scholarly journals Insights into EB1 structure and the role of its C-terminal domain for discriminating microtubule tips from the lattice

2011 ◽  
Vol 22 (16) ◽  
pp. 2912-2923 ◽  
Author(s):  
Rubén M. Buey ◽  
Renu Mohan ◽  
Kris Leslie ◽  
Thomas Walzthoeni ◽  
John H. Missimer ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Binding Proteins ◽  
Coiled Coil ◽  
Actin Binding ◽  
Terminal Domain ◽  
X Ray Scattering ◽  
C Terminus ◽  
Stable Association ◽  
Chemical Cross Linking

End-binding proteins (EBs) comprise a conserved family of microtubule plus end–tracking proteins. The concerted action of calponin homology (CH), linker, and C-terminal domains of EBs is important for their autonomous microtubule tip tracking, regulation of microtubule dynamics, and recruitment of numerous partners to microtubule ends. Here we report the detailed structural and biochemical analysis of mammalian EBs. Small-angle X-ray scattering, electron microscopy, and chemical cross-linking in combination with mass spectrometry indicate that EBs are elongated molecules with two interacting CH domains, an arrangement reminiscent of that seen in other microtubule- and actin-binding proteins. Removal of the negatively charged C-terminal tail did not affect the overall conformation of EBs; however, it increased the dwell times of EBs on the microtubule lattice in microtubule tip–tracking reconstitution experiments. An even more stable association with the microtubule lattice was observed when the entire negatively charged C-terminal domain of EBs was replaced by a neutral coiled-coil motif. In contrast, the interaction of EBs with growing microtubule tips was not significantly affected by these C-terminal domain mutations. Our data indicate that long-range electrostatic repulsive interactions between the C-terminus and the microtubule lattice drive the specificity of EBs for growing microtubule ends.

The Role of Actin-Binding Proteins Vinculin, Filamin, and Fibronectin in Intracellular and Intercellular Linkages in Cardiac Muscle

1985 ◽  
pp. 215-221 ◽  
Author(s):  
Victor E. Koteliansky ◽  
Vladimir P. Shirinsky ◽  
Gennady N. Gneushev ◽  
Michail A. Chernousov
Keyword(s):  
Cardiac Muscle ◽  
Binding Proteins ◽  
Actin Binding ◽  
Actin Binding Proteins

scholarly journals Molecular basis of the dual role of the Mlh1-Mlh3 endonuclease in MMR and in meiotic crossover formation

2021 ◽  
Vol 118 (23) ◽  
pp. e2022704118
Author(s):  
Jingqi Dai ◽  
Aurore Sanchez ◽  
Céline Adam ◽  
Lepakshi Ranjha ◽  
Giordano Reginato ◽  
...  
Keyword(s):  
Meiotic Recombination ◽  
Molecular Basis ◽  
Crystal Packing ◽  
Critical Role ◽  
Holliday Junctions ◽  
Dual Roles ◽  
Terminal Domain ◽  
C Terminus ◽  
Meiotic Crossover

In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ’s dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.

Role of actin-binding proteins in cytoskeletal dynamics

1991 ◽  
Vol 19 (4) ◽  
pp. 1016-1020 ◽  
Author(s):  
A. G. Weeds ◽  
J. Gooch ◽  
M. Hawkins ◽  
B. Pope ◽  
M. Way
Keyword(s):  
Binding Proteins ◽  
Actin Binding ◽  
Actin Binding Proteins ◽  
Cytoskeletal Dynamics

Withaferin A binds covalently to the N-terminal domain of annexin A2

2012 ◽  
Vol 393 (10) ◽  
pp. 1151-1163 ◽  
Author(s):  
Gabriel Ozorowski ◽  
Christopher M. Ryan ◽  
Julian P. Whitelegge ◽  
Hartmut Luecke
Keyword(s):  
Actin Polymerization ◽  
Inhibitory Effect ◽  
Annexin A2 ◽  
Actin Dynamics ◽  
Withaferin A ◽  
Core Domain ◽  
Cancer Pathways ◽  
Terminal Domain ◽  
C Terminus

