A Computational Approach to Unravel Structural Preferences among HPRT Family Members.

VALSECCHI, WM; FERNÁNDEZ BETTELLI, L; MOSCATO, F; ROSSI, RC; DELFINO, JM; FERNÁNDEZ, ML

Córdoba

Congreso; LI Reunión Anual de la Sociedad Argentina de Biofísica; 2023

In the field of molecular biology, proteins belonging to the hypoxanthine phosphoribosyl-transferase (HPRT) family serve a pivotal role in the purine recycling pathway, contributing to nucleotide synthesis. Given their critical involvement in cellular metabolism, they are considered promising targets for drug development. Structurally, most members of this family assemble as homo-tetramers, with two distinct structural groups known as D1T1 and D1T1'. Although the three-dimensional structures of these groups are superimposable in terms of both their monomers and dimers, they exhibit differing tetrameric arrangements. We have previously shown that the human variant (HsHPRT, D1T1' group) is insensitive to certain drugs used as inhibitors of the T. cruzi HPRT variant (TcHPRT, D1T1 group) 1. Additionally, we conducted a characterization to elucidate the underlying factors governing the oligomeric arrangements in each species. Nonetheless, numerous questions remain unanswered: Why does HsHPRT prefer the D1T1' arrangement over D1T1, despite having similar surface features on both faces? Will attempting to impose a D1T1 structure using the HsHPRT sequence result in a stable tetramer or lead to disassembly? Likewise, what happens when we generate a D1T1' structure from TcHPRT sequence? What explains the TcHPRT C-terminal region's (CTR) inability to form helical or sheet-like structures, while still clearly defining the dimer/tetramer oligomeric state? Is the NTR of HsHPRT as essential for its tetrameric arrangement as the CTR is for TcHPRT? Does a dimeric form of HsHPRT display enhanced activity similar to TcHPRT? To uncover the mechanisms behind each variant's preference for a given arrangement, we employed computational techniques. We assessed AlphaFold-Multimer ability to predict the tetrameric structures of both D1T1 and D1T1' groups solely from HsHPRT and TcHPRT sequence data. Our findings demonstrated that AlphaFold accurately predicts the tetrameric configurations of both proteins. Notably, for TcHPRT, the prediction indicated disorder in the 30-amino acid CTR, which is consistent with the absence of crystallographic data. Furthermore, we conducted molecular dynamics (MD) simulations to assess tetramer stability. For HsHPRT, the MD simulations revealed a stable tetramer, despite including high mobility loops pertaining to the active site.References:1 Santos J, et al. (2023) Archives of Biochemistry and Biophysics. doi/10.1016/j.abb.2023.109550