Architecture of human Rag GTPase heterodimers and their complex with mTORC1

© 2019 American Association for the Advancement of Science. All rights reserved. The Rag guanosine triphosphatases (GTPases) recruit the master kinase mTORC1 to lysosomes to regulate cell growth and proliferation in response to amino acid availability. The nucleotide state of Rag heterodimers is critical for their association with mTORC1. Our cryo–electron microscopy structure of RagA/RagC in complex with mTORC1 shows the details of RagA/RagC binding to the RAPTOR subunit of mTORC1 and explains why only the RagAGTP/RagCGDPnucleotide state binds mTORC1. Previous kinetic studies suggested that GTP binding to one Rag locks the heterodimer to prevent GTP binding to the other. Our crystal structures and dynamics of RagA/RagC show the mechanism for this locking and explain how oncogenic hotspot mutations disrupt this process. In contrast to allosteric activation by RHEB, Rag heterodimer binding does not change mTORC1 conformation and activates mTORC1 by targeting it to lysosomes

Probing the Solution Structure of IκB Kinase (IKK) Subunit γ and Its Interaction with Kaposi Sarcoma-associated Herpes Virus Flice-interacting Protein and IKK Subunit β by EPR Spectroscopy.

Viral flice-interacting protein (vFLIP), encoded by the oncogenic Kaposi sarcoma-associated herpes virus (KSHV), constitutively activates the canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) pathway. This is achieved through subversion of the IκB kinase (IKK) complex (or signalosome), which involves a physical interaction between vFLIP and the modulatory subunit IKKγ. Although this interaction has been examined both in vivo and in vitro, the mechanism by which vFLIP activates the kinase remains to be determined. Because IKKγ functions as a scaffold, recruiting both vFLIP and the IKKα/β subunits, it has been proposed that binding of vFLIP could trigger a structural rearrangement in IKKγ conducive to activation. To investigate this hypothesis we engineered a series of mutants along the length of the IKKγ molecule that could be individually modified with nitroxide spin labels. Subsequent distance measurements using electron paramagnetic resonance spectroscopy combined with molecular modeling and molecular dynamics simulations revealed that IKKγ is a parallel coiled-coil whose response to binding of vFLIP or IKKβ is localized twisting/stiffening and not large-scale rearrangements. The coiled-coil comprises N- and C-terminal regions with distinct registers accommodated by a twist: this structural motif is exploited by vFLIP, allowing it to bind and subsequently activate the NF-κB pathway. In vivo assays confirm that NF-κB activation by vFLIP only requires the N-terminal region up to the transition between the registers, which is located directly C-terminal of the vFLIP binding site

Molecular architecture of SAS-5 enables construction of a daughter centriole

In dividing cells, centrioles are duplicated once per cell cycle in a semi-conservative manner. A daughter centriole forms perpendicularly to the mother in a process templated by cartwheel-like structures. Cartwheels are found in centrioles of most eukaryotes, and are regarded as the key factor in establishing the nine-fold symmetry of centrioles. Cartwheels comprise the self-oligomerising protein SAS-6, recruitment of which to the mother centriole is mediated by direct binding to protein SAS-5 (also known as Ana2 or STIL). Although SAS-5 is an essential protein for centriole duplication, depletion of which completely terminates centrosome-dependent cell division, its exact role in this process has remained obscure. Using X-ray crystallography and a range of biophysical techniques, we have determined the molecular architecture of SAS-5. We show that SAS-5 forms a complex oligomeric structure, mediated by two self-associating domains: a trimeric coiled coil and a novel globular dimeric Implico domain. Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function. We propose that SAS-5 provides multivalent attachment sites that are critical for promoting assembly of SAS-6 into a cartwheel, and thus centriole formation.</p

