Active site conformational dynamics in human uridine phosphorylase 1

Abstract

Uridine phosphorylase (UPP) is a central enzyme in the pyrimidine salvage pathway, catalyzing the reversible phosphorolysis of uridine to uracil and ribose-1-phosphate. Human UPP activity has been a focus of cancer research due to its role in activating fluoropyrimidine nucleoside chemotherapeutic agents such as 5-fluorouracil (5-FU) and capecitabine. Additionally, specific molecular inhibitors of this enzyme have been found to raise endogenous uridine concentrations, which can produce a cytoprotective effect on normal tissues exposed to these drugs. Here we report the structure of hUPP1 bound to 5-FU at 2.3 A resolution. Analysis of this structure reveals new insights as to the conformational motions the enzyme undergoes in the course of substrate binding and catalysis. The dimeric enzyme is capable of a large hinge motion between its two domains, facilitating ligand exchange and explaining observed cooperativity between the two active sites in binding phosphate-bearing substrates. Further, a loop toward the back end of the uracil binding pocket is shown to flexibly adjust to the varying chemistry of different compounds through an "induced-fit" association mechanism that was not observed in earlier hUPP1 structures. The details surrounding these dynamic aspects of hUPP1 structure and function provide unexplored avenues to develop novel inhibitors of this protein with improved specificity and increased affinity. Given the recent emergence of new roles for uridine as a neuron protective compound in ischemia and degenerative diseases, such as Alzheimer's and Parkinson's, inhibitors of hUPP1 with greater efficacy, which are able to boost cellular uridine levels without adverse side-effects, may have a wide range of therapeutic applications.

Figure 1. Structural comparison of hUPP1 with varying ligands.

Overlay of the structures of hUPP1 bound to 5-FU, BAU, or ligand-free (APO) reveals the high degree of retention of the global fold of the enzyme when binding either substrate or inhibitor. The position of the two 5-FU molecules within the symmetric active sites at the dimer interface is also shown. In this illustration, the green/yellow monomers are least-squares aligned (R.M.S.D.s shown in angstroms) and the resulting displacement of the backbone traces of the partnering chains (arrows) reveals the interdomain flexibility of hUPP1. Between aligned monomers binding either 5-FU or BAU, there is a noticeable structural difference only in the conformation of a loop proximate to the active site (magenta).

Figure 2. 5-Fluorouracil binding to hUPP1.

(A) 5-FU is coordinated by residues restricted to the individual monomers of the hUPP1 dimer, in contrast to the binding of BAU that traverses the dimer interface. As expected, Gln217 and Arg219, the key uridine-discriminating residues, form multiple hydrogen bonds with one face of the uracil base. This face also includes a well-coordinated, buried water molecule that associates with 5-FU and creates stabilizing bonds with both Gln217 and Arg275. Additional favourable interactions may be formed by both the backbone carbonyl and side chain hydroxyl groups of Thr141, although the geometry observed in the crystal structure is not consistent with hydrogen bonding. The fluorine moiety resides in a hydrophobic pocket created by Leu272, Leu273 and Ile281, and forms a hydrogen bond with Ser142. Electron density from a 2F_o-F_c map contoured at 1.5σ is shown for the ligand (blue wire). (B) Surface representation from the same perspective emphasizes the depth and fit of the active site for the pyrimidine substrate. The position of Phe213, which was omitted from (A) for clarity, is also illustrated. This residue caps the active site and forms hydrophobic, herringbone stacking interactions with the uracil ring. (C) Schematic map of the contacts between hUPP1 and 5-FU as analyzed by LigPlot .

Figure 3. Conformational dynamics of a hUPP1 active site loop.

(A) Comparison of the structure of the loop lining the back of the hUPP1 active site when bound to 5-FU (green), BAU (lime), or ligand-free (yellow), reveals that this region is somewhat mobile and able to close around substrate upon its binding. (B) Overlay of the BAU molecule with the known structures of hUPP1 shows that the benzyl moiety of this inhibitor displaces Ile281 from its normal substrate binding position to accommodate the extra bulkiness of this molecule. It is notable how similar the BAU-bound and ligand-free conformations of hUPP1 are, suggesting that BAU fits the naturally occurring structure of the protein in the absence of substrate. (C) While the new structure of hUPP1 reveals some degree of flexibility in the back-side active site loop, the conformational range of this region of hUPP1 is substantially less than that of the equivalent part of E. coli UPP, which closes more tightly when bound to 5-FU (yellow) and opens wider in the absence of ligand (orange) when compared with its BAU-inhibited structure (red). The increased rigidity of the human enzyme is likely due to the insertion of two additional residues into this loop region, including a proline (inset).

Figure 4. Inter-domain flexibility of hUPP1.

Illustration highlights conformational changes at the dimer interface proximate to the active site, overlaying the 5-FU-bound structure (gold), the BAU-bound structure (orange), and ligand-free structure (red). Despite a lack of molecular contacts between residues from the partnering subunit and the 5-FU ligand, the critical residues for binding natural substrates adopt conformations close to those seen in the BAU-bound structure, where they are stabilized by the formation of favourable molecular interactions, and not the conformations revealed in the ligand-free structure. The location of the phosphate ion from the BAU-bound structure is shown for orientation, but not found to be occupied in the 5-FU-bound structure.