGDH, and (ii) the inhibitor UDP-Xyl can bind to both the SBS and NBS to inhibit. This hypothesis will be tested by the following specific aims: 1) we will determine how the NBS and SBS allosteric sites interact to enhance the allosteric inhibition by UDP-Xyl; 2) we will determine the relationship between a putative low barrier hydrogen bond (LBHB) and the stability of the NBS and SBS allosteric sites; and 3) we will identify the structural features that couple the intrinsically disordered C-terminus (ID-tail) of hUGDH to the favorable formation of E. The research proposed in this application is innovative because it focuses on the allosteric inhibition of hUGDH as a global mechanism for controlling glucuronidation, and uses our recent discoveries of: (i) the novel NBS allosteric site; (ii) a putitive low barrier hydrogen bond in the allosteric mechanism; and (iii) the entropic force generated by the intrinsically disordered C-terminus. Since these features are recent discoveries from my lab, this research is distinct from previous attempts that tried to control glucuronidation. The expected outcomes of this work are significant. A detailed description of the allosteric mechanism of hUGDH will serve as a foundation for the design of a class of allosteric inhibitors that will act as global regulators of glucuronidation. And more broadly, because of the persistence of long (>30 residues) intrinsically disordered segments in the human proteome, (>44% of all human proteins), learning how the entropic force generated by the disordered terminus of hUGDH modifies the protein function will have a broad impact.