scholarly journals Suppressor mutation analysis combined with 3D modeling explains cohesin’s capacity to hold and release DNA

2018 ◽  
Vol 115 (21) ◽  
pp. E4833-E4842 ◽  
Author(s):  
Xingya Xu ◽  
Ryuta Kanai ◽  
Norihiko Nakazawa ◽  
Li Wang ◽  
Chikashi Toyoshima ◽  
...  
Keyword(s):  
Functional Organization ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Dna Evidence ◽  
Ring Model ◽  
Suppressor Mutations ◽  
Sister Chromatids ◽  
Cohesin Subunit ◽  
Extragenic Suppressors ◽  
Release Model

Cohesin is a fundamental protein complex that holds sister chromatids together. Separase protease cleaves a cohesin subunit Rad21/SCC1, causing the release of cohesin from DNA to allow chromosome segregation. To understand the functional organization of cohesin, we employed next-generation whole-genome sequencing and identified numerous extragenic suppressors that overcome either inactive separase/Cut1 or defective cohesin in the fission yeast Schizosaccharomyces pombe. Unexpectedly, Cut1 is dispensable if suppressor mutations cause disorders of interfaces among essential cohesin subunits Psm1/SMC1, Psm3/SMC3, Rad21/SCC1, and Mis4/SCC2, the crystal structures of which suggest physical and functional impairment at the interfaces of Psm1/3 hinge, Psm1 head–Rad21, or Psm3 coiled coil–Rad21. Molecular-dynamics analysis indicates that the intermolecular β-sheets in the cohesin hinge of cut1 suppressor mutants remain intact, but a large mobility change occurs at the coiled coil bound to the hinge. In contrast, suppressors of rad21-K1 occur in either the head ATPase domains or the Psm3 coiled coil that interacts with Rad21. Suppressors of mis4-G1326E reside in the head of Psm3/1 or the intragenic domain of Mis4. These may restore the binding of cohesin to DNA. Evidence is provided that the head and hinge of SMC subunits are proximal, and that they coordinate to form arched coils that can hold or release DNA by altering the angles made by the arched coiled coils. By combining molecular modeling with suppressor sequence analysis, we propose a cohesin structure designated the “hold-and-release” model, which may be considered as an alternative to the prevailing “ring” model.

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

2019 ◽  
Vol 116 (22) ◽  
pp. 10889-10898 ◽  
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.

scholarly journals Folding of cohesin’s coiled coil is important for Scc2/4-induced association with chromosomes

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Naomi J Petela ◽  
Andres Gonzalez Llamazares ◽  
Sarah Dixon ◽  
Bin Hu ◽  
Byung-Gil Lee ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Entire Length ◽  
Mutant Phenotype ◽  
Physiological Importance ◽  
Sister Chromatids ◽  
Dna Loop ◽  
Smc Proteins

Cohesin’s association with and translocation along chromosomal DNAs depend on an ATP hydrolysis cycle driving the association and subsequent release of DNA. This involves DNA being ‘clamped’ by Scc2 and ATP-dependent engagement of cohesin’s Smc1 and Smc3 head domains. Scc2’s replacement by Pds5 abrogates cohesin’s ATPase and has an important role in halting DNA loop extrusion. The ATPase domains of all SMC proteins are separated from their hinge dimerisation domains by 50-nm-long coiled coils, which have been observed to zip up along their entire length and fold around an elbow, thereby greatly shortening the distance between hinges and ATPase heads. Whether folding exists in vivo or has any physiological importance is not known. We present here a cryo-EM structure of the apo form of cohesin that reveals the structure of folded and zipped-up coils in unprecedented detail and shows that Scc2 can associate with Smc1’s ATPase head even when it is fully disengaged from that of Smc3. Using cysteine-specific crosslinking, we show that cohesin’s coiled coils are frequently folded in vivo, including when cohesin holds sister chromatids together. Moreover, we describe a mutation (SMC1D588Y) within Smc1’s hinge that alters how Scc2 and Pds5 interact with Smc1’s hinge and that enables Scc2 to support loading in the absence of its normal partner Scc4. The mutant phenotype of loading without Scc4 is only explicable if loading depends on an association between Scc2/4 and cohesin’s hinge, which in turn requires coiled coil folding.

