Project description:Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and a type 1 transmembrane domain protein, receptor activity-modifying protein 1 (RAMP1). Here we report the structure of the human CGRP receptor in complex with CGRP and the Gs-protein heterotrimer at 3.3 Å global resolution, determined by Volta phase-plate cryo-electron microscopy. The receptor activity-modifying protein transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilizes CLR extracellular loop 2. RAMP1 makes only limited direct contact with CGRP, consistent with its function in allosteric modulation of CLR. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly in the positioning of the extracellular domain of CLR. This work provides insights into the control of G-protein-coupled receptor function.
Project description:The U2/U6 snRNA complex is a conserved and essential component of the active spliceosome that interacts with the pre-mRNA substrate and essential protein splicing factors to promote splicing catalysis. Here we have elucidated the solution structure of a 111-nucleotide U2/U6 complex using an approach that integrates SAXS, NMR, and molecular modeling. The U2/U6 structure contains a three-helix junction that forms an extended "Y" shape. The U6 internal stem-loop (ISL) forms a continuous stack with U2/U6 Helices Ib, Ia, and III. The coaxial stacking of Helix Ib on the U6 ISL is a configuration that is similar to the Domain V structure in group II introns. Interestingly, essential features of the complex--including the U80 metal binding site, AGC triad, and pre-mRNA recognition sites--localize to one face of the molecule. This observation suggests that the U2/U6 structure is well-suited for orienting substrate and cofactors during splicing catalysis.
Project description:U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7?Å resolution led to an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 small nuclear RNAs (snRNAs). The structure reveals striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5'-splice site during catalytic activation, forms a hairpin stabilized by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP, but its putative catalytic histidine is held away from the ?-phosphate by hydrogen bonding to a tyrosine in the amino-terminal domain of Prp8. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre.
Project description:U6 snRNA is a catalytic RNA responsible for pre-mRNA splicing reactions and undergoes various post-transcriptional modifications during its maturation process. The 3'-oligouridylation of U6 snRNA by the terminal uridylyltransferase, TUT1, provides the Lsm-binding site in U6 snRNA for U4/U6 di-snRNP formation and this ensures pre-mRNA splicing. Here, we present the crystal structure of human TUT1 (hTUT1) complexed with U6 snRNA, representing the post-uridylation of U6 snRNA by hTUT1. The N-terminal ZF-RRM and catalytic palm clamp the single-stranded AUA motif between the 5'-short stem and the 3'-telestem of U6 snRNA, and the ZF-RRM specifically recognizes the AUA motif. The ZF and the fingers hold the telestem, and the 3'-end of U6 snRNA is placed in the catalytic pocket of the palm for oligouridylation. The oligouridylation of U6 snRNA depends on the internal four-adenosine tract in the 5'-part of the telestem of U6 snRNA, and hTUT1 adds uridines until the internal adenosine tract can form base-pairs with the 3'-oligouridine tract. Together, the recognition of the specific structure and sequence of U6 snRNA by the multi-domain TUT1 protein and the intrinsic sequence and structure of U6 snRNA ensure the oligouridylation of U6 snRNA.
Project description:Chaperonins play an important role in folding newly synthesized or translocated proteins in all organisms. The bacterial chaperonin GroEL has served as a model system for the understanding of these proteins. In comparison, its human homolog, known as mitochondrial heat shock protein family member D1 (HSPD1) is poorly understood. Here, we present the structure of HSPD1 in the apo state determined by cryo-electron microscopy (cryo-EM). Unlike GroEL, HSPD1 forms mostly single ring assemblies in the absence of co-chaperonin (HSPE1). Comparison with GroEL shows a rotation and increased flexibility of the apical domain. Together with published structures of the HSPD1/HSPE1 co-chaperonin complex, this work gives insight into the structural changes that occur during the catalytic cycle. This new understanding of HSPD1 structure and its rearrangements upon complex formation may provide new insights for the development of HSPD1-targeting treatments against a diverse range of diseases including glioblastoma.
