Project description:The endothelin ETB receptor is a promiscuous G-protein coupled receptor that is activated by vasoactive peptide endothelins. ETB signaling induces reactive astrocytes in the brain and vasorelaxation in vascular smooth muscle. Consequently, ETB agonists are expected to be drugs for neuroprotection and improved anti-tumor drug delivery. Here, we report the cryo-electron microscopy structure of the endothelin-1-ETB-Gi complex at 2.8 Å resolution, with complex assembly stabilized by a newly established method. Comparisons with the inactive ETB receptor structures revealed how endothelin-1 activates the ETB receptor. The NPxxY motif, essential for G-protein activation, is not conserved in ETB, resulting in a unique structural change upon G-protein activation. Compared with other GPCR-G-protein complexes, ETB binds Gi in the shallowest position, further expanding the diversity of G-protein binding modes. This structural information will facilitate the elucidation of G-protein activation and the rational design of ETB agonists.
Project description:The exocyst is an evolutionarily conserved octameric protein complex that mediates the tethering of post-Golgi secretory vesicles to the plasma membrane during exocytosis and is implicated in many cellular processes such as cell polarization, cytokinesis, ciliogenesis and tumor invasion. Using cryo-EM and chemical cross-linking MS (CXMS), we solved the structure of the Saccharomyces cerevisiae exocyst complex at an average resolution of 4.4 Å. Our model revealed the architecture of the exocyst and led to the identification of the helical bundles that mediate the assembly of the complex at its core. Sequence analysis suggests that these regions are evolutionarily conserved across eukaryotic systems. Additional cell biological data suggest a mechanism for exocyst assembly that leads to vesicle tethering at the plasma membrane.
Project description:The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of Rapamycin Complex 1 (TORC1) in yeast1. Functionally, the SEAC has been divided into two subcomplexes: SEACIT, which has GAP activity and inhibits TORC1, and SEACAT, which regulates SEACIT2. This system is conserved in mammals: the GATOR complex, consisting of GATOR1 (SEACIT) and GATOR2 (SEACAT), transmits amino acid3 and glucose4 signals to mTORC1. Despite its importance, the structure of SEAC/GATOR, and thus molecular understanding of its function, is lacking. Here, we solve the cryo-EM structure of the native eight-subunit SEAC. The SEAC has a modular structure in which a COPII-like cage corresponding to SEACAT binds two flexible wings, which correspond to SEACIT. The wings are tethered to the core via Sea3, which forms part of both modules. The GAP mechanism of GATOR1 is conserved in SEACIT, and GAP activity is unaffected by SEACAT in vitro. In vivo, the wings are essential for recruitment of the SEAC to the vacuole, primarily via the EGO complex. Our results indicate that rather than being a direct inhibitor of SEACIT, SEACAT acts as a scaffold for the binding of TORC1 regulators.
Project description:The CST-Polα/primase complex is essential for telomere maintenance and functions to counteract resection at double-strand breaks. We report a 4.6-Å resolution cryo-EM structure of human CST-Polα/primase, captured prior to catalysis in a recruitment state stabilized by chemical cross-linking. Our structure reveals an evolutionarily conserved interaction between the C-terminal domain of the catalytic POLA1 subunit and an N-terminal expansion in metazoan CTC1. Cross-linking mass spectrometry and negative-stain EM analysis provide insight into CST binding by the flexible POLA1 N-terminus. Finally, Coats plus syndrome disease mutations previously characterized to disrupt formation of the CST-Polα/primase complex map to protein-protein interfaces observed in the recruitment state. Together, our results shed light on the architecture and stoichiometry of the metazoan fill-in machinery.
Project description:Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone-RF3 bound to a non-hydrolyzable GTP-analog-have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3. DOI:http://dx.doi.org/10.7554/eLife.00411.001.
Project description:ATR (ataxia telangiectasia-mutated and Rad3-related) protein kinase and ATRIP (ATR-interacting protein) form a complex and play a critical role in response to replication stress and DNA damage. Here, we determined the cryo-electron microscopy (EM) structure of the human ATR-ATRIP complex at 4.7 Å resolution and built an atomic model of the C-terminal catalytic core of ATR (residues 1 521-2 644) at 3.9 Å resolution. The complex adopts a hollow "heart" shape, consisting of two ATR monomers in distinct conformations. The EM map for ATRIP reveals 14 HEAT repeats in an extended "S" shape. The conformational flexibility of ATR allows ATRIP to properly lock the N-termini of the two ATR monomers to favor ATR-ATRIP complex formation and functional diversity. The isolated "head-head" and "tail-tail" each adopts a pseudo 2-fold symmetry. The catalytic pockets face outward and substrate access is not restricted by inhibitory elements. Our studies provide a structural basis for understanding the assembly of the ATR-ATRIP complex and a framework for characterizing ATR-mediated DNA repair pathways.
Project description:Trastuzumab and pertuzumab are monoclonal antibodies that bind to distinct subdomains of the extracellular domain of human epidermal growth factor receptor 2 (HER2). Adding these monoclonal antibodies to the treatment regimen of HER2-positive breast cancer has changed the paradigm for treatment in that form of cancer. Synergistic activity has been observed with the combination of these two antibodies leading to hypotheses regarding the mechanism(s) and to the development of bispecific antibodies to maximize the clinical effect further. Although the individual crystal structures of HER2-trastuzumab and HER2-pertuzumab revealed the distinct binding sites and provided the structural basis for their anti-tumor activities, detailed structural information on the HER2-trastuzumab-pertuzumab complex has been elusive. Here we present the cryo-EM structure of HER2-trastuzumab-pertuzumab at 4.36 Å resolution. Comparison with the binary complexes reveals no cooperative interaction between trastuzumab and pertuzumab, and provides key insights into the design of novel, high-avidity bispecific molecules with potentially greater clinical efficacy.