TY - JOUR
T1 - Activation of ClpP protease by ADEP antibiotics
T2 - Insights from hydrogen exchange mass spectrometry
AU - Sowole, Modupeola A.
AU - Alexopoulos, John A.
AU - Cheng, Yi Qiang
AU - Ortega, Joaquin
AU - Konermann, Lars
N1 - Funding Information:
Funding for this work was provided by grants from the Natural Sciences and Engineering Research Council of Canada , the Canada Foundation for Innovation , and the Canada Research Chairs program to L.K. and from the Canadian Institutes of Health Research to J.O. ( MOP-82930 ).
PY - 2013/11/15
Y1 - 2013/11/15
N2 - 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.
AB - 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.
KW - caseinolytic protease P
KW - conformational dynamics
KW - electrospray ionization
KW - hydrophobic clustering
KW - ligand binding
UR - http://www.scopus.com/inward/record.url?scp=84886642731&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2013.08.005
DO - 10.1016/j.jmb.2013.08.005
M3 - Article
C2 - 23948506
AN - SCOPUS:84886642731
SN - 0022-2836
VL - 425
SP - 4508
EP - 4519
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 22
ER -