Protein quality control is an essential cellular function mainly executed by a vast array of different proteases and molecular chaperones. One of the bacterial high temperature requirement A (HtrA) protein family members, the homo-oligomeric DegP protease, plays a crucial role in the Escherichia coli protein quality control machinery by removing unfolded proteins or preventing their aggregation and chaperoning them to their final folded state within the periplasm. DegP contains two regulatory PDZ domains, which play key roles in substrate recognition and in the transformation of DegP between inactive hexameric and proteolytic active cage-like structures. Here, we analyze the interaction and dynamics of the DegP PDZ domains underlying this transformation by high-resolution NMR spectroscopy complemented with biochemical cleavage assays. We identify an interdomain molecular lock, which controls the interactions between the two PDZ domains, regulated by fine-tuned temperature-dependent protein dynamics, and which is potentially conserved in proteins harboring tandem PDZ domains.