Structural and functional characterisation of Uric Acid Permease, a fungal nucleobase transporter, with novel substrates

The development of novel antifungals capable of eliminating Aspergillus fungal infections and improving patient outcomes in Aspergillosis is essential. This work has focussed on Uric Acid Permease, a nucleobase-ascorbate transporter from Aspergillus nidulans, as a potential antifungal carrier. The existing crystal structure of UapA-G411VΔ1-11 in complex with xanthine (PDB 5I6C)1, has provided invaluable information about the substrate binding site, and allowed for the structure-guided design of derivatised xanthine analogues. Competitive uptake assays were used to obtain the binding affinity of each of the analogues for UapA in Aspergillus nidulans. 3-benzylxanthine (5) was identified as a novel ligand of UapA, binding with 35x higher affinity (KI = 0.2 μM) than the native substrates xanthine (KM = 7 μM) and uric acid (KI = 7 μM). This work describes the functional characterisation of 3-benzylxanthine (5) using fungal growth assays, and fluorescence microscopy. The effect of this substrate on UapA-WT and the thermostabilised mutants UapA-G411VΔ1-11 and UapA-Q408EΔ1-11 was assessed using cellular thermal shift assays (CETSA), fluorescence size-exclusion chromatography (FSEC), CPM-based thermostability assays and nano-differential scanning fluorimetry (nano-DSF). While these approaches had their challenges, they all demonstrated that 3-benzylxanthine (5) did not significantly destabilise UapA-WT or the thermostabilised mutants UapA-G411VΔ1-11 and UapA-Q408EΔ1-11. The later part of this work describes attempts to structurally characterize the binding interaction between 3-benzylxanthine (5) and UapA using X-ray crystallography, hydrogen-deuterium mass spectrometry (HDX-MS) and cryo-electron microscopy (cryo-EM). While the structure of UapA-WT or its mutants with 3-benzylxanthine (5) has not yet been resolved, preliminary results from hydrogen-deuterium mass spectrometry (HDX-MS) suggest that 3-benzylxanthine (5) may bind an outward or occluded conformation of UapA-Q408EΔ1-11. Structural characterisation of UapA with 3-benzylxanthine (5) using cryo-EM is ongoing. Once resolved, this will form the basis of future structure-guided xanthine analogue design initiatives.Open Acces