A folded conformation of MukBEF and Cohesin

AbstractStructural maintenance of chromosomes (SMC)-kleisin complexes organize chromosomal DNAs in all domains of life, where they have key roles in chromosome segregation, DNA repair and regulation of gene expression. They function through topological entrapment and active translocation of DNA, but the underlying conformational changes are largely unclear. Using structural biology, mass spectrometry and cross-linking, we investigated the architecture of two evolutionarily distant SMC-kleisin complexes: proteobacterial MukBEF and eukaryotic cohesin. We show that both contain a dynamic coiled-coil discontinuity, the elbow, near the middle of their arms that permits a folded conformation. Bending at the elbow brings into proximity the hinge dimerization domain and the head/kleisin module, situated at opposite ends of the arms. Our findings favor SMC activity models that include a large conformational change in the arms, such as a relative movement between DNA contact sites during DNA loading and translocation.

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Suppressor screening reveals common kleisin–hinge interaction in condensin and cohesin, but different modes of regulation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1902699116 ◽

2019 ◽

Vol 116 (22) ◽

pp. 10889-10898 ◽

Cited By ~ 6

Author(s):

Xingya Xu ◽

Mitsuhiro Yanagida

Keyword(s):

Chromatin Remodeling ◽

Chromosome Segregation ◽

Coiled Coil ◽

Coiled Coils ◽

Whole Genome ◽

Chromatid Cohesion ◽

Suppression Mechanism ◽

Elimination Pathway ◽

Extragenic Suppressors ◽

Structural Maintenance Of Chromosomes

Cohesin and condensin play fundamental roles in sister chromatid cohesion and chromosome segregation, respectively. Both consist of heterodimeric structural maintenance of chromosomes (SMC) subunits, which possess a head (containing ATPase) and a hinge, intervened by long coiled coils. Non-SMC subunits (Cnd1, Cnd2, and Cnd3 for condensin; Rad21, Psc3, and Mis4 for cohesin) bind to the SMC heads. Here, we report a large number of spontaneous extragenic suppressors for fission yeast condensin and cohesin mutants, and their sites were determined by whole-genome sequencing. Mutants of condensin’s non-SMC subunits were rescued by impairing the SUMOylation pathway. Indeed, SUMOylation of Cnd2, Cnd3, and Cut3 occurs in midmitosis, and Cnd3 K870 SUMOylation functionally opposes Cnd subunits. In contrast, cohesin mutants rad21 and psc3 were rescued by loss of the RNA elimination pathway (Erh1, Mmi1, and Red1), and loader mutant mis4 was rescued by loss of Hrp1-mediated chromatin remodeling. In addition, distinct regulations were discovered for condensin and cohesin hinge mutants. Mutations in the N-terminal helix bundle [containing a helix–turn–helix (HTH) motif] of kleisin subunits (Cnd2 and Rad21) rescue virtually identical hinge interface mutations in cohesin and condensin, respectively. These mutations may regulate kleisin’s interaction with the coiled coil at the SMC head, thereby revealing a common, but previously unknown, suppression mechanism between the hinge and the kleisin N domain, which is required for successful chromosome segregation. We propose that in both condensin and cohesin, the head (or kleisin) and hinge may interact and collaboratively regulate the resulting coiled coils to hold and release chromosomal DNAs.

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A rod conformation of the Pyrococcus furiosus Rad50 coiled coil

10.1101/2020.06.24.160820 ◽

2020 ◽

Author(s):

Young-Min Soh ◽

Jerome Basquin ◽

Stephan Gruber

Keyword(s):

Pyrococcus Furiosus ◽

Coiled Coil ◽

Vital Role ◽

Coiled Coils ◽

Double Strand Breaks ◽

Dna Double Strand Breaks ◽

Dimerization Domain ◽

Strand Breaks ◽

Domains Of Life ◽

Bacterial Homolog

AbstractThe Rad50-Mre11 nuclease complex plays a vital role in DNA repair in all domains of life. It recognizes and processes DNA double-strand breaks. Rad50 proteins fold into an extended structure with a ~20-60 nm long coiled coil connecting a globular ABC ATPase domain with a zinc hook dimerization domain. A published structure of an archaeal Rad50 zinc hook shows coiled coils pointing away from each other. Here we present the crystal structure of an alternate conformation displaying co-aligned coiled coils. Archaeal Rad50 may thus switch between rod-shaped and ring-like conformations as recently proposed for a bacterial homolog.