scholarly journals Differential Suppression of priA2::kan Phenotypes in Escherichia coli K-12 by Mutations in priA, lexA, and dnaC

Genetics ◽  
1996 ◽  
Vol 143 (1) ◽  
pp. 5-13 ◽  
Author(s):  
Steven J Sandler ◽  
Hardeep S Samra ◽  
Alvin J Clark
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Mutant Allele ◽  
Wild Type ◽  
Suppressor Mutations ◽  
Dnab Helicase ◽  
K 12 ◽  
Extragenic Suppressors ◽  
Assembly Activity

Abstract First identified as an essential component of the ϕX174 in vitro DNA replication system, PriA has ATPase, helicase, translocase, and primosome-assembly activities. priA1::kan strains of Escherichia coli are sensitive to UV irradiation, deficient in homologous recombination following transduction, and filamentous. priA2::kan strains have eightfold higher levels of uninduced SOS expression than wild type. We show that (1) priA1::kan strains have eightfold higher levels of uninduced SOS expression, (2) priA2::kan strains are UVS and Rec−, (3) lexA3 suppresses the high basal levels of SOS expression of a priA2::kan strain, and (4) plasmid-encoded priA300 (K230R), a mutant allele retaining only the primosome-assembly activity of priA+, restores both UVR and Rec+ phenotypes to a priA2::kan strain. Finally, we have isolated 17 independent UVR Rec+ revertants of priA2::kan strains that carry extragenic suppressors. All 17 map in the C-terminal half of the dnaC gene. DnaC loads the DnaB helicase onto DNA as a prelude for primosome assembly and DNA replication. We conclude that priA's primosome-assembly activity is essential for DNA repair and recombination and that the dnaC suppressor mutations allow these processes to occur in the absence of priA.

scholarly journals Coiled-coil networking shapes cell molecular machinery

2012 ◽  
Vol 23 (19) ◽  
pp. 3911-3922 ◽  
Author(s):  
Yongqiang Wang ◽  
Xinlei Zhang ◽  
Hong Zhang ◽  
Yi Lu ◽  
Haolong Huang ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Critical Role ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Protein Protein Interactions ◽  
Full Spectrum ◽  
Kinetochore Assembly ◽  
Genome Wide ◽  
Molecular Machinery ◽  
Systematic Understanding

The highly abundant α-helical coiled-coil motif not only mediates crucial protein–protein interactions in the cell but is also an attractive scaffold in synthetic biology and material science and a potential target for disease intervention. Therefore a systematic understanding of the coiled-coil interactions (CCIs) at the organismal level would help unravel the full spectrum of the biological function of this interaction motif and facilitate its application in therapeutics. We report the first identified genome-wide CCI network in Saccharomyces cerevisiae, which consists of 3495 pair-wise interactions among 598 predicted coiled-coil regions. Computational analysis revealed that the CCI network is specifically and functionally organized and extensively involved in the organization of cell machinery. We further show that CCIs play a critical role in the assembly of the kinetochore, and disruption of the CCI network leads to defects in kinetochore assembly and cell division. The CCI network identified in this study is a valuable resource for systematic characterization of coiled coils in the shaping and regulation of a host of cellular machineries and provides a basis for the utilization of coiled coils as domain-based probes for network perturbation and pharmacological applications.

scholarly journals ARCIMBOLDOon coiled coils

2018 ◽  
Vol 74 (3) ◽  
pp. 194-204 ◽  
Author(s):  
Iracema Caballero ◽  
Massimo Sammito ◽  
Claudia Millán ◽  
Andrey Lebedev ◽  
Nicolas Soler ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Coiled Coils ◽  
Translational Symmetry ◽  
Command Line ◽  
Phase Problem ◽  
Final Solution ◽  
Resolution Limit ◽  
Helical Structures ◽  
Small Model ◽  
Test Pool

