scholarly journals Structural Basis of Eco1-Mediated Cohesin Acetylation

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
William C. H. Chao ◽  
Benjamin O. Wade ◽  
Céline Bouchoux ◽  
Andrew W. Jones ◽  
Andrew G. Purkiss ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Substrate Specificity ◽  
Binding Site ◽  
Conformational Changes ◽  
Sister Chromatid ◽  
Molecular Basis ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Chromatid Cohesion

Abstract Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension.

scholarly journals Structural analysis of the LDL receptor–interacting FERM domain in the E3 ubiquitin ligase IDOL reveals an obscured substrate-binding site

2020 ◽  
Vol 295 (39) ◽  
pp. 13570-13583
Author(s):  
Luca Martinelli ◽  
Athanassios Adamopoulos ◽  
Patrik Johansson ◽  
Paul T. Wan ◽  
Jenny Gunnarsson ◽  
...  
Keyword(s):  
Binding Site ◽  
Ubiquitin Ligase ◽  
Substrate Recognition ◽  
E3 Ubiquitin Ligase ◽  
Full Length ◽  
Scattering Data ◽  
Lipid Lowering ◽  
Structural Basis ◽  
Ferm Domain

Hepatic abundance of the low-density lipoprotein receptor (LDLR) is a critical determinant of circulating plasma LDL cholesterol levels and hence development of coronary artery disease. The sterol-responsive E3 ubiquitin ligase inducible degrader of the LDLR (IDOL) specifically promotes ubiquitination and subsequent lysosomal degradation of the LDLR and thus controls cellular LDL uptake. IDOL contains an extended N-terminal FERM (4.1 protein, ezrin, radixin, and moesin) domain, responsible for substrate recognition and plasma membrane association, and a second C-terminal RING domain, responsible for the E3 ligase activity and homodimerization. As IDOL is a putative lipid-lowering drug target, we investigated the molecular details of its substrate recognition. We produced and isolated full-length IDOL protein, which displayed high autoubiquitination activity. However, in vitro ubiquitination of its substrate, the intracellular tail of the LDLR, was low. To investigate the structural basis for this, we determined crystal structures of the extended FERM domain of IDOL and multiple conformations of its F3ab subdomain. These reveal the archetypal F1-F2-F3 trilobed FERM domain structure but show that the F3c subdomain orientation obscures the target-binding site. To substantiate this finding, we analyzed the full-length FERM domain and a series of truncated FERM constructs by small-angle X-ray scattering (SAXS). The scattering data support a compact and globular core FERM domain with a more flexible and extended C-terminal region. This flexibility may explain the low activity in vitro and suggests that IDOL may require activation for recognition of the LDLR.

scholarly journals Structural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling

2021 ◽  
Author(s):  
Hauke S. Hillen ◽  
Elena Lavdovskaia ◽  
Franziska Nadler ◽  
Elisa Hanitsch ◽  
Andreas Linden ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Large Subunit ◽  
Structural Basis ◽  
Catalytic Core ◽  
Mitochondrial Ribosomes ◽  
Mitochondrial Ribosome ◽  
Translation Systems

Ribosome biogenesis is an essential process that requires auxiliary factors to promote folding and assembly of ribosomal proteins and RNA. In particular, maturation of the peptidyl transferase center (PTC), the catalytic core of the ribosome, is mediated by universally conserved GTPases, but the molecular basis is poorly understood. Here, we define the mechanism of GTPase-driven maturation of the human mitochondrial ribosomal large subunit (mtLSU) using a combination of endogenous complex purification, in vitro reconstitution and cryo-electron microscopy (cryo-EM). Structures of transient native mtLSU assembly intermediates that accumulate in GTPBP6-deficient cells reveal how the biogenesis factors GTPBP5, MTERF4 and NSUN4 facilitate PTC folding. Subsequent addition of recombinant GTPBP6 reconstitutes late mtLSU biogenesis in vitro and shows that GTPBP6 triggers a molecular switch by releasing MTERF4-NSUN4 and GTPBP5 accompanied by the progression to a near-mature PTC state. In addition, cryo-EM analysis of GTPBP6-treated mature mitochondrial ribosomes reveals the structural basis for the dual-role of GTPBP6 in ribosome biogenesis and recycling. Together, these results define the molecular basis of dynamic GTPase-mediated PTC maturation during mitochondrial ribosome biogenesis and provide a framework for understanding step-wise progression of PTC folding as a critical quality control checkpoint in all translation systems.

scholarly journals Molecular Basis of Substrate Recognition of Endonuclease Q from the Euryarchaeon Pyrococcus furiosus

