John V. Lin King

Transient receptor potential (TRP) ion channels are sophisticated signaling machines that detect a wide variety of environmental and physiological signals. Every cell in the body expresses one or more members of the extended TRP channel family, which consists of over 30 subtypes, each likely possessing distinct pharmacological, biophysical, and/or structural attributes. While the function of some TRP subtypes remains enigmatic, those involved in sensory signaling are perhaps best characterized and have served as models for understanding how these excitatory ion channels serve as polymodal signal integrators. With the recent resolution revolution in cryo-electron microscopy, these and other TRP channel subtypes are now yielding their secrets to detailed atomic analysis, which is beginning to reveal structural underpinnings of stimulus detection and gating, ion permeation, and allosteric mechanisms governing signal integration. These insights are providing a framework for designing and evaluating modality-specific pharmacological agents for treating sensory and other TRP channel-associated disorders.

Presequence protease (PreP), a 117 kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3-4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, these structures reveal the key stages of the PreP catalytic cycle and how the binding of substrates or PreP inhibitor drives a rigid body motion of the protein for substrate binding and catalysis. Together, our studies provide key mechanistic insights into M16C metalloproteases for future therapeutic innovations.