scholarly journals The CryoEM Structure of the Ribosome Maturation Factor Rea1

2018 ◽  
Author(s):  
Piotr Sosnowski ◽  
Linas Urnavicius ◽  
Andreas Boland ◽  
Robert Fagiewicz ◽  
Johan Busselez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Atpase Activity ◽  
Conformational Changes ◽  
Ribosomal Subunit ◽  
Force Generation ◽  
Docking Site ◽  
Aaa Atpase ◽  
Helical Bundle ◽  
Maturation Factor ◽  
Assembly Factors

AbstractThe biogenesis of the 60S ribosomal subunit is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualize the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide induced conformational changes that create a docking site for the substrate binding MIDAS domain of Rea1 on the AAA+ ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.

scholarly journals The CryoEM structure of the Saccharomyces cerevisiae ribosome maturation factor Rea1

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Piotr Sosnowski ◽  
Linas Urnavicius ◽  
Andreas Boland ◽  
Robert Fagiewicz ◽  
Johan Busselez ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Conformational Changes ◽  
Force Generation ◽  
Docking Site ◽  
Ribosomal Subunits ◽  
Aaa Atpase ◽  
Helical Bundle ◽  
Maturation Factor ◽  
Assembly Factors

The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualise the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide-induced conformational changes that create a docking site for the substrate binding MIDAS domain on the AAA +ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.

scholarly journals Identification of a 521-Kilodalton Protein (Gli521) Involved in Force Generation or Force Transmission for Mycoplasma mobile Gliding

2005 ◽  
Vol 187 (10) ◽  
pp. 3502-3510 ◽  
Author(s):  
Shintaro Seto ◽  
Atsuko Uenoyama ◽  
Makoto Miyata
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Conformational Changes ◽  
Peptide Bond ◽  
Gliding Motility ◽  
Force Generation ◽  
Mycoplasma Species ◽  
Membrane Protrusion ◽  
Force Transmission ◽  
Inhibitory Effects

ABSTRACT Several mycoplasma species are known to glide on solid surfaces such as glass in the direction of the membrane protrusion, but the mechanism underlying this movement is unknown. To identify a novel protein involved in gliding, we raised monoclonal antibodies against a detergent-insoluble protein fraction of Mycoplasma mobile, the fastest glider, and screened the antibodies for inhibitory effects on gliding. Five monoclonal antibodies stopped the movement of gliding mycoplasmas, keeping them on the glass surface, and all of them recognized a large protein in immunoblotting. This protein, named Gli521, is composed of 4,738 amino acids, has a predicted molecular mass of 520,559 Da, and is coded downstream of a gene for another gliding protein, Gli349, which is known to be responsible for glass binding during gliding. Edman degradation analysis indicated that the N-terminal region is processed at the peptide bond between the amino acid residues at positions 43 and 44. Analysis of gliding mutants isolated previously revealed that the Gli521 protein is missing in a nonbinding mutant, m9, where the gli521 gene is truncated by a nonsense mutation at the codon for the amino acid at position 1170. Immunofluorescence and immunoelectron microscopy indicated that Gli521 localizes all around the base of the membrane protrusion, at the “neck,” as previously observed for Gli349. Analysis of the inhibitory effects of the anti-Gli521 antibody on gliding motility revealed that this protein is responsible for force generation or force transmission, a role distinct from that of Gli349, and also suggested conformational changes of Gli349 and Gli521 during gliding.

scholarly journals Structural basis for inhibition of the AAA-ATPase Drg1 by diazaborine

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Michael Prattes ◽  
Irina Grishkovskaya ◽  
Victor-Valentin Hodirnau ◽  
Ingrid Rössler ◽  
Isabella Klein ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Atp Hydrolysis ◽  
Ribosomal Subunit ◽  
Covalent Bond ◽  
Resistance Mechanisms ◽  
Drug Binding ◽  
Structural Basis ◽  
Aaa Atpase ◽  
Maturation Factor ◽  
Key Factor

