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.

Keywords: alanine:glyoxylate aminotransferases; conformational plasticity; protein evolution; protein fitness; structural dynamics.

FIGURE 1

Structure of AGT‐Mi. (a) Cartoon representation of the structure of AGT‐Mi superimposed on that of AGT‐Ma (PDB: 5F9S). The two subunits of AGT‐Mi are shown in dark and light blue while for AGT‐Ma are shown in white and dark gray. The regions that were not included in the model of AGT‐Mi, due to disorder, are colored in red in the structure of AGT‐Ma. Except for the N‐terminus, the other disordered regions are numbered from 1 to 3 and correspond to residue stretches 97–102, 121–146, and 170–177, respectively. (b) Two AGT‐Ma structures solved at different resolution and in different space groups are colored according to temperature factor (B‐factors) values (PDBs: 5OG0 and 5F9S on the left and right side, respectively). Both structures show high B‐factors in the regions that are disordered in AGT‐Mi. (c) Analysis of normalized B‐factors over the range of all available AGT‐Ma structures in different space groups. High B‐factors values correspond to the disordered regions (1, 2, and 3) in AGT‐Mi. These regions are not involved in crystal contacts; therefore, the observed disorder is not a consequence of crystal packing. The B‐factor of the structure of the sigle mutant I340M (PDB: 2YOB) is the lowest, suggesting a destabilizing effect of the P11L mutation balanced by a stabilizing effect of the I340M mutation

FIGURE 2

Molecular dynamics simulations. (a) Plot of the root mean square fluctuation (RMSF) as a function of residue number: AGT‐Ma (purple) and AGT‐Mi (green). The difference ΔRMSF (RMSF AGT‐Mi – RMSF AGT‐Ma) is mapped on the structure of AGT‐Ma as thickness of the tube‐like representation (obtained by Chimera). The RMSF values used were obtained as the average of five trajectories. The regions of interest are marked with the same colors in the structure and in the plot. The increased mobility of the N‐terminus (in pink) and especially of the residues 48–64 (in magenta) of AGT‐Mi may cause a domino effect that is propagated through the C‐terminus (in yellow) and three structural regions (indicated with a, b, and c) that are directly interacting with PLP and with the disordered regions 1, 2, and 3 (in red). Details on the MD method are reported in Table S2. (b) Free energy landscape as a function of the Φ and Ψ dihedral angles of the backbone of the high B‐factor region 121–133. This region forms a β‐strand that contacts the flexible C‐terminus. (c) AGT‐Ma conformational frustration plot. Green and red colors indicate couples of minimal and maximal frustrated residues, respectively. The configurational frustration index evaluates how the energetic contribution to protein stability of the interaction between residues i and j compares to different possible local environments. The interaction is considered frustrated when other conformations with higher stabilization energy are possible. Areas of high frustration are localized at the interface of the highly fluctuating regions, connecting the N‐terminus to the disordered regions in AGT‐Mi (blue arrow). The same coloring of panel A is used to highlight the different portions of AGT1 in the structure and in the correlation plot. (d) Free Energy Surface (FES) of the five combined simulations for AGT‐Ma and AGT‐Mi. The first 5 ns of each simulation were removed from the computations to remove any bias due to the equilibration of the system. The FES was generated in a similar fashion as in Strodel et al. and using RMSD and gyration radius as the two coordinate systems. On the right, a representative structure of AGT‐Mi in the highlighted region of panel B (with high RMSD and high gyration radius) is shown in pink, superposed with the structure of AGT‐Ma (5F9S) that is depicted with the same coloring pattern of panel A. The loss of secondary structure and displacement (blue arrows) is evident in parts of the disordered regions and in the C‐terminal domain. The displacement also affects the cofactor position (inset)

FIGURE 3

Library construction and biochemical and biophysical characterization of the variants. (a) Consensus logo of the three frustrated regions under study. The sequences (110–130) from the non‐redundant database were collected by blast search and aligned using MUSCLE algorithm on Jalview. The logo was generated using Logo maker (https://logomaker.readthedocs.io/en/latest/). In bold black numbers, the positions that were mutated. In black, the amino acids of the human AGT1 sequence, while in red, the amino acids considered for the library. (b) The mutated residues are plotted over the AGT‐Ma structures in different colors. In orange those with activity below AGT‐Mi, in blue those with activity between AGT‐Mi and AGT‐Ma, and in green those above AGT‐Ma. (c) Residual activity of the indicated single variants as compared to AGT‐Ma. Inset panel C) Residual activity of the indicated double variants as compared to AGT‐Ma. Experiments have been performed in duplicate, and for each single experiments, three technical triplicates have been used. Data are represented as mean values ± SD. (d) Heat map of residual activity of selected mutants obtained after incubating the lysate at different temperatures for 10 min. The color code is the same of panel c. (e) Thermal stability curves of the selected variants represented in panel d. Color code is the same of panel c. Experiments have been performed in duplicate, and for each single experiment, three technical replicates have been used

FIGURE 4

Expression, specific activity, and interactors of AGT‐Ma, AGT‐Mi, and selected variants in human cells. (a) and (b) HEK293 cells expressing the indicated species were harvested and lysed. In panel a, soluble protein levels were quantified by western‐blot. Then, 10 μg of soluble lysate of each sample was subjected to SDS‐PAGE, immunoblotted with anti‐AGT from rabbit (1:10,000), and detected by chemiluminescence. The histogram is representative of immunoblot band volume (as mean ± SEM of at least three independent experiments). In panel b, the specific activity for the transamination reaction was measured by incubating 90 μg of soluble lysate with 0.5 M l‐alanine, 10 mM glyoxylate, and 200 μM PLP at 25°C in 100 mM KP, pH 7.4. c,d) Interactome of AGT‐Ma and AGT‐Mi. Panel c shows that AGT1 protein levels were enriched in AGT‐Ma and AGT‐Mi transfected samples after immunoprecipitation using a pan anti‐AGT1 antibody. Control samples (Ctrl) showed undetectable levels of AGT; Panel d shows Venn diagram of significant interactions compared to controls between AGT‐Ma and AGT‐Mi and the interaction network of AGT‐Ma and AGT‐Mi isoforms. Nodes are representative of proteins described with gene name, edges' width is proportional to SAINT score versus control samples

FIGURE 5

Scheme representing the fitness window of AGT1 as resulting from the balance between structural stability (blue curve) and frustration (red curve)