Molecular architecture of SAS-5 enables construction of a daughter centriole

In dividing cells, centrioles are duplicated once per cell cycle in a semi-conservative manner. A daughter centriole forms perpendicularly to the mother in a process templated by cartwheel-like structures. Cartwheels are found in centrioles of most eukaryotes, and are regarded as the key factor in establishing the nine-fold symmetry of centrioles. Cartwheels comprise the self-oligomerising protein SAS-6, recruitment of which to the mother centriole is mediated by direct binding to protein SAS-5 (also known as Ana2 or STIL). Although SAS-5 is an essential protein for centriole duplication, depletion of which completely terminates centrosome-dependent cell division, its exact role in this process has remained obscure. Using X-ray crystallography and a range of biophysical techniques, we have determined the molecular architecture of SAS-5. We show that SAS-5 forms a complex oligomeric structure, mediated by two self-associating domains: a trimeric coiled coil and a novel globular dimeric Implico domain. Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function. We propose that SAS-5 provides multivalent attachment sites that are critical for promoting assembly of SAS-6 into a cartwheel, and thus centriole formation.</p

Cryo-EM Structure of the Human FLCN-FNIP2-Rag-Ragulator Complex

mTORC1 controls anabolic and catabolic processes in response to nutrients through the Rag GTPase heterodimer, which is regulated by multiple upstream protein complexes. One such regulator, FLCN-FNIP2, is a GTPase activating protein (GAP) for RagC/D, but despite its important role, how it activates the Rag GTPase heterodimer remains unknown. We used cryo-EM to determine the structure of FLCN-FNIP2 in a complex with the Rag GTPases and Ragulator. FLCN-FNIP2 adopts an extended conformation with two pairs of heterodimerized domains. The Longin domains heterodimerize and contact both nucleotide binding domains of the Rag heterodimer, while the DENN domains interact at the distal end of the structure. Biochemical analyses reveal a conserved arginine on FLCN as the catalytic arginine finger and lead us to interpret our structure as an on-pathway intermediate. These data reveal features of a GAP-GTPase interaction and the structure of a critical component of the nutrient-sensing mTORC1 pathway

Structural analysis of the G-Box domain of the microcephaly protein CPAP suggests a role in centriole architecture

Centrioles are evolutionarily conserved eukaryotic organelles composed of a protein scaffold surrounded by sets of microtubules organized with a 9-fold radial symmetry. CPAP, a centriolar protein essential for microtubule recruitment, features a C-terminal domain of unknown structure, the G-box. A missense mutation in the G-box reduces affinity for the centriolar shuttling protein STIL and causes primary microcephaly. Here, we characterize the molecular architecture of CPAP and determine the G-box structure alone and in complex with a STIL fragment. The G-box comprises a single elongated β sheet capable of forming supramolecular assemblies. Structural and biophysical studies highlight the conserved nature of the CPAP-STIL complex. We propose that CPAP acts as a horizontal “strut” that joins the centriolar scaffold with microtubules, whereas G-box domains form perpendicular connections

Structural basis for the docking of mTORC1 on the lysosomal surface

© 2019 American Association for the Advancement of Science. All rights reserved. The mTORC1 (mechanistic target of rapamycin complex 1) protein kinase regulates growth in response to nutrients and growth factors. Nutrients promote its translocation to the lysosomal surface, where its Raptor subunit interacts with the Rag guanosine triphosphatase (GTPase)-Ragulator complex. Nutrients switch the heterodimeric Rag GTPases among four different nucleotide-binding states, only one of which (RagA/B•GTP-RagC/D•GDP) permits mTORC1 association. We used cryo-electron microscopy to determine the structure of the supercomplex of Raptor with Rag-Ragulator at a resolution of 3.2 angstroms. Our findings indicate that the Raptor a-solenoid directly detects the nucleotide state of RagA while the Raptor "claw" threads between the GTPase domains to detect that of RagC. Mutations that disrupted Rag-Raptor binding inhibited mTORC1 lysosomal localization and signaling. By comparison with a structure of mTORC1 bound to its activator Rheb, we developed a model of active mTORC1 docked on the lysosome