Activation of ClpP Protease by ADEP Antibiotics: Insights from Hydrogen Exchange Mass Spectrometry

• Published in: Journal of molecular biology 2013-11, Vol.425 (22), p.4508-4519
• Main Authors: Sowole, Modupeola A., Alexopoulos, John A., Cheng, Yi-Qiang, Ortega, Joaquin, Konermann, Lars
• Format: Article
• Language: English
• Subjects: Allosteric Regulation; Amino Acid Sequence; caseinolytic protease P; conformational dynamics; Depsipeptides - chemistry; Depsipeptides - metabolism; Depsipeptides - pharmacology; electrospray ionization; Endopeptidase Clp - chemistry; Endopeptidase Clp - metabolism; Enzyme Activation - drug effects; Hydrogen - chemistry; Hydrogen Bonding; Hydrophobic and Hydrophilic Interactions; hydrophobic clustering; Kinetics; ligand binding; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Protein Binding; Protein Conformation; Protein Multimerization
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2013.08.005

The bacterial protease ClpP consists of 14 subunits that assemble into two stacked heptameric rings. The central degradation chamber can be accessed via axial pores. In free ClpP, these pores are obstructed by the N-terminal regions of the seven subunits at either end of the barrel. Acyldepsipeptides (ADEPs) are antibacterial compounds that bind in hydrophobic clefts surrounding the pore region, causing the pores to open up. The ensuing uncontrolled degradation of intracellular proteins is responsible for the antibiotic activity of ADEPs. Recently published X-ray structures yielded conflicting models regarding the conformation adopted by the N-terminal regions in the open state. Here, we use hydrogen/deuterium exchange (HDX) mass spectrometry to obtain complementary insights into the ClpP behavior with and without ADEP1. Ligand binding causes rigidification of the equatorial belt, accompanied by destabilization in the vicinity of the binding clefts. The N-terminal regions undergo rapid deuteration with only minor changes after ADEP1 binding, revealing a lack of stable H-bonding. Our data point to a mechanism where the pore opening mechanism is mediated primarily by changes in the packing of N-terminal nonpolar side chains. We propose that a “hydrophobic plug” causes pore blockage in ligand-free ClpP. ADEP1 binding provides new hydrophobic anchor points that nonpolar N-terminal residues can interact with. In this way, ADEP1 triggers the transition to an open conformation, where nonpolar moieties are clustered around the rim of the pore. This proposed mechanism helps reconcile the conflicting models that had been put forward earlier. [Display omitted] •ADEP binding to the ClpP protease complex induces opening of axial pores.•The pore opening mechanism remains under discussion.•We use HDX/mass spectrometry to monitor changes in ClpP structure and dynamics upon ADEP binding.•Binding stabilizes the equatorial belt but causes only minor HDX changes in the pore region.•The pore opening mechanism is attributed to changes in the packing of nonpolar residues.