Allosteric regulation of proteolytic machines unveiled by the synergy between cryo-EM and solution NMR spectroscopy.

Mechanistic studies of biomolecular machines involved in intracellular protein degradation-such as the caseinolytic protease P, ATPases associated with diverse cellular activities (AAA+) motors, and the high-temperature requirement A family of enzymes-are of great interest as they are implicated in a host of human diseases. The function of these systems is dependent on both their fine-tuned three-dimensional structure and the conformational dynamics that modulate this structure. Their large sizes, inherent conformational plasticity, and oligomeric heterogeneity dictate that their mechanism of action cannot be deciphered by any one method. Synergistic application of methyl-transverse relaxation optimized spectroscopy (methyl-TROSY), nuclear magnetic resonance (NMR), and single-particle electron cryomicroscopy (cryo-EM) has uniquely positioned researchers to tackle the outstanding questions in this area of structural biology. Cryo-EM enables structural characterization and modeling of the large and conformationally heterogeneous complexes involved in protein degradation, while methyl-TROSY NMR enables monitoring structural transitions and conformational dynamics of these systems in response to various stimuli in solution at atomic resolution. This review highlights how combining these two approaches offers a distinct and powerful means to unravel allosteric pathways within complex, multipartite biomolecular machines.