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Miyako Shiraishi ◽  
Shigenori Iwai
Keyword(s):  
Dna Repair ◽  
Bacillus Subtilis ◽  
Substrate Specificity ◽  
Molecular Basis ◽  
Pyrococcus Furiosus ◽  
Substrate Recognition ◽  
Base Flipping ◽  
Content Type ◽  
Cleavage Activity

ABSTRACT Endonuclease Q (EndoQ), a DNA repair endonuclease, was originally identified in the hyperthermophilic euryarchaeon Pyrococcus furiosus in 2015. EndoQ initiates DNA repair by generating a nick on DNA strands containing deaminated bases and an abasic site. Although EndoQ is thought to be important for maintaining genome integrity in certain bacteria and archaea, the underlying mechanism catalyzed by EndoQ remains unclear. Here, we provide insights into the molecular basis of substrate recognition by EndoQ from P. furiosus (PfuEndoQ) using biochemical approaches. Our results of the substrate specificity range and the kinetic properties of PfuEndoQ demonstrate that PfuEndoQ prefers the imide structure in nucleobases along with the discovery of its cleavage activity toward 5,6-dihydrouracil, 5-hydroxyuracil, 5-hydroxycytosine, and uridine in DNA. The combined results for EndoQ substrate binding and cleavage activity analyses indicated that PfuEndoQ flips the target base from the DNA duplex, and the cleavage activity is highly dependent on spontaneous base flipping of the target base. Furthermore, we find that PfuEndoQ has a relatively relaxed substrate specificity; therefore, the role of EndoQ in restriction modification systems was explored. The activity of the EndoQ homolog from Bacillus subtilis was found not to be inhibited by the uracil glycosylase inhibitor from B. subtilis bacteriophage PBS1, whose genome is completely replaced by uracil instead of thymine. Our findings suggest that EndoQ not only has additional functions in DNA repair but also could act as an antiviral enzyme in organisms with EndoQ. IMPORTANCE Endonuclease Q (EndoQ) is a lesion-specific DNA repair enzyme present in certain bacteria and archaea. To date, it remains unclear how EndoQ recognizes damaged bases. Understanding the mechanism of substrate recognition by EndoQ is important to grasp genome maintenance systems in organisms with EndoQ. Here, we find that EndoQ from the euryarchaeon Pyrococcus furiosus recognizes the imide structure in nucleobases by base flipping, and the cleavage activity is enhanced by the base pair instability of the target base, along with the discovery of its cleavage activity toward 5,6-dihydrouracil, 5-hydroxyuracil, 5-hydroxycytosine, and uridine in DNA. Furthermore, a potential role of EndoQ in Bacillus subtilis as an antiviral enzyme by digesting viral genome is demonstrated.

Structural basis of cofactor-mediated stabilization and substrate recognition of the α-tubulin acetyltransferase αTAT1

2015 ◽  
Vol 467 (1) ◽  
pp. 103-113 ◽  
Author(s):  
Satoru Yuzawa ◽  
Sachiko Kamakura ◽  
Junya Hayase ◽  
Hideki Sumimoto
Keyword(s):  
Catalytic Domain ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Stable Complex ◽  
Amino Acid Residues ◽  
Substrate Selection ◽  
Post Translational Modification ◽  
Acetyl Group

The functions of microtubules are controlled in part by tubulin post-translational modification including acetylation of Lys40 in α-tubulin. αTAT1 (α-tubulin acetyltransferase 1), an enzyme evolutionarily conserved among eukaryotes, has recently been identified as the major α-tubulin Lys40 acetyltransferase, in which AcCoA (acetyl-CoA) serves as an acetyl group donor. The regulation and substrate recognition of this enzyme, however, have not been fully understood. In the present study, we show that AcCoA and CoA each form a stable complex with human αTAT1 to maintain the protein integrity both in vivo and in vitro. The invariant residues Arg132 and Ser160 in αTAT1 participate in the stable interaction not only with AcCoA but also with CoA, which is supported by analysis of the present crystal structures of the αTAT1 catalytic domain in complex with CoA. Alanine substitution for Arg132 or Ser160 leads to a drastic misfolding of the isolated αTAT1 catalytic domain in the absence of CoA and AcCoA but not in the presence of excess amounts of either cofactor. A mutant αTAT1 carrying the R132A or S160A substitution is degraded much faster than the wild-type protein when expressed in mammalian Madin–Darby canine kidney cells. Furthermore, alanine-scanning experiments using Lys40-containing peptides reveal that α-tubulin Ser38 is crucial for substrate recognition of αTAT1, whereas Asp39, Ile42, the glycine stretch (amino acid residues 43–45) and Asp46 are also involved. The requirement for substrate selection is totally different from that in various histone acetyltransferases, which appears to be consistent with the inability of αTAT1 to acetylate histones.