ARCIMBOLDOsolves the phase problem by combining the location of small model fragments usingPhaserwith density modification and autotracing usingSHELXE. Mainly helical structures constitute favourable cases, which can be solved using polyalanine helical fragments as search models. Nevertheless, the solution of coiled-coil structures is often complicated by their anisotropic diffraction and apparent translational noncrystallographic symmetry. Long, straight helices have internal translational symmetry and their alignment in preferential directions gives rise to systematic overlap of Patterson vectors. This situation has to be differentiated from the translational symmetry relating different monomers.ARCIMBOLDO_LITEhas been run on single workstations on a test pool of 150 coiled-coil structures with 15–635 amino acids per asymmetric unit and with diffraction data resolutions of between 0.9 and 3.0 Å. The results have been used to identify and address specific issues when solving this class of structures usingARCIMBOLDO. Features fromPhaserv.2.7 onwards are essential to correct anisotropy and produce translation solutions that will pass the packing filters. As the resolution becomes worse than 2.3 Å, the helix direction may be reversed in the placed fragments. Differentiation between true solutions and pseudo-solutions, in which helix fragments were correctly positioned but in a reverse orientation, was found to be problematic at resolutions worse than 2.3 Å. Therefore, after every new fragment-placement round, complete or sparse combinations of helices in alternative directions are generated and evaluated. The final solution is once again probed by helix reversal, refinement and extension. To conclude, density modification andSHELXEautotracing incorporating helical constraints is also exploited to extend the resolution limit in the case of coiled coils and to enhance the identification of correct solutions. This study resulted in a specialized mode withinARCIMBOLDOfor the solution of coiled-coil structures, which overrides the resolution limit and can be invoked from the command line (keyword coiled_coil) orARCIMBOLDO_LITEtask interface inCCP4i.

scholarly journals α/β coiled coils

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Marcus D Hartmann ◽  
Claudia T Mendler ◽  
Jens Bassler ◽  
Ioanna Karamichali ◽  
Oswin Ridderbusch ◽  
...  
Keyword(s):  
Repeat Unit ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Heptad Repeat ◽  
Protein Fold ◽  
Local Formation ◽  
Unexpected Formation ◽  
New Type ◽  
Backbone Structure ◽  
Level Of Understanding

Coiled coils are the best-understood protein fold, as their backbone structure can uniquely be described by parametric equations. This level of understanding has allowed their manipulation in unprecedented detail. They do not seem a likely source of surprises, yet we describe here the unexpected formation of a new type of fiber by the simple insertion of two or six residues into the underlying heptad repeat of a parallel, trimeric coiled coil. These insertions strain the supercoil to the breaking point, causing the local formation of short β-strands, which move the path of the chain by 120° around the trimer axis. The result is an α/β coiled coil, which retains only one backbone hydrogen bond per repeat unit from the parent coiled coil. Our results show that a substantially novel backbone structure is possible within the allowed regions of the Ramachandran space with only minor mutations to a known fold.

scholarly journals Integration of Rotation and Piston Motions in Coiled-Coil Signal Transduction

2007 ◽  
Vol 189 (16) ◽  
pp. 6048-6056 ◽  
Author(s):  
Rong Gao ◽  
David G. Lynn
Keyword(s):  
Coiled Coil ◽  
Dynamic Environment ◽  
Coiled Coils ◽  
Signal Integration ◽  
Signaling Mechanisms ◽  
Protein Receptor ◽  
Single Protein ◽  
Environmental Responses ◽  
Multiple Signaling ◽  
Host Signals

ABSTRACT A coordinated response to a complex and dynamic environment requires an organism to simultaneously monitor and interpret multiple signaling cues. In bacteria and some eukaryotes, environmental responses depend on the histidine autokinases (HKs). For example, VirA, a large integral membrane HK from Agrobacterium tumefaciens, regulates the expression of virulence genes in response to signals from multiple molecular classes (phenol, pH, and sugar). The ability of this pathogen to perceive inputs from different known host signals within a single protein receptor provides an opportunity to understand the mechanisms of signal integration. Here we exploited the conserved domain organization of the HKs and engineered chimeric kinases to explore the signaling mechanisms of phenol sensing and pH/sugar integration. Our data implicate a piston-assisted rotation of coiled coils for integration of multiple inputs and regulation of critical responses during pathogenesis.