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Dynamic architecture of the Escherichia coli structural maintenance of chromosomes (SMC) complex, MukBEF

Nucleic Acids Research ◽

10.1093/nar/gkz696 ◽

2019 ◽

Vol 47 (18) ◽

pp. 9696-9707 ◽

Cited By ~ 4

Author(s):

Karthik V Rajasekar ◽

Rachel Baker ◽

Gemma L M Fisher ◽

Jani R Bolla ◽

Jarno Mäkelä ◽

...

Keyword(s):

Escherichia Coli ◽

Chromosome Segregation ◽

Conformational Changes ◽

Atp Hydrolysis ◽

Nucleotide Binding ◽

Dynamic Architecture ◽

Protein Conformational Changes ◽

C And N ◽

Atp Binding And Hydrolysis ◽

Structural Maintenance Of Chromosomes

Abstract Ubiquitous Structural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organize and individualize chromosomes, thereby facilitating chromosome segregation. They utilize cycles of adenosine triphosphate (ATP) binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contact sites. By analysing changes in the architecture and stoichiometry of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis, we demonstrate directly the formation of dimer of MukBEF dimer complexes, dependent on dimeric MukF kleisin. Using truncated and full length MukB, in combination with MukEF, we show that engagement of the MukB ATPase heads on nucleotide binding directs the formation of dimers of heads-engaged dimer complexes. Complex formation requires functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively, and MukE, which organizes the complexes by stabilizing binding of MukB heads to MukF. In the absence of head engagement, a MukF dimer bound by MukE forms complexes containing only a dimer of MukB. Finally, we demonstrate that cells expressing MukBEF complexes in which MukF is monomeric are Muk−, with the complexes failing to associate with chromosomes.

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Dynamic architecture of the Escherichia coli Structural Maintenance of Chromosomes (SMC) complex, MukBEF

10.1101/547786 ◽

2019 ◽

Cited By ~ 2

Author(s):

Karthik V. Rajasekar ◽

Minzhe Tang ◽

Rachel Baker ◽

Katarzyna Zawadzka ◽

Oliwia Koczy ◽

...

Keyword(s):

Escherichia Coli ◽

Chromosome Segregation ◽

Conformational Changes ◽

Atp Hydrolysis ◽

Dynamic Architecture ◽

Functional Interactions ◽

Protein Conformational Changes ◽

C And N ◽

Atp Binding And Hydrolysis ◽

Structural Maintenance Of Chromosomes

AbstractStructural Maintenance of Chromosomes (SMC) complexes use a proteinaceous ring-shaped architecture to organise chromosomes, thereby facilitating chromosome segregation. They utilise cycles of ATP binding and hydrolysis to transport themselves rapidly with respect to DNA, a process requiring protein conformational changes and multiple DNA contacts. We have analysed changes in the architecture of the Escherichia coli SMC complex, MukBEF, as a function of nucleotide binding to MukB and subsequent ATP hydrolysis. This builds upon previous work showing that MukF kleisin directs formation of a MukBEF tripartite ring as a consequence of functional interactions between the C- and N-terminal domains of MukF with the MukB head and neck, respectively (Zawadzka et al., 2018). Using both model truncated substrates and complexes containing full length MukB, we now demonstrate formation of MukBEF ‘dimers of dimers’, dependent on MukF dimerization, MukB head-engagement and MukE, which plays an essential role in organizing MukBEF complexes.

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Myosinome: A Database of Myosins from Select Eukaryotic Genomes to Facilitate Analysis of Sequence-Structure-Function Relationships

Bioinformatics and Biology Insights ◽

10.4137/bbi.s9902 ◽

2012 ◽

Vol 6 ◽

pp. BBI.S9902 ◽

Cited By ~ 3

Author(s):

Divya P. Syamaladevi ◽

Margaret S Sunitha ◽

S. Kalaimathy ◽

Chandrashekar C. Reddy ◽

Mohammed Iftekhar ◽

...

