Loss of stability and unfolding cooperativity in hPGK1 upon gradual structural perturbation of its N-terminal domain hydrophobic core

Abstract

Phosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with cancer development and rare genetic diseases that affect several of its features. To investigate how mutations affect hPGK1 folding landscape and interaction networks, we have introduced mutations at a buried site in the N-terminal domain (F25 mutants) that either created cavities (F25L, F25V, F25A), enhanced conformational entropy (F25G) or introduced structural strain (F25W) and evaluated their effects using biophysical experimental and theoretical methods. All F25 mutants folded well, but showed reduced unfolding cooperativity, kinetic stability and altered activation energetics according to the results from thermal and chemical denaturation analyses. These alterations correlated well with the structural perturbation caused by mutations in the N-terminal domain and the destabilization caused in the interdomain interface as revealed by H/D exchange under native conditions. Importantly, experimental and theoretical analyses showed that these effects are significant even when the perturbation is mild and local. Our approach will be useful to establish the molecular basis of hPGK1 genotype–phenotype correlations due to phosphorylation events and single amino acid substitutions associated with disease.

Conformational stability of peroxidase from the latex of Artocarpus lakoocha: influence of pH, chaotropes, and temperature

“…Applying this formula will provide m 1 , m 2 , and m 3 in addition to the free energy differences between the native form and the intermediate, and unfolded states. T is the experimental temperature (298.15 K), and R is the ideal gas constant ( Pacheco-García et al., 2022 ). X N , X 1 , and X U are the mole fractions of the proteins in their native, intermediate, and unfolded states, respectively, and Q is the partition function.…”

Conformational Stability of Peroxidase from latex ofArtocarpus lakoocha: Influence of pH, Chaotropes, and Temperature

“…m N and m U represent the slopes of the native and unfolded state baselines, while m denaturants describe the unfolding cooperativity. R is the ideal gas constant, and T is the experimental temperature (298.15 K) (Pacheco-García et al, 2022). This model provides a simplified approach for comparing the cooperativity of reversible chemical unfolding in A. lakoocha peroxidase.…”

Analysis of Structural Changes in the Protein near the Phosphorylation Site

“…It is still an open question whether the changes in protein geometry are significant after modification [29,30]. For instance, Juan Luis Pacheco-García et al note the loss of stability in human isoform 1 caused by a disruption of the hydrophobic core in the N-terminal domain due to amino acid substitutions or post-translational modification [31]. There are also studies that indicate minor structural changes resulting from protein modifications [30,[32][33][34] Fuxiao Xin and Predrag Radivojac showed that the proportion of significant conformational changes (RMSD>2 Å) after glycosylation is estimated to be only 7%, and after phosphorylation, it is approximately 13% [30].…”