Biochemical characterization of PGAM5: Filaments stability and dynamic

Phosphoglycerate mutase 5 (PGAM5) is a histidine-dependent serine/threonine protein phosphatase involved in regulating mitochondrial dynamics, apoptosis and mitophagy. Located in the inner mitochondrial membrane facing the intermembrane space, its dimer formation is mediated by F244 at the C-terminus, followed by higher-order multimeric complex assembly driven by the WDXNWD allosteric motif at the N-terminus, a key feature required for its phosphatase activity. In this study we aimed to biochemically characterize the untagged PGAM5_49-289 (without the mitochondrial target signal) and evaluate its filament stability. By assessing enzymatic activity using pNPP (0 – 40 mM) as substrate, secondary structure using Circular dichroism (CD), tertiary structure using fluoroscence analysis and transmission electron microscopy (TEM) images across various experiment conditions, including temperature (30 - 45^oC), pH (6.0 – 8.0), denaturating agents (urea 0 – 8 M) and salt concentration (50 – 600 mM of NaCl), we systematically mapped untagged PGAM5_49-289 stability. PGAM5_49-289 (2 μM) optimal enzymatic activity at acidic pH 6.3 and elevated temperatures 45^oC. PGAM5_49-289 (10 μM) showed an alpha-helix profile structure and thermal stability of 60^oC analyzed by CD. Analysis by TEM in cooper grid, with carbon film Lacey, 400 mesh showed that at under optimal pH 6.3 and low NaCl (100 mM) concentrations, PGAM5_49-289 (0.05 - 0.1 mg/mL) assembled into large filament of dodecamer structures alongside smaller species; conversely, basic pH 8.0 and high NaCl (300 mM) concentrations prompted monomerization and aggregation. Interestingly, the his-tag at the N-terminus of PGAM5_49-289 exhibited a mixture of oligomeric to monomeric species, whereas untagged PGAM5_49-289 eluted exclusively as a single peak in the void volume of size exclusion column. These findings contrast with previously published data, highlighting that subtle structural modifications and environmental parameters strongly dictate PGAM5 assembly, stability and function. Finally, this study provides fundamental structural and biochemical insights into PGAM5 oligomerization, establishing a crucial framework for interpreting its functional dynamics in mitochondrial regulation.