Electrostatic interactions drive native‐like aggregation of human alanine:glyoxylate aminostransferase

Abstract

Protein aggregate formation is the basis of several misfolding diseases, including those displaying loss-of-function pathogenesis. Although aggregation is often attributed to the population of intermediates exposing hydrophobic surfaces, the contribution of electrostatic forces has recently gained attention. Here, we combined computational and in vitro studies to investigate the aggregation process of human peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme involved in glyoxylate detoxification. We demonstrated that AGT is susceptible to electrostatic aggregation due to its peculiar surface charge anisotropy and that PLP binding counteracts the self-association process. The two polymorphic mutations P11L and I340M exert opposite effects. The P11L substitution enhances the aggregation tendency, probably by increasing surface charge anisotropy, while I340M plays a stabilizing role. In light of these results, we examined the effects of the most common missense mutations leading to primary hyperoxaluria type I (PH1), a rare genetic disorder associated with abnormal calcium oxalate precipitation in the urinary tract. All of them endow AGT with a strong electrostatic aggregation propensity. Moreover, we predicted that pathogenic mutations of surface residues could alter charge distribution, thus inducing aggregation under physiological conditions. A global model describing the AGT aggregation process is provided. Overall, the results indicate that the contribution of electrostatic interactions in determining the fate of proteins and the effect of amino acid substitutions should not be underestimated and provide the basis for the development of new therapeutic strategies for PH1 aimed at increasing AGT stability.

Insights into the pathogenesis of primary hyperoxaluria type I from the structural dynamics of alanine:glyoxylate aminotransferase variants

“…Our study has provided insight into the changes in AGT native state stability between two polymorphic variants (WT and LM) and the most common PH1-causing mutation (LM-G170R). Previous in vitro analyses on the thermal (kinetically-controlled) denaturation of these three as well as many other PH1 variants have allowed to propose native state destabilization as a plausible cause of protein misfolding in PH1 [22,28,[43][44][45]. However, we must note that these changes in kinetic (or thermal) stability truly reflect changes in the activation free energy barrier for denaturation [29] (that often require quite long extrapolations to physiological temperature; [22,28,46]) that may arise from stability effects on either the native or the transition state for irreversible denaturation.…”

Biochemical Characterization of Aspergillus fumigatus AroH, a Putative Aromatic Amino Acid Aminotransferase

“…The electrostatic potential maps of Aro8 and AroH were calculated by the adaptive Poisson-Boltzmann solver 1.3 tool, using the non-linear Poisson–Boltzmann equation, as previously reported (Dindo et al, 2017 ). The graphical interface of the tool is integrated in UCSF CHIMERA version 1.1 (Pettersen et al, 2004 ).…”

Evaluation of the impact of antibody fragments on aggregation of intact molecules via size exclusion chromatography coupled with native mass spectrometry

“… 6 , 10 Attraction between complementary electrostatic patches could destabilize protein molecule and foster their association. 6 , 11 External stimuli such as temperature, light, pH, and ionic strength may induce the formation of more of these aggregation-prone features and exacerbate the inter-molecular interactions. 7 , 12–14 …”