Final Report Summary - MTOR_COMPLEXES (Structural and biophysical characterization of the human mTOR kinase and its signaling complexes)
Multi-protein complexes containing the Serine/Threonine kinase mammalian target of rapamycin (mTOR) are essential regulators of cell growth and metabolism (mTOR complex 1, mTORC1), cell survival and cytoskeletal organization (mTORC2). mTORC1/2 signaling is tightly regulated and aberrant mTORC1/2 function is linked to human disorders like metabolic disease, diabetes, cancer and neurodegeneration. The core components of mTORC1 are mTOR, Raptor and mLST8. mTORC2 consist of mTOR, Rictor, mLST8, mSIN1 and Protor. A detailed knowledge of the molecular architecture of mTORCs is crucial to understand mTORC1/2 assembly, signaling and regulation. Currently, structural information of mTORCs is limited to a low-resolution (26 Å) reconstruction of mTORC1 by cryo electron microscopy (cryo-EM) and the crystal structure of the mTOR kinase domain in complex with mLST8 at 3.2 Å resolution. To understand complex assembly and regulation, the major objective of the ongoing research project is to obtain structural information of mTORC1 and mTORC2 at intermediate to high resolution. The expected key results are (a) expression and purification protocols for mTORC1/2 and (b) intermediate-resolution structures describing the domain architecture of the individual subunits and their arrangement in mTORC1/2.
To achieve the research goals, the project was organized in three main phases: (1) design, purification and biophysical characterization of mTORC1/2 subunits, (2) identification of relevant mTOR sub-complexes for structure determination and (3) structure determination of mTORC1/2. Since the beginning of the project the researcher cloned all mTORC1/2 components into expression vectors suitable for pro- and eukaryotic expression systems. Subsequently, the researcher identified the multi-gene expression system MultiBac® (Baculovirus expression system) as suitable for the production of intact mTORC1 and continued to establish mTORC1 expression and purification strategies. She optimized the Baculovirus production procedure, screened different expression cell lines and tested the effect of mTOR kinase inhibitors on the expression levels of mTORC1 in insect cells. Moreover, the researcher systematically screened cell lysis conditions, tested different affinity purification resins and optimized the size exclusion chromatography set-up. Ultimately, she was able to develop expression and purification protocols that yield 0.2-0.5 mg mTORC1. The purified mTORC1 was catalytically active as determined in kinase activity assays using the mTORC1 substrate 4E-BP1. Biophysical characterization by static light scattering confirmed that mTORC1 is a dimer with the expected molecular weight of 950 kDa. Additionally, analysis by negative stain EM confirmed the rhomboid, dimeric shape and the overall dimensions of human mTORC1.
To obtain crystals for structure determination by X-ray crystallography, mTORC1 was subjected to high-throughput crystallization screening experiments. Although promising conditions were identified, several rounds of optimization did not yield diffraction quality crystals. To meet the project objectives the researcher additionally focused on an integrative structure determination approach combining cross-linking mass spectrometry (XL-MS) with negative stain and cryo-EM. XL-MS experiments identified regions in mTOR and Raptor that are in close proximity to each other providing distance restraints for structure determination at intermediate resolution. Moreover, the cross-linking results were consistent with a proposed head-to-tail dimer arrangement. In parallel, the researcher further optimized sample quality for data collection and tested stabilization protocols for particle reconstruction by cryo-EM.
The main achievement of the research project is the development of reproducible expression and purification protocols that yield biologically active, intact mTORC1. Sample quality is suitable for structure determination by crystallography and cryo-EM, although the total mTORC1 yields currently limit extensive crystallization screening. Importantly, human mTORC1 purified from insect cells clearly resembles mTORC1 extracted from human cell lines and the fully assembled complex therefore represents a highly relevant target for structure determination. So far, human mTORC1 was available only from human cell lines stably expressing the Raptor component. The cost and time effective preparation protocols for biologically active human mTORC1 from insect cells are therefore an important prerequisite for successful structure determination at higher resolution in the future. Moreover, preliminary experiments indicate that the established expression and purification protocols are directly transferrable to mTORC2 and will be relevant for structure determination of this complex. The researcher will continue to work on structure determination of mTORC1 focusing on EM techniques in collaboration with the N. Ban Laboratory at the ETH in Zurich. The expected final results are single particle reconstructions of mTORC1 at intermediate resolution (less than 10 Å) providing insight into complex assembly and function.
To achieve the research goals, the project was organized in three main phases: (1) design, purification and biophysical characterization of mTORC1/2 subunits, (2) identification of relevant mTOR sub-complexes for structure determination and (3) structure determination of mTORC1/2. Since the beginning of the project the researcher cloned all mTORC1/2 components into expression vectors suitable for pro- and eukaryotic expression systems. Subsequently, the researcher identified the multi-gene expression system MultiBac® (Baculovirus expression system) as suitable for the production of intact mTORC1 and continued to establish mTORC1 expression and purification strategies. She optimized the Baculovirus production procedure, screened different expression cell lines and tested the effect of mTOR kinase inhibitors on the expression levels of mTORC1 in insect cells. Moreover, the researcher systematically screened cell lysis conditions, tested different affinity purification resins and optimized the size exclusion chromatography set-up. Ultimately, she was able to develop expression and purification protocols that yield 0.2-0.5 mg mTORC1. The purified mTORC1 was catalytically active as determined in kinase activity assays using the mTORC1 substrate 4E-BP1. Biophysical characterization by static light scattering confirmed that mTORC1 is a dimer with the expected molecular weight of 950 kDa. Additionally, analysis by negative stain EM confirmed the rhomboid, dimeric shape and the overall dimensions of human mTORC1.
To obtain crystals for structure determination by X-ray crystallography, mTORC1 was subjected to high-throughput crystallization screening experiments. Although promising conditions were identified, several rounds of optimization did not yield diffraction quality crystals. To meet the project objectives the researcher additionally focused on an integrative structure determination approach combining cross-linking mass spectrometry (XL-MS) with negative stain and cryo-EM. XL-MS experiments identified regions in mTOR and Raptor that are in close proximity to each other providing distance restraints for structure determination at intermediate resolution. Moreover, the cross-linking results were consistent with a proposed head-to-tail dimer arrangement. In parallel, the researcher further optimized sample quality for data collection and tested stabilization protocols for particle reconstruction by cryo-EM.
The main achievement of the research project is the development of reproducible expression and purification protocols that yield biologically active, intact mTORC1. Sample quality is suitable for structure determination by crystallography and cryo-EM, although the total mTORC1 yields currently limit extensive crystallization screening. Importantly, human mTORC1 purified from insect cells clearly resembles mTORC1 extracted from human cell lines and the fully assembled complex therefore represents a highly relevant target for structure determination. So far, human mTORC1 was available only from human cell lines stably expressing the Raptor component. The cost and time effective preparation protocols for biologically active human mTORC1 from insect cells are therefore an important prerequisite for successful structure determination at higher resolution in the future. Moreover, preliminary experiments indicate that the established expression and purification protocols are directly transferrable to mTORC2 and will be relevant for structure determination of this complex. The researcher will continue to work on structure determination of mTORC1 focusing on EM techniques in collaboration with the N. Ban Laboratory at the ETH in Zurich. The expected final results are single particle reconstructions of mTORC1 at intermediate resolution (less than 10 Å) providing insight into complex assembly and function.