Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery

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 proteolysisNational Institutes of Health (U.S.) (NIH Grant GM-101988)Brown UniversityNational Science Foundation (U.S.) (CAREER award)National Institute of General Medical Sciences (U.S.) (Grant P41 GM103403)United States. Dept. of Energy (Contract DE-AC02-06CH11357

Restriction of the Conformational Dynamics of the Cyclic Acyldepsipeptide Antibiotics Improves Their Antibacterial 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.National Science Foundation (U.S.) (NSF CAREER Award)Brown UniversityNational Institutes of Health (U.S.) (NIH Grant GM-101988

Toward a Method of Collaborative, Evidence-Based Response to Desertification

Over generalized narratives about how desertified ecosystems will respond to restoration actions may result in wasted resources, missed opportunities, or accelerated degradation. Evidence-based collaborative adaptive management (CAM) could solve this problem by providing site-specific information that is trusted by users and enables learning opportunities. Although calls for CAM are increasing, many recommendations remain abstract and difficult to operationalize in specific projects. We review some general challenges for managing desertification in rangelands and draw upon recommendations in the recent literature to develop a 6-step method of CAM to address desertification. The method draws upon our ongoing experiences and makes novel connections between CAM concepts and technologies including ecological sites, state-and-transition models, ecological state mapping, and web-based knowledge systems. The development of a broadly-applicable and flexible methodology for CAM could increase the frequency and success of projects and provide sorely needed knowledge to guide locally-tailored responses to desertification

A Simple Fragment of Cyclic Acyldepsipeptides Is Necessary and Sufficient for ClpP Activation and Antibacterial Activity

The development of new antibacterial agents, particularly those with unique biological targets, is essential to keep pace with the inevitable emergence of drug resistance in pathogenic bacteria. We identified the minimal structural component of the cyclic acyldepsipeptide (ADEP) antibiotics that exhibits antibacterial activity. We found that N-acyldifluorophenylalanine fragments function via the same mechanism of action as ADEPs, as evidenced by the requirement of ClpP for the fragments' antibacterial activity, the ability of fragments to activate Bacillus subtilis ClpP in vitro, and the capacity of an N-acyldifluorophenylalanine affinity matrix to capture ClpP from B. subtilis cell lysates. N-acyldifluorophenylalanine fragments are much simpler in structure than the full ADEPs and are also highly amenable to structural diversification. Thus, the stage has been set for the development of non-peptide activators of ClpP that can be used as antibacterial agents.National Science Foundation (U.S.)United States. National Institutes of Health (GM-101988

Tactical Themes for Rangeland Research

The problems threatening the conservation and management of rangeland, over one-half of the world’s terrestrial surfaces, are significant and growing. Current assessments of drivers and externalities shaping these problems have resulted in strategies intended to result in sustainable development of these lands and their resources. However, how can individual scientists and individual research programs support the needed strategies and goals? What can we realistically contribute and accomplish? We believe that technology can connect individual scientists and their science to the problems manifest in rangelands over the world, in a more rapid exchange than has occurred in the past. Recognition of local challenges, innovations, and scientific tests of the effectiveness of our technological solutions to these problems can keep pace with rapid change and help us adapt to that change. However, to do this, we have to invest in a process of connecting science to landscapes. Our tactics are to link, openly and collaboratively, the scientific method to discrete, specific, managed landscapes. We term these collective tactics, our fundamental research theme, “Landscape Portals”. All of the elements of this theme exist currently, to various degrees, but they lack cohesion and interactive, real-time connections. Future investment requires two basic, tactical scientific behaviors: a post-normal application of science in support of land management by hypothesis and a scientific method modified to accommodate a data intensive scientific inquiry directed towards adaptive management. These behaviors support our “Landscape Portals” theme: science conducted in a highly interactive, transparent, data enriched, locally relevant, globally connected, popularly translated, and ecologically robust manner

ICEF2004-960 MICROPHONES AND KNOCK SENSORS FOR FEEDBACK CONTROL OF HCCI ENGINES

ABSTRACT Homogeneous charge compression ignition (HCCI) engines lack direct in-cylinder CA50 engine crank position in CAD at 50% heat release CAD crank angle degrees HCCI homogeneous charge compression ignition µ sample mean ∇ differencing operator, ∇Y t = Y t −Y t−1 P t predicted (at engine position t) value of a series φ fuel-air equivalence ratio PID proportional-integral-derivative control law RPM revolutions per minute SI spark-ignited TDC top-dead-center of the compression stroke V voltage Y t time t values of data series WN(µ,σ 2 ) normally-distributed white noise process with mean µ and variance σ