Keyword(s):

Conformational Changes ◽

Atp Hydrolysis ◽

Homo Sapiens ◽

Relevant Literature ◽

Myosin Ii ◽

Coiled Coil ◽

Structural Features ◽

Model Organisms ◽

Congenital Diseases ◽

C Elegans

Myosins are one of the largest protein superfamilies with 24 classes. They have conserved structural features and catalytic domains yet show huge variation at different domains resulting in a variety of functions. Myosins are molecules driving various kinds of cellular processes and motility until the level of organisms. These are ATPases that utilize the chemical energy released by ATP hydrolysis to bring about conformational changes leading to a motor function. Myosins are important as they are involved in almost all cellular activities ranging from cell division to transcriptional regulation. They are crucial due to their involvement in many congenital diseases symptomatized by muscular malfunctions, cardiac diseases, deafness, neural and immunological dysfunction, and so on, many of which lead to death at an early age. We present Myosinome, a database of selected myosin classes (myosin II, V, and VI) from five model organisms. This knowledge base provides the sequences, phylogenetic clustering, domain architectures of myosins and molecular models, structural analyses, and relevant literature of their coiled-coil domains. In the current version of Myosinome, information about 71 myosin sequences belonging to three myosin classes (myosin II, V, and VI) in five model organisms ( Homo Sapiens, Mus musculus, D. melanogaster, C. elegans and S. cereviseae) identified using bioinformatics surveys are presented, and several of them are yet to be functionally characterized. As these proteins are involved in congenital diseases, such a database would be useful in short-listing candidates for gene therapy and drug development. The database can be accessed from http://caps.ncbs.res.in/myosinome .

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Assembly of the elongated collagen prolyl 4-hydroxylase α2β2 heterotetramer around a central α2 dimer

Biochemical Journal ◽

10.1042/bcj20161000 ◽

2017 ◽

Vol 474 (5) ◽

pp. 751-769 ◽

Cited By ~ 7

Author(s):

M. Kristian Koski ◽

Jothi Anantharajan ◽

Petri Kursula ◽

Prathusha Dhavala ◽

Abhinandan V. Murthy ◽

...

Keyword(s):

Three Dimensional ◽

Coiled Coil ◽

Β Subunit ◽

Dimerization Domain ◽

Α Subunit ◽

Protein Dimer ◽

Symmetric Molecule ◽

Asymmetric Shape ◽

Proline Rich Peptide ◽

Α Subunits

Collagen prolyl 4-hydroxylase (C-P4H), an α2β2 heterotetramer, is a crucial enzyme for collagen synthesis. The α-subunit consists of an N-terminal dimerization domain, a central peptide substrate-binding (PSB) domain, and a C-terminal catalytic (CAT) domain. The β-subunit [also known as protein disulfide isomerase (PDI)] acts as a chaperone, stabilizing the functional conformation of C-P4H. C-P4H has been studied for decades, but its structure has remained elusive. Here, we present a three-dimensional small-angle X-ray scattering model of the entire human C-P4H-I heterotetramer. C-P4H is an elongated, bilobal, symmetric molecule with a length of 290 Å. The dimerization domains from the two α-subunits form a protein–protein dimer interface, assembled around the central antiparallel coiled-coil interface of their N-terminal α-helices. This region forms a thin waist in the bilobal tetramer. The two PSB/CAT units, each complexed with a PDI/β-subunit, form two bulky lobes pointing outward from this waist region, such that the PDI/β-subunits locate at the far ends of the βααβ complex. The PDI/β-subunit interacts extensively with the CAT domain. The asymmetric shape of two truncated C-P4H-I variants, also characterized in the present study, agrees with this assembly. Furthermore, data from these truncated variants show that dimerization between the α-subunits has an important role in achieving the correct PSB–CAT assembly competent for catalytic activity. Kinetic assays with various proline-rich peptide substrates and inhibitors suggest that, in the competent assembly, the PSB domain binds to the procollagen substrate downstream from the CAT domain.

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New Insights into the Spring-Loaded Conformational Change of Influenza Virus Hemagglutinin

Journal of Virology ◽

10.1128/jvi.76.9.4456-4466.2002 ◽

2002 ◽

Vol 76 (9) ◽

pp. 4456-4466 ◽

Cited By ~ 42

Author(s):

Jennifer A. Gruenke ◽

R. Todd Armstrong ◽

William W. Newcomb ◽

Jay C. Brown ◽

Judith M. White

Keyword(s):

Influenza Virus ◽

Conformational Change ◽

Conformational Changes ◽

Limited Proteolysis ◽

Coiled Coil ◽

Fusion Peptide ◽

Wild Type ◽

Influenza Virus Hemagglutinin ◽

Target Membrane ◽

Mutant Forms

ABSTRACT Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix “spring-loaded” conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only ∼50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.

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