scholarly journals Coiled-Coils: The Molecular Zippers that Self-Assemble Protein Nanostructures

2020 ◽  
Vol 21 (10) ◽  
pp. 3584 ◽  
Author(s):  
Won Min Park
Keyword(s):  
Modular Design ◽  
Coiled Coil ◽  
Molecular Complexes ◽  
Building Blocks ◽  
Structural Features ◽  
Coiled Coils ◽  
Design Strategies ◽  
Protein Motifs ◽  
Self Assembling ◽  
Current Design

Coiled-coils, the bundles of intertwined helical protein motifs, have drawn much attention as versatile molecular toolkits. Because of programmable interaction specificity and affinity as well as well-established sequence-to-structure relationships, coiled-coils have been used as subunits that self-assemble various molecular complexes in a range of fields. In this review, I describe recent advances in the field of protein nanotechnology, with a focus on programming assembly of protein nanostructures using coiled-coil modules. Modular design approaches to converting the helical motifs into self-assembling building blocks are described, followed by a discussion on the molecular basis and principles underlying the modular designs. This review also provides a summary of recently developed nanostructures with a variety of structural features, which are in categories of unbounded nanostructures, discrete nanoparticles, and well-defined origami nanostructures. Challenges existing in current design strategies, as well as desired improvements for controls over material properties and functionalities for applications, are also provided.

Hierarchical nanomechanics of vimentin alpha helical coiled-coil proteins

2006 ◽  
Vol 978 ◽  
Author(s):  
Theodor Ackbarow ◽  
Markus J. Buehler
Keyword(s):  
Atomistic Simulation ◽  
Coiled Coil ◽  
Building Blocks ◽  
Tensile Tests ◽  
Coiled Coils ◽  
Atomistic Modeling ◽  
Alpha Helices ◽  
Hierarchical Assembly ◽  
Leading Role ◽  
Elementary Building

AbstractCoiled-coil alpha-helical dimers are the elementary building blocks of intermediate filaments (IFs), an important component of the cell's cytoskeleton. Therefore, IFs play a leading role in the mechanical integrity of the cells. Here we use atomistic simulation to carry out tensile tests on coiled-coils as well as on single alpha-helices of the 2B segment of the vimentin dimer that has been shown to control the large-deformation behavior of cells. We compare the characteristic force-strain curves of both structures and suggest explanations for the differences on this fundamental level of hierarchical assembly. We further systematically explore the strain rate dependence of the mechanical properties of the vimentin coiled-coil protein. We develop a simple continuum model capable of reproducing the atomistic modeling results. The model enables us to extrapolate to much lower deformation rates approaching those used in experiment.

scholarly journals Dynamic allostery of protein alpha helical coiled-coils

2005 ◽  
Vol 3 (6) ◽  
pp. 125-138 ◽  
Author(s):  
Rhoda J Hawkins ◽  
Tom C.B McLeish
Keyword(s):  
Free Energy ◽  
Ligand Binding ◽  
Molecular Motor ◽  
Coiled Coil ◽  
Coiled Coils ◽  
Coarse Grained ◽  
Static Structure ◽  
Change In The Mean ◽  
The Individual ◽  
Vibrational Free Energy

Alpha helical coiled-coils appear in many important allosteric proteins such as the dynein molecular motor and bacteria chemotaxis transmembrane receptors. As a mechanism for transmitting the information of ligand binding to a distant site across an allosteric protein, an alternative to conformational change in the mean static structure is an induced change in the pattern of the internal dynamics of the protein. We explore how ligand binding may change the intramolecular vibrational free energy of a coiled-coil, using parameterized coarse-grained models, treating the case of dynein in detail. The models predict that coupling of slide, bend and twist modes of the coiled-coil transmits an allosteric free energy of ∼2 k B T , consistent with experimental results. A further prediction is a quantitative increase in the effective stiffness of the coiled-coil without any change in inherent flexibility of the individual helices. The model provides a possible and experimentally testable mechanism for transmission of information through the alpha helical coiled-coil of dynein.

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