Structural basis for the dimerization-dependent CRISPR-Cas12f nuclease

2020 ◽  
Author(s):  
Renjian Xiao ◽  
Zhuang Li ◽  
Shukun Wang ◽  
Ruijie Han ◽  
Leifu Chang
Keyword(s):  
Genome Editing ◽  
Conformational Changes ◽  
Dna Cleavage ◽  
Substrate Recognition ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Target Dna ◽  
Type V ◽  
Underlying Substrate

ABSTRACTCas12f, also known as Cas14, is an exceptionally small type V-F CRISPR-Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 Å and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR-Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.

scholarly journals Assembly and cryo-EM structures of RNA-specific measles virus nucleocapsids provide mechanistic insight into paramyxoviral replication

2019 ◽  
Vol 116 (10) ◽  
pp. 4256-4264 ◽  
Author(s):  
Ambroise Desfosses ◽  
Sigrid Milles ◽  
Malene Ringkjøbing Jensen ◽  
Serafima Guseva ◽  
Jacques-Philippe Colletier ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Measles Virus ◽  
Rna Binding ◽  
Structural Basis ◽  
Particle Assembly ◽  
Interaction Sites ◽  
Assembly Kinetics ◽  
High Resolution Structure ◽  
Binding Groove

Assembly of paramyxoviral nucleocapsids on the RNA genome is an essential step in the viral cycle. The structural basis of this process has remained obscure due to the inability to control encapsidation. We used a recently developed approach to assemble measles virus nucleocapsid-like particles on specific sequences of RNA hexamers (poly-Adenine and viral genomic 5′) in vitro, and determined their cryoelectron microscopy maps to 3.3-Å resolution. The structures unambiguously determine 5′ and 3′ binding sites and thereby the binding-register of viral genomic RNA within nucleocapsids. This observation reveals that the 3′ end of the genome is largely exposed in fully assembled measles nucleocapsids. In particular, the final three nucleotides of the genome are rendered accessible to the RNA-dependent RNA polymerase complex, possibly enabling efficient RNA processing. The structures also reveal local and global conformational changes in the nucleoprotein upon assembly, in particular involving helix α6 and helix α13 that form edges of the RNA binding groove. Disorder is observed in the bound RNA, localized at one of the two backbone conformational switch sites. The high-resolution structure allowed us to identify putative nucleobase interaction sites in the RNA-binding groove, whose impact on assembly kinetics was measured using real-time NMR. Mutation of one of these sites, R195, whose sidechain stabilizes both backbone and base of a bound nucleic acid, is thereby shown to be essential for nucleocapsid-like particle assembly.

scholarly journals Two Human Orthologues of Eco1/Ctf7 Acetyltransferases Are Both Required for Proper Sister-Chromatid Cohesion

2005 ◽  
Vol 16 (8) ◽  
pp. 3908-3918 ◽  
Author(s):  
Fajian Hou ◽  
Hui Zou
Keyword(s):  
Sister Chromatid ◽  
Cell Cycle Regulation ◽  
Sister Chromatid Cohesion ◽  
Genetic Studies ◽  
Conserved Domain ◽  
Chromatid Cohesion ◽  
The Difference ◽  
Cycle Regulation ◽  
Acetyltransferase Domain

Genetic studies in yeast and Drosophila have uncovered a conserved acetyltransferase involved in sister-chromatid cohesion. Here, we described the two human orthologues, previously named EFO1/ESCO1 and EFO2/ESCO2. Similar to their yeast (Eco1/Ctf7 and Eso1) and fly (deco) counterparts, both proteins feature a conserved C-terminal domain consisting of a H2C2 zinc finger motif and an acetyltransferase domain that is able to catalyze autoacetylation reaction in vitro. However, no similarity can be detected outside of the conserved domain. RNA interference depletion experiment revealed that EFO1/ESCO1 and EFO2/ESCO2 were not redundant and that both were required for proper sister-chromatid cohesion. The difference between EFO1 and EFO2 also is reflected in their cell cycle regulation. In mitosis, EFO1 is phosphorylated, whereas EFO2 is degraded. Furthermore, both proteins associate with chromosomes, and the chromosome binding depends on the diverse N-terminal domains. We propose that EFO1 and EFO2 are targeted to different chromosome structures to help establish or maintain sister-chromatid cohesion.

scholarly journals The acetyltransferase activity of San stabilizes the mitotic cohesin at the centromeres in a shugoshin-independent manner