AbstractThe hexameric AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis and initiates cytoplasmic maturation of the large ribosomal subunit by releasing the shuttling maturation factor Rlp24. Drg1 monomers contain two AAA-domains (D1 and D2) that act in a concerted manner. Rlp24 release is inhibited by the drug diazaborine which blocks ATP hydrolysis in D2. The mode of inhibition was unknown. Here we show the first cryo-EM structure of Drg1 revealing the inhibitory mechanism. Diazaborine forms a covalent bond to the 2′-OH of the nucleotide in D2, explaining its specificity for this site. As a consequence, the D2 domain is locked in a rigid, inactive state, stalling the whole Drg1 hexamer. Resistance mechanisms identified include abolished drug binding and altered positioning of the nucleotide. Our results suggest nucleotide-modifying compounds as potential novel inhibitors for AAA-ATPases.

scholarly journals Conformational heterogeneity of the AAA ATPase p97 characterized by single particle cryo-EM

2014 ◽  
Vol 70 (a1) ◽  
pp. C853-C853
Author(s):  
Driss Mountassif ◽  
Lucien Fabre ◽  
Kaustuv Basu ◽  
Mihnea Bostina ◽  
Slavica Jonic ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Conformational Changes ◽  
Single Particle ◽  
Molecular Mechanisms ◽  
Protein Complexes ◽  
Normal Mode Analysis ◽  
Single Amino Acid ◽  
Mode Analysis ◽  
Conformational Heterogeneity ◽  
Aaa Atpase

p97, a member of the AAA (ATPase Associated with various Activities) ATPase family, is essential and centrally involved in a wide variety of cellular processes. Single amino-acid substitutions in p97 have been associated with the severe degenerative disorder of Inclusion Body Myopathy associated with Paget disease of bone and Frontotemporal Dementia (IBMPFD) as well as amytropic leteral sclerosis (ALS). Current models propose that p97 acts as a motor transmitting the energy from the ATPase cycle to conformational changes of substrate protein complexes causing segregation, remodeling or translocation. Mutations at the interface between the N and the D1 domains impact the ATPase activity and the conformation of D2 on the opposite side of the protein complex, suggesting intermolecular communication. Because of limited structural information, the molecular mechanisms on how p97 drives its activities and the molecular basis for transmission of information within the molecule remain elusive. Structural heterogeneity is observed in vitro and is likely relevant for the in vivo biological function of p97. Single particle cryo-EM is the method of choice to study a flexible complex. The technique allows study in solution and also deals with sample heterogeneity by image classification. We have set-up the characterization of the conformational heterogeneity in WT and disease relevant p97 mutant using multi-likelihood classification and Hybrid Electron Microscopy Normal Mode Analysis HEMNMA. The multi-likelihood analysis shows a link between the conformations of the N and D2 domains. HEMNMA allows the analysis of the asymmetry of the conformational changes. Together these studies describe the structural flexibility of p97 and the coupling of the ATPase activity with conformational changes in health and in disease. Study of this model system also allows the development of new methods to understand the conformational heterogeneity of other protein complexes.

scholarly journals Conformational switches control early maturation of the eukaryotic small ribosomal subunit

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mirjam Hunziker ◽  
Jonas Barandun ◽  
Olga Buzovetsky ◽  
Caitlin Steckler ◽  
Henrik Molina ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Conformational Changes ◽  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Ribosome Assembly ◽  
Small Ribosomal Subunit ◽  
Early Maturation ◽  
External Transcribed Spacer ◽  
Assembly Factors

Eukaryotic ribosome biogenesis is initiated with the transcription of pre-ribosomal RNA at the 5’ external transcribed spacer, which directs the early association of assembly factors but is absent from the mature ribosome. The subsequent co-transcriptional association of ribosome assembly factors with pre-ribosomal RNA results in the formation of the small subunit processome. Here we show that stable rRNA domains of the small ribosomal subunit can independently recruit their own biogenesis factors in vivo. The final assembly and compaction of the small subunit processome requires the presence of the 5’ external transcribed spacer RNA and all ribosomal RNA domains. Additionally, our cryo-electron microscopy structure of the earliest nucleolar pre-ribosomal assembly - the 5’ external transcribed spacer ribonucleoprotein – provides a mechanism for how conformational changes in multi-protein complexes can be employed to regulate the accessibility of binding sites and therefore define the chronology of maturation events during early stages of ribosome assembly.