Abstract Annexin A2 (AnxA2), a 38-kDa member of the Ca2+-binding annexin family, has been implicated in numerous cancer pathways. Withaferin A (WithfA), a natural plant compound, has been reported previously to bind covalently to Cys133 of the AnxA2 core domain leading to a reduction of the invasive capabilities of cancer cells by altering their cytoskeleton. We show here that AnxA2 has an inhibitory effect on actin polymerization, and a modification with WithfA significantly increases this inhibitory role of AnxA2. Using mass spectrometry and single-site mutants, we localized the WithfA-AnxA2 interaction to the N-terminal domain of AnxA2 where WithfA binds covalently to Cys9. Whereas binding to F-actin filaments has been mapped to the C terminus of AnxA2, our results suggest that the N-terminal domain modified by WithfA may also play a role in the AnxA2-actin interaction. The binding of WithfA may regulate the AnxA2-mediated actin dynamics in two distinct ways: (i) the increase of F-actin bundling activity by the Anx2/p11 heterotetramer and (ii) the decrease of actin polymerization as a result of the increased affinity of AnxA2 to the barbed end of actin microfilaments. We demonstrate the susceptibility of Cys9 of AnxA2 to chemical modifications and exclude Cys133 as a binding site for WithfA.

scholarly journals The Dense-Core Vesicle Maturation Protein CCCP-1 Binds RAB-2 and Membranes through its C-terminal Domain

2017 ◽  
Author(s):  
Jérôme Cattin-Ortolá ◽  
Irini Topalidou ◽  
Annie Dosey ◽  
Alexey J. Merz ◽  
Michael Ailion
Keyword(s):  
Structure Function ◽  
Function Analysis ◽  
Coiled Coil ◽  
Lipid Binding ◽  
Small Gtpase ◽  
Dense Core ◽  
Binding Motif ◽  
Dense Core Vesicle ◽  
Terminal Domain ◽  
C Terminus

AbstractDense-core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled-coil protein CCCP-1 was identified as a component of the DCV biogenesis pathway in the nematode C. elegans. CCCP-1 binds the small GTPase RAB-2 and colocalizes with it at the trans-Golgi. Here we report a structure-function analysis of CCCP-1 to identify domains of the protein important for its localization, binding to RAB-2, and function in DCV biogenesis. We find that the CCCP-1 C-terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP-1 localization and for binding to RAB-2, and is required for the function of CCCP-1 in DCV biogenesis. Additionally, CCCP-1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid-binding motif. We conclude that CCCP-1 is a coiled-coil protein that binds an activated Rab and localizes to the Golgi via its C-terminus, properties similar to members of the golgin family of proteins. CCCP-1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.Synopsis statementCCCP-1 is a coiled-coil protein important for dense-core vesicle (DCV) biogenesis. A structure-function analysis of CCCP-1 shows that its C-terminal domain is required for (1) localization to membrane compartments near the trans-Golgi, (2) binding to activated RAB-2, (3) function in DCV biogenesis, and (4) direct binding to membranes. CCCP-1 has an elongated shape and forms oligomers. These findings suggest that CCCP-1 resembles members of the golgin family of proteins that act as membrane tethers.

scholarly journals Molecular basis of F-actin regulation and sarcomere assembly via myotilin

PLoS Biology ◽  
2021 ◽  
Vol 19 (4) ◽  
pp. e3001148
Author(s):  
Julius Kostan ◽  
Miha Pavšič ◽  
Vid Puž ◽  
Thomas C. Schwarz ◽  
Friedel Drepper ◽  
...  
Keyword(s):  
Structural Model ◽  
Structural Integrity ◽  
Striated Muscle ◽  
Actin Binding ◽  
Conformational Ensembles ◽  
X Ray Scattering ◽  
Sarcomere Assembly ◽  
Flanking Regions ◽  
Competition Assays

Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs—the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin’s structural role in Z-discs.

Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation

1991 ◽  
Vol 99 (4) ◽  
pp. 823-836
Author(s):  
S.J. Atkinson ◽  
M. Stewart
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Coiled Coil ◽  
Periodic Variation ◽  
Heptad Repeat ◽  
C Terminus ◽  
N Terminus ◽  
Low Ionic Strength ◽  
Myosin Rod

We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM. The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule. Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M. Constructs in which the ‘skip’ residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly. Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region. The failure of deletions from the molecule's N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin.

Acute cell volume changes in anisotonic media affect F-actin content of HL-60 cells

1991 ◽  
Vol 261 (6) ◽  
pp. C1154-C1161 ◽  
Author(s):  
K. R. Hallows ◽  
C. H. Packman ◽  
P. A. Knauf
Keyword(s):  
Cell Volume ◽  
Volume Regulation ◽  
Binding Proteins ◽  
Critical Concentration ◽  
Inverse Function ◽  
Actin Binding ◽  
Absolute Amount ◽  
Actin Assembly ◽  
Volume Changes

To investigate the possible role of the cytoskeleton in volume regulatory responses of human promyelocytic leukemic (HL-60) cells, we monitored and modulated the F-actin content of these cells undergoing volume regulation in anisotonic media. Initial volume changes of HL-60 cells suspended in hypertonic media followed a Van't Hoff relationship, and intracellular F-actin content during volume regulatory responses in anisotonic media changed concomitantly as an inverse function of the volume shifts. These F-actin changes were shown to be an explicit function of cell volume and not tonicity of the medium. The data fit with the idea that changes in affinity of actin-binding proteins (ABPs) for actin and/or changes in the overall effective critical concentration of actin occur during acute cell volume changes, producing shifts in the relative amounts of G- and F-actin. Treatment of HL-60 cells with dihydrocytochalasin B (DHB), which perturbs cellular actin assembly, lowered resting levels of intracellular F-actin but did not prevent volume-associated F-actin changes in anisotonic media. Despite the lowered F-actin levels, HL-60 cells in the presence of DHB still undergo normal volume regulatory responses. Thus the absolute amount of intracellular F-actin does not appear to be critical for volume regulation in HL-60 cells.

scholarly journals Structural characterization suggests models for monomeric and dimeric forms of full-length ezrin

2016 ◽  
Vol 473 (18) ◽  
pp. 2763-2782 ◽  
Author(s):  
Juanita M. Phang ◽  
Stephen J. Harrop ◽  
Anthony P. Duff ◽  
Anna V. Sokolova ◽  
Ben Crossett ◽  
...  
Keyword(s):  
Active Form ◽  
Coiled Coil ◽  
Full Length ◽  
Heptad Repeat ◽  
Actin Binding ◽  
Stable Complex ◽  
Globular Domain ◽  
Erm Proteins ◽  
Metazoan Evolution ◽  
Terminal Domain

Ezrin is a member of the ERM (ezrin–radixin–moesin) family of proteins that have been conserved through metazoan evolution. These proteins have dormant and active forms, where the latter links the actin cytoskeleton to membranes. ERM proteins have three domains: an N-terminal FERM [band Four-point-one (4.1) ERM] domain comprising three subdomains (F1, F2, and F3); a helical domain; and a C-terminal actin-binding domain. In the dormant form, FERM and C-terminal domains form a stable complex. We have determined crystal structures of the active FERM domain and the dormant FERM:C-terminal domain complex of human ezrin. We observe a bistable array of phenylalanine residues in the core of subdomain F3 that is mobile in the active form and locked in the dormant form. As subdomain F3 is pivotal in binding membrane proteins and phospholipids, these transitions may facilitate activation and signaling. Full-length ezrin forms stable monomers and dimers. We used small-angle X-ray scattering to determine the solution structures of these species. As expected, the monomer shows a globular domain with a protruding helical coiled coil. The dimer shows an elongated dumbbell structure that is twice as long as the monomer. By aligning ERM sequences spanning metazoan evolution, we show that the central helical region is conserved, preserving the heptad repeat. Using this, we have built a dimer model where each monomer forms half of an elongated antiparallel coiled coil with domain-swapped FERM:C-terminal domain complexes at each end. The model suggests that ERM dimers may bind to actin in a parallel fashion.

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