Structural characterisation of human protein complexes in nutrient signalling by X-ray crystallography, cryo-EM and cross-linking mass spectrometry

Nutrient availability regulates cellular growth and metabolism through modulation of the mTORC1 activity. Aberrant activation of this serine/threonine kinase complex has been implicated in a host of metabolic disorders such as cancer and type 2 diabetes. Much work has been done to understand the processes involved in metabolic disease pathogenesis and to develop treatment strategies. However, limited effectiveness of available therapies targeting mTORC1 highlights the need for better understanding of the mechanistic aspects of regulation of this pathway, as well as complex crosstalk and feedback mechanisms towards other systems. In this work I have focused on high-resolution structural characterisation of proteins and protein-protein interactions involved in glucose metabolism and amino acid sensing pathway, as well as development of a cross-linking mass spectrometry method for studying transient and dynamic protein interactions. I have solved a structure of a naturally occurring R42H mutation in the hexokinase 2 that was previously shown to decrease enzyme activity and subsequently lead to development of glucose intolerance in Mexican cavefish. With help of differential scanning fluorimetry I have shown that this mutation destabilises the protein without a measurable effect on the overall substrate and product binding. However, it is possible that the mutation influences the overall flexibility and regulation of the enzyme’s activity, which requires further investigation. Characterisation of RagAC-Raptor biding through generation of several nucleotide-binding mimicking mutation combinations allowed in vitro assembly of the full lysosomal mTORC1 complex for single particle cryo-EM structure determination which is in agreement with currently proposed models of the mega complex. Using the established RagAC system I have developed a protocol for in vitro zero-length cross-linking, enrichment and identification of cross-linked peptides with 1,1-carbonyldiimidazole (CDI) and in vivo incorporation of photo-leucine and photo-methionine incorporation for cross-linking studies of similar dynamic systems. The wide range of cross-link distances (7- 140 Å) obtained in the CDI cross-linking experiments have revealed a likely conformational heterogeneity and flexibility of the complex in solution, highlighting the weaknesses of this method for studying heterogeneous mixtures and dynamic protein complexes. Although on a peptide level there was a substantial overlap, the identification of the cross-link site differed considerably between different software, therefore the results of similar studies should be interpreted with caution. The analysis of photo-leucine and photo-methionine incorporation rates revealed overall values on par with previous studies (of up to 10% and 30% respectively). More than 85% efficiency of UV activation, with some variation depending on the residue location, is promising for further studies. However, reduced protein production, large variations between different residue positions, and poor identification of modifications on less abundant peptides should be considered when designing cross-linking experiments with photo-amino acids.