Caseinolytic peptidase P (ClpP), a double-ring peptidase with 14 subunits, collaborates with ATPases associated with diverse activities (AAA+) partners to execute ATP-dependent protein degradation. Although many ClpP enzymes self-assemble into catalytically active homo-tetradecamers able to cleave small peptides, the Mycobacterium tuberculosis enzyme consists of discrete ClpP1 and ClpP2 heptamers that require a AAA+ partner and protein-substrate delivery or a peptide agonist to stabilize assembly of the active tetradecamer. Here, we show that cyclic acyldepsipeptides (ADEPs) and agonist peptides synergistically activate ClpP1P2 by mimicking AAA+ partners and substrates, respectively, and determine the structure of the activated complex. Our studies establish the basis of heteromeric ClpP1P2 assembly and function, reveal tight coupling between the conformations of each ring, show that ADEPs bind only to one ring but appear to open the axial pores of both rings, provide a foundation for rational drug development, and suggest strategies for studying the roles of individual ClpP1 and ClpP2 rings in Clp-family proteolysis.AAA+ proteases | allosteric coupling | pathogen drug target

Trends of grizzly bear (Ursus arctos) populations are most sensitive to female survival; thus, understanding rates and causes of grizzly bear mortality is critical for their conservation. Survival rates were estimated and causes of mortalities investigated for 388 grizzly bears radiocollared for research purposes in 13 study areas in the Rocky and Columbia mountains of Alberta, British Columbia, Montana, Idaho, and Washington between 1975 and 1997. People killed 77-85% of the 99 grizzly bears known or suspected to have died while they were radiocollared. In jurisdictions that permitted grizzly bear hunting, legal harvest accounted for 39-44% of the mortalities. Other major causes of mortality included control killing for being close to human habitation or property, self-defense, and malicious killings. The mortality rate due to hunting was higher (P = 0.006) for males than females, and subadult males had a higher probability (P = 0.007) of being killed as problem animals than did adult males or females. Adult females had a higher (P = 0.009) mortality rate from natural causes than males. Annual survival rates of subadult males (0.74-0.81) were less than other sex-age classes. Adult male survival rates varied between 0.84 and 0.89 in most areas. Survival of females appeared highest (0.95-0.96) in 2 areas dominated by multiple-use land and were lower (0.91) in an area dominated by parks, although few bears were killed within park boundaries. Without radiotelemetry, management agencies would have been unaware of about half (46-51%) of the deaths of radiocollared grizzly bears. The importance of well-managed multiple-use land to grizzly bear conservation should be recognized, and land-use plans for these areas should ensure no human settlement and low levels of recreational activity.

The cyclic acyldepsipeptide (ADEP) antibiotics are a new class of antibacterial agents that kill bacteria via a mechanism that is distinct from all clinically used drugs. These molecules bind and dysregulate the activity of the ClpP peptidase. The potential of these antibiotics as antibacterial drugs has been enhanced by the elimination of pharmacological liabilities through medicinal chemistry efforts. Here, we demonstrate that the ADEP conformation observed in the ADEP–ClpP crystal structure is fortified by transannular hydrogen bonding and can be further stabilized by judicious replacement of constituent amino acids within the peptidolactone core structure with more conformationally constrained counterparts. Evidence supporting constraint of the molecule into the bioactive conformer was obtained by measurements of deuterium-exchange kinetics of hydrogens that were proposed to be engaged in transannular hydrogen bonds. We show that the rigidified ADEP analogs bind and activate ClpP at lower concentrations in vitro. Remarkably, these compounds have up to 1200-fold enhanced antibacterial activity when compared to those with the peptidolactone core structure common to two ADEP natural products. This study compellingly demonstrates how rational modulation of conformational dynamics may be used to improve the bioactivities of natural products.