ABSTRACT
mTORC1 is a protein kinase complex that controls cellular growth in response to nutrient availability. Amino acid signals are transmitted toward mTORC1 via the Rag/Gtr GTPases and their upstream regulators. An important regulator is LAMTOR, which localizes Rag/Gtr on the lysosomal/vacuole membrane. In human cells, LAMTOR consists of five subunits, but in yeast, only three or four. Currently, it is not known how variation of the subunit stoichiometry may affect its structural organization and biochemical properties. Here, we report a 3.1 Å-resolution structural model of the Gtr-Lam complex in Schizosaccharomyces pombe. We found that SpGtr shares conserved architecture as HsRag, but the intersubunit communication that coordinates nucleotide loading on the two subunits differs. In contrast, SpLam contains distinctive structural features, but its GTP-specific GEF activity toward SpGtr is evolutionarily conserved. Our results revealed unique evolutionary paths of the protein components of the mTORC1 pathway.
Subject(s)
Monomeric GTP-Binding Proteins , Schizosaccharomyces , Humans , Mechanistic Target of Rapamycin Complex 1/metabolism , Amino Acids/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Monomeric GTP-Binding Proteins/chemistryABSTRACT
The mechanistic target of rapamycin (mTOR) complex is responsible for coordinating nutrient availability with eukaryotic cell growth. Amino acid signals are transmitted towards mTOR via the Rag/Gtr heterodimers. Due to the obligatory heterodimeric architecture of the Rag/Gtr GTPases, investigating their biochemical properties has been challenging. Here, we describe an updated assay that allows us to probe the guanine nucleotide-binding affinity and kinetics to the Gtr heterodimers in Saccharomyces cerevisiae. We first identified the structural element that Gtr2p lacks to enable crosslinking. By using a sequence conservation-based mutation, we restored the crosslinking between Gtr2p and the bound nucleotides. Using this construct, we determined the nucleotide-binding affinities of the Gtr heterodimer, and found that it operates under a different form of intersubunit communication than human Rag GTPases. Our study defines the evolutionary divergence of the Gtr/Rag-mTOR axis of nutrient sensing.
Subject(s)
Monomeric GTP-Binding Proteins , Saccharomyces cerevisiae , Humans , Guanine/metabolism , Guanine Nucleotides/metabolism , Mechanistic Target of Rapamycin Complex 1/metabolism , Monomeric GTP-Binding Proteins/metabolism , Nucleotides/metabolism , Saccharomyces cerevisiae/metabolism , TOR Serine-Threonine Kinases/metabolism , GTP Phosphohydrolases/metabolismABSTRACT
The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide, reaching near fixation in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and on cells rendered permissive by ectopic expression of human ACE2 or of ACE2 orthologs from various mammals, including Chinese rufous horseshoe bat and Malayan pangolin. D614G did not alter S protein synthesis, processing, or incorporation into SARS-CoV-2 particles, but D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts an interprotomer contact and that the conformation is shifted toward an ACE2 binding-competent state, which is modeled to be on pathway for virion membrane fusion with target cells. Consistent with this more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated.
Subject(s)
Betacoronavirus/physiology , Betacoronavirus/ultrastructure , Spike Glycoprotein, Coronavirus/physiology , Spike Glycoprotein, Coronavirus/ultrastructure , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/immunology , Betacoronavirus/pathogenicity , COVID-19 , Cells, Cultured , Coronavirus Infections/virology , Female , Genetic Variation , HEK293 Cells , Humans , Male , Models, Molecular , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , Protein Conformation , Protein Processing, Post-Translational , Receptors, Coronavirus , Receptors, Virus/metabolism , SARS-CoV-2 , Species SpecificityABSTRACT
The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and cells rendered permissive by ectopic expression of various mammalian ACE2 orthologs. Nonetheless, D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts a critical interprotomer contact and that this dramatically shifts the S protein trimer conformation toward an ACE2-binding and fusion-competent state. Consistent with the more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated. These results indicate that D614G adopts conformations that make virion membrane fusion with the target cell membrane more probable but that D614G retains susceptibility to therapies that disrupt interaction of the SARS-CoV-2 S protein with the ACE2 receptor.