Project description:The thyroglobulin (TG) protein is essential to thyroid hormone synthesis, plays a vital role in the regulation of metabolism, development and growth and serves as intraglandular iodine storage. Its architecture is conserved among vertebrates. Synthesis of triiodothyronine (T3) and thyroxine (T4) hormones depends on the conformation, iodination and post-translational modification of TG. Although structural information is available on recombinant and deglycosylated endogenous human thyroglobulin (hTG) from patients with goiters, the structure of native, fully glycosylated hTG remained unknown. Here, we present the cryo-electron microscopy structure of native and fully glycosylated hTG from healthy thyroid glands to 3.2 Å resolution. The structure provides detailed information on hormonogenic and glycosylation sites. We employ liquid chromatography-mass spectrometry (LC-MS) to validate these findings as well as other post-translational modifications and proteolytic cleavage sites. Our results offer insights into thyroid hormonogenesis of native hTG and provide a fundamental understanding of clinically relevant mutations.
Project description:DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase complex composed of a catalytic subunit (DNA-PKcs) and KU70/80 heterodimer bound to DNA. DNA-PK holoenzyme plays a critical role in non-homologous end joining (NHEJ), the major DNA repair pathway. Here, we determined cryo-electron microscopy structure of human DNA-PK holoenzyme at 6.6 Å resolution. In the complex structure, DNA-PKcs, KU70, KU80 and DNA duplex form a 650-kDa heterotetramer with 1:1:1:1 stoichiometry. The N-terminal ?-solenoid (?2 800 residues) of DNA-PKcs adopts a double-ring fold and connects the catalytic core domain of DNA-PKcs and KU70/80-DNA. DNA-PKcs and KU70/80 together form a DNA-binding tunnel, which cradles ?30-bp DNA and prevents sliding inward of DNA-PKcs along with DNA duplex, suggesting a mechanism by which the broken DNA end is protected from unnecessary processing. Structural and biochemical analyses indicate that KU70/80 and DNA coordinately induce conformational changes of DNA-PKcs and allosterically stimulate its kinase activity. We propose a model for activation of DNA-PKcs in which allosteric signals are generated upon DNA-PK holoenzyme formation and transmitted to the kinase domain through N-terminal HEAT repeats and FAT domain of DNA-PKcs. Our studies suggest a mechanism for recognition and protection of broken DNA ends and provide a structural basis for understanding the activation of DNA-PKcs and DNA-PK-mediated NHEJ pathway.
Project description:GLUT4 is the primary glucose transporter in adipose and skeletal muscle tissues. Its cellular trafficking is regulated by insulin signaling. Failed or reduced plasma membrane localization of GLUT4 is associated with diabetes. Here, we report the cryo-EM structures of human GLUT4 bound to a small molecule inhibitor cytochalasin B (CCB) at resolutions of 3.3 Å in both detergent micelles and lipid nanodiscs. CCB-bound GLUT4 exhibits an inward-open conformation. Despite the nearly identical conformation of the transmembrane domain to GLUT1, the cryo-EM structure reveals an extracellular glycosylation site and an intracellular helix that is invisible in the crystal structure of GLUT1. The structural study presented here lays the foundation for further mechanistic investigation of the modulation of GLUT4 trafficking. Our methods for cryo-EM analysis of GLUT4 will also facilitate structural determination of many other small size solute carriers.
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:Mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) plays an essential role in regulating cell proliferation through phosphorylating AGC protein kinase family members, including AKT, PKC and SGK1. The functional core complex consists of mTOR, mLST8, and two mTORC2-specific components, Rictor and mSin1. Here we investigated the intermolecular interactions within mTORC2 complex and determined its cryo-electron microscopy structure at 4.9 Å resolution. The structure reveals a hollow rhombohedral fold with a 2-fold symmetry. The dimerized mTOR serves as a scaffold for the complex assembly. The N-terminal half of Rictor is composed of helical repeat clusters and binds to mTOR through multiple contacts. mSin1 is located close to the FRB domain and catalytic cavity of mTOR. Rictor and mSin1 together generate steric hindrance to inhibit binding of FKBP12-rapamycin to mTOR, revealing the mechanism for rapamycin insensitivity of mTORC2. The mTOR dimer in mTORC2 shows more compact conformation than that of mTORC1 (rapamycin sensitive), which might result from the interaction between mTOR and Rictor-mSin1. Structural comparison shows that binding of Rictor and Raptor (mTORC1-specific component) to mTOR is mutually exclusive. Our study provides a basis for understanding the assembly of mTORC2 and a framework to further characterize the regulatory mechanism of mTORC2 pathway.