Dissecting the role of interprotomer cooperativity in the activation of oligomeric high-temperature requirement A2 protein

Significance The HtrA2 enzyme removes toxic proteins in mitochondria, ensuring proper cellular function. HtrA2 malfunction is associated with neurodegenerative disorders such as Parkinson’s disease and essential tremor. HtrA2 is composed of three copies of a protein chain, and the ways in which these chains communicate to form a functional enzyme are not well understood. Here, we have used NMR spectroscopy in concert with a strategy for producing trimers containing different types of chains to elucidate the mechanisms by which the HtrA2 enzyme is regulated. Our data lead to a model explaining how HtrA2 suppresses proteolysis of nonsubstrate proteins and, furthermore, how a single point mutation in HtrA2 that leads to neurodegeneration in mice gives rise to an inactive enzyme. The human high-temperature requirement A2 (HtrA2) mitochondrial protease is critical for cellular proteostasis, with mutations in this enzyme closely associated with the onset of neurodegenerative disorders. HtrA2 forms a homotrimeric structure, with each subunit composed of protease and PDZ (PSD-95, DLG, ZO-1) domains. Although we had previously shown that successive ligand binding occurs with increasing affinity, and it has been suggested that allostery plays a role in regulating catalysis, the molecular details of how this occurs have not been established. Here, we use cysteine-based chemistry to generate subunits in different conformational states along with a protomer mixing strategy, biochemical assays, and methyl-transverse relaxation optimized spectroscopy–based NMR studies to understand the role of interprotomer allostery in regulating HtrA2 function. We show that substrate binding to a PDZ domain of one protomer increases millisecond-to-microsecond timescale dynamics in neighboring subunits that prime them for binding substrate molecules. Only when all three PDZ-binding sites are substrate bound can the enzyme transition into an active conformation that involves significant structural rearrangements of the protease domains. Our results thus explain why when one (or more) of the protomers is fixed in a ligand-binding–incompetent conformation or contains the inactivating S276C mutation that is causative for a neurodegenerative phenotype in mouse models of Parkinson’s disease, transition to an active state cannot be formed. In this manner, wild-type HtrA2 is only active when substrate concentrations are high and therefore toxic and unregulated proteolysis of nonsubstrate proteins can be suppressed.