scholarly journals Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader

2021 ◽  
Author(s):  
Christl Gaubitz ◽  
Xingchen Liu ◽  
Joshua Pajak ◽  
Nicholas P. Stone ◽  
Janelle A. Hayes ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Conformational Changes ◽  
Large Scale ◽  
Atp Hydrolysis ◽  
Nuclear Antigen ◽  
Clamp Loader ◽  
Aaa Atpase ◽  
Sliding Clamp ◽  
Sliding Clamps ◽  
Factor C

Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the S. cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale 'crab-claw' expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC's switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

Infrared spectra of N-acetyl-L-α-amino-acid N'-methylamides with aliphatic side chains in non-polar solvents

1981 ◽  
Vol 46 (3) ◽  
pp. 772-780 ◽  
Author(s):  
Jorga Smolíková ◽  
Jan Pospíšek ◽  
Karel Bláha
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Infrared Spectra ◽  
Room Temperature ◽  
Side Chains ◽  
Polar Solvents

Infrared spectra of the L-alanine (I), L-leucine (II), L-valine (III) and L-tert-leucine (IV) N-acetyl N'-methylamides were measured. Amides I-IV are not self associated in tetrachlormethane in the concentration 2 . 10-5 mol l-1 at room temperature and in tetrachloroethylene in the concentration 1.5 . 10-4 mol l-1 at temperatures above 65° C. True conformational changes are observable only with the least flexible amide IV which exists at room temperature in a C5 conformation. This conformational type is also highly populated in the valine derivative III, but is less important in the alanine and leucine derivatives I and II in which the intramolecularly bonded C7 and the distorted hydrogen-nonbonded conformations contribute seriously.

scholarly journals Determinants That Control the Specific Interactions between TAB1 and p38α

2006 ◽  
Vol 26 (10) ◽  
pp. 3824-3834 ◽  
Author(s):  
Huamin Zhou ◽  
Min Zheng ◽  
Jianming Chen ◽  
Changchuan Xie ◽  
Anand R. Kolatkar ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Conformational Changes ◽  
Transforming Growth Factor ◽  
Mutational Analysis ◽  
Specific Binding ◽  
Point Mutations ◽  
Transforming Growth Factor Β ◽  
Mitogen Activated Protein Kinase ◽  
Docking Site ◽  
C Terminus

ABSTRACT Previous studies have revealed that transforming growth factor-β-activated protein kinase 1 (TAB1) interacts with p38α and induces p38α autophosphorylation. Here, we examine the sequence requirements in TAB1 and p38α that drive their interaction. Deletion and point mutations in TAB1 reveal that a proline residue in the C terminus of TAB1 (Pro412) is necessary for its interaction with p38α. Furthermore, a cryptic D-domain-like docking site was identified adjacent to the N terminus of Pro412, putting Pro412 in the φB+3 position of the docking site. Through mutational analysis, we found that the previously identified hydrophobic docking groove in p38α is involved in this interaction, whereas the CD domain and ED domain are not. Furthermore, chimeric analysis with p38β (which does not bind to TAB1) revealed a previously unidentified locus of p38α comprising Thr218 and Ile275 that is essential for specific binding of p38α to TAB1. Converting either of these residues to the corresponding amino acid of p38β abolishes p38α interaction with TAB1. These p38α mutants still can be fully activated by p38α upstream activating kinase mitogen-activated protein kinase kinase 6, but their basal activity and activation in response to some extracellular stimuli are reduced. Adjacent to Thr218 and Ile275 is a site where large conformational changes occur in the presence of docking-site peptides derived from p38α substrates and activators. This suggests that TAB1-induced autophosphorylation of p38α results from conformational changes that are similar but unique to those seen in p38α interactions with its substrates and activating kinases.

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