2007 ◽  
Vol 177 (4) ◽  
pp. 587-597 ◽  
Author(s):  
Fajian Hou ◽  
Chih-Wen Chu ◽  
Xiangduo Kong ◽  
Kyoko Yokomori ◽  
Hui Zou
Keyword(s):  
Cell Cycle ◽  
Sister Chromatid ◽  
Sister Chromatid Cohesion ◽  
Independent Manner ◽  
Acetyltransferase Activity ◽  
Chromatid Cohesion ◽  
Interphase Cells ◽  
Gain Access

Proper sister chromatid cohesion is critical for maintaining genetic stability. San is a putative acetyltransferase that is important for sister chromatid cohesion in Drosophila melanogaster, but not in budding yeast. We showed that San is critical for sister chromatid cohesion in HeLa cells, suggesting that this mechanism may be conserved in metazoans. Furthermore, although a small fraction of San interacts with the NatA complex, San appears to mediate cohesion independently. San exhibits acetyltransferase activity in vitro, and its activity is required for sister chromatid cohesion in vivo. In the absence of San, Sgo1 localizes correctly throughout the cell cycle. However, cohesin is no longer detected at the mitotic centromeres. Furthermore, San localizes to the cytoplasm in interphase cells; thus, it may not gain access to chromosomes until mitosis. Moreover, in San-depleted cells, further depletion of Plk1 rescues the cohesion along the chromosome arms, but not at the centromeres. Collectively, San may be specifically required for the maintenance of the centromeric cohesion in mitosis.

scholarly journals Structural basis of adenylyl cyclase 9 activation

2021 ◽  
Author(s):  
Chao Qi ◽  
Pia Lavriha ◽  
Ved Mehta ◽  
Basavraj Khanppnavar ◽  
Inayathulla Mohammed ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Binding Site ◽  
Adenylyl Cyclase ◽  
Conformational Changes ◽  
Structural Basis ◽  
Structural Studies ◽  
Nucleotide Analogue ◽  
C Terminus ◽  
Membrane Bound ◽  
A Site

Adenylyl cyclase 9 (AC9) is a membrane-bound enzyme that converts ATP into cAMP. The enzyme is weakly activated by forskolin, fully activated by the G protein Gαs subunit and is autoinhibited by the AC9 C-terminus. Although our recent structural studies of the AC9-Gαs complex provided the framework for understanding AC9 autoinhibition, the conformational changes that AC9 undergoes in response to activator binding remains poorly understood. Here, we present the cryo-EM structures of AC9 in several distinct states: (i) AC9 bound to a nucleotide inhibitor MANT-GTP, (ii) bound to an artificial activator (DARPin C4) and MANT-GTP, (iii) bound to DARPin C4 and a nucleotide analogue ATPαS, (iv) bound to Gαs and MANT-GTP. The artificial activator DARPin C4 partially activates AC9 by binding at a site that overlaps with the Gαs binding site. Together with the previously observed occluded and forskolin-bound conformations, structural comparisons of AC9 in the four new conformations show that secondary structure rearrangements in the region surrounding the forskolin binding site are essential for AC9 activation.

scholarly journals Molecular basis for the ATPase-powered substrate translocation by the Lon AAA+ protease

2020 ◽  
Author(s):  
Kaiming Zhang ◽  
Shanshan Li ◽  
Kan-Yen Hsieh ◽  
Shih-Chieh Su ◽  
Grigore D. Pintilie ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Conformational Changes ◽  
Molecular Basis ◽  
Structural Basis ◽  
Cryo Electron Microscopy ◽  
Adenosine Triphosphatases ◽  
Translocation Mechanism ◽  
Atp Binding And Hydrolysis ◽  
Proteolytic Chamber ◽  
Specific Nucleotide

AbstractThe Lon AAA+ (adenosine triphosphatases associated with diverse cellular activities) protease (LonA) converts ATP-fuelled conformational changes into sufficient mechanical force to drive translocation of the substrate into a hexameric proteolytic chamber. To understand the structural basis for the substrate translocation process, we have determined the cryo-electron microscopy (cryo-EM) structure of Meiothermus taiwanensis LonA (MtaLonA) at 3.6 Å resolution in a substrate-engaged state. Substrate interactions are mediated by the dual pore-loops of the ATPase domains, organized in spiral staircase arrangement from four consecutive protomers in different ATP-binding and hydrolysis states; a closed AAA+ ring is nevertheless maintained by two disengaged ADP-bound protomers transiting between the lowest and highest position. The structure reveals a processive rotary translocation mechanism mediated by LonA-specific nucleotide-dependent allosteric coordination among the ATPase domains, which is induced by substrate binding.

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