Structure-function studies of the Tuberous Sclerosis Complex protein complex and its partners

Abstract

Tuberous Sclerosis Complex (TSC) is a progressive multi-organ system disease, for which a unifying feature is benign tumour growth across, but not limited to, brain, skin, heart, lungs, and kidney tissues. TSC was genetically linked to mutations in the tumour suppressor genes TSC1 and TSC2 in the late 1980s, the gene products of which bind a constitutive third member, TBC1D7 to form the TSC protein complex (TSCC). It is known that TSCC is a key negative regulator of mTOR complex 1 (mTORC1) in eukaryotes, a principal controller of growth and biogenesis, and performs this function by inhibiting the mTORC1 activator RHEB, a small GTPase. Physiologically, TSCC integrates multiple upstream growth- and stress-related signals to regulate the activation state of mTORC1 at the lysosomal surface. Our knowledge of TSCC biological and physiological activity has been illuminated by decades of biological, biochemical and cellular data, however, TSCC remained structurally uncharacterised as a holo-complex. Structural knowledge of TSCC is crucial for our understanding of structure-function relationships governing TSCC activity. We aimed to structurally characterise TSCC using cryo-electron microscopy (cryo-EM), a method uniquely positioned for structural biology of large, dynamic protein complexes. We detail methods by which human TSCC may be recombinantly expressed in mammalian cells and purified to a quality sufficient to pursue structure determination using single-particle cryo-EM, from which we further report the molecular architecture of human TSCC at intermediate resolution. We also explore the interactions of TSCC with its partner proteins, including its GTPase target RHEB, the cytosolic localisation adaptor 14-3-3, and lysosomally-enriched negatively charged lipids, and make strides towards structural studies of these co-complexes. Finally, through our exploration and optimisation of TSCC-partner complexes for cryo-EM, we recognise that sample- and grid-preparation are major bottlenecks for structure determination of complexes in their native state from in vitro purification and assembly. To this end, we show that TSCC can be produced in cell-free reactions, highlighting its potential as a viable alternative for cryo-EM sample production. Additionally, we further detail a proof-of-concept, on-grid affinity purification workflow which improves sample-to-grid efficiency, using a 3D-printed flow-cell device. Such a workflow can be leveraged to support and streamline future structural studies of TSCC, purified directly from lysate or other native sources, and may be extended to the study of other difficult-to-produce mammalian protein complexes. Further development of affinity purification methods may yet become valuable a tool for unpicking the structural biology of TSCC in its physiologically relevant states.