A Disease-Associated Mutation Impedes PPIA through Allosteric Dynamics Modulation

Abstract

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron degeneration. Peptidylprolyl cis-trans isomerase A (PPIA) is a molecular chaperone involved in protein folding, and its dysfunction has been linked to ALS pathogenesis, as proline is recognized as a key residue for maintaining proper folding of ALS-related proteins. A recent study identified a K76E mutation in PPIA in sporadic ALS patients, but its effect on protein function and structure remain unclear. In this study, we used biochemical and biophysical techniques to investigate the structural and functional consequences of the K76E mutation. Our results show that K76E significantly reduces enzyme activity without affecting structure, monodispersity, or substrate recognition. Significant effects of K76E mutation were identified by relaxation dispersion NMR experiments, showing that K76E disrupts key protein dynamics and alters an allosteric network essential for isomerase activity. Corroborated by theoretical kinetic analysis, these dynamics data, revealing the exchange process for K76E to be approximately 1 order of magnitude slower than that of the wild type, explain the reduced cis-trans isomerase activity of the K76E mutant. These findings suggest that the pathogenic effect of K76E arises primarily from impaired protein dynamics rather than direct structural disruption. Our study provides new insights into the molecular mechanisms underlying ALS-associated mutations and their impact on protein function.

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(A) PPIase activity assessed by monitoring the refolding of RNase T1. Fluorescence increases during RNase T1 refolding in the absence of PPIA (black) and in the presence of wild-type PPIA (red) or K76E (blue). Shaded areas represent the standard deviation (N = 3). (B) Apparent rate constants derived from fitting the refolding curves (up to 200 s) to a single exponential function. Statistical significance between wild-type PPIA and K76E was assessed using Welch’s t-test. ** indicates p < 0.01.

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(A) Overlay of ¹H–¹⁵N correlation spectra of wild-type PPIA (black) and K76E (red). (B) Chemical shift perturbation (CSP) calculated from ¹H–¹⁵N correlation spectra of wild-type PPIA and K76E. Red dashed line indicates the threshold value (mean +1 SD). (C) Residues exhibiting CSP above the threshold are mapped as red spheres onto the structure of PPIA (PDB ID: 1RMH). The K76 side chain is shown as a magenta stick. The putative substrate binding site is indicated by a gray dashed line.

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(A) Exchange rate constants (k _ex) for wild-type PPIA (black) and K76E (red) obtained from fitting ¹⁵N CPMG relaxation dispersion data to the Luz-Meiboom equation for the fast exchange regime. Error bars represent fitting errors. Some of the resonances in K76E mutant showed severe line broadening and thus are omitted in the CPMG analysis. (B) Exchange rate constants (k _ex) mapped onto the structure of PPIA (PDB ID: 1RMH). Residues are shown as spheres colored according to the k _ex value (stepwise color scale shown below). The putative substrate binding site is indicated by a gray dashed line. (C) Five-state kinetic model used for simulations of RNase T1 refolding catalyzed by PPIA. (D) Simulated RNase T1 refolding curves in the absence of PPIA (black), presence of wild-type PPIA (red), and presence of K76E (blue), based on the kinetic model in (C) and parameters in Table S1.