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The last 5 years have seen a series of advances in the application of isothermal titration microcalorimetry (ITC) and interpretation of ITC data. ITC has played an invaluable role in understanding multiprotein complex formation including proteolysis‐targeting chimeras (PROTACS), and mitochondrial autophagy receptor Nix interaction with LC3 and GABARAP. It has also helped elucidate complex allosteric communication in protein complexes like trp RNA‐binding attenuation protein (TRAP) complex. Advances in kinetics analysis have enabled the calculation of kinetic rate constants from pre‐existing ITC data sets. Diverse strategies have also been developed to study enzyme kinetics and enzyme‐inhibitor interactions. ITC has also been applied to study small molecule solvent and solute interactions involved in extraction, separation, and purification applications including liquid‐liquid separation and extractive distillation. Diverse applications of ITC have been developed from the analysis of protein instability at different temperatures, determination of enzyme kinetics in suspensions of living cells to the adsorption of uremic toxins from aqueous streams. [ABSTRACT FROM AUTHOR]

The 300-kDa ClpP1P2 protease from Mycobacterium tuberculosis collaborates with the AAA+ (ATPases associated with a variety of cellular activities) unfoldases, ClpC1 and ClpX, to degrade substrate proteins. Unlike in other bacteria, all of the components of the Clp system are essential for growth and virulence of mycobacteria, and their inhibitors show promise as antibiotics. MtClpP1P2 is unique in that it contains a pair of distinct ClpP1 and ClpP2 rings and also requires the presence of activator peptides, such as benzoyl-leucylleucine (Bz-LL), for function. Understanding the structural basis for this requirement has been elusive but is critical for the rational design and improvement of antituberculosis (anti-TB) therapeutics that target the Clp system. Here, we present a combined biophysical and biochemical study to explore the structure-dynamics-function relationship in MtClpP1P2. Electron cryomicroscopy (cryo-EM) structures of apo and acyldepsipeptide-bound MtClpP1P2 explain their lack of activity by showing loss of a key β-sheet in a sequence known as the handle region that is critical for the proper formation of the catalytic triad. Methyl transverse relaxation-optimized spectroscopy (TROSY)- based NMR, cryo-EM, and biochemical assays show that, on binding Bz-LL or covalent inhibitors, MtClpP1P2 undergoes a conformational change from an inactive compact state to an active extended structure that can be explained by a modified Monod-Wyman-Changeux model. Our study establishes a critical role for the handle region as an on/off switch for function and shows extensive allosteric interactions involving both intra- and interring communication that regulate MtClpP1P2 activity and that can potentially be exploited by small molecules to target M. tuberculosis. [ABSTRACT FROM AUTHOR]