Structural dynamics shape the fitness window of alanine:glyoxylate aminotransferase

Abstract

The conformational landscape of a protein is constantly expanded by genetic variations that have a minimal impact on the function(s) while causing subtle effects on protein structure. The wider the conformational space sampled by these variants, the higher the probabilities to adapt to changes in environmental conditions. However, the probability that a single mutation may result in a pathogenic phenotype also increases. Here we present a paradigmatic example of how protein evolution balances structural stability and dynamics to maximize protein adaptability and preserve protein fitness. We took advantage of known genetic variations of human alanine:glyoxylate aminotransferase (AGT1), which is present as a common major allelic form (AGT‐Ma) and a minor polymorphic form (AGT‐Mi) expressed in 20% of Caucasian population. By integrating crystallographic studies and molecular dynamics simulations, we show that AGT‐Ma is endowed with structurally unstable (frustrated) regions, which become disordered in AGT‐Mi. An in‐depth biochemical characterization of variants from an anticonsensus library, encompassing the frustrated regions, correlates this plasticity to a fitness window defined by AGT‐Ma and AGT‐Mi. Finally, co‐immunoprecipitation analysis suggests that structural frustration in AGT1 could favor additional functions related to protein–protein interactions. These results expand our understanding of protein structural evolution by establishing that naturally occurring genetic variations tip the balance between stability and frustration to maximize the ensemble of conformations falling within a well‐defined fitness window, thus expanding the adaptability potential of the protein.

Identification of Human Alanine–Glyoxylate Aminotransferase Ligands as Pharmacological Chaperones for Variants Associated with Primary Hyperoxaluria Type 1

“…It can be observed that most of the compounds active on wild-type AGT are also active on the G14R variant. The slight differences observed between the two forms can be ascribed to subtle active site changes that the mutation causes through the loop 24-32 and helix 48-62 connected to the PLP binding pocket, as previously mentioned. , …”

“… 25 More recently, molecular dynamics simulations have shown that helix 48-64 samples alternative conformations inducing a fluctuation that propagates to the active site. 26 It can be hypothesized that the mutation of Gly41 could affect the conformation of both the loop 24-32 and the helix 48-64, thus accounting for the active site variations. We also observed that the IC 50 values of the analogues were lower than that of compound 1 , with the only exception being compound 4 on the G41R variant.…”

“…The slight differences observed between the two forms can be ascribed to subtle active site changes that the mutation causes through the loop 24-32 and helix 48-62 connected to the PLP binding pocket, as previously mentioned. 25 , 26 …”

“…Nevertheless, previous analyses have suggested that the mutation of Gly41 could give rise to subtle active site changes through the loop 24-32 connecting the N-terminus to the PLP binding pocket . More recently, molecular dynamics simulations have shown that helix 48-64 samples alternative conformations inducing a fluctuation that propagates to the active site . It can be hypothesized that the mutation of Gly41 could affect the conformation of both the loop 24-32 and the helix 48-64, thus accounting for the active site variations.…”

“…The slight differences observed between the two forms can be ascribed to subtle active site changes that the mutation causes through the loop 24-32 and helix 48-62 connected to the PLP binding pocket, as previously mentioned. 25,26 Test of Synthesized AGT Ligands on Wild-Type and Mutant AGT Expressed in Mammalian Cells. We tested the effect of synthesized compounds on AGT expression and specific activity in CHO-GO-AGTwt and CHO-GO-G41R cells (Figure 6A,B).…”