scholarly journals Architecture of the Mto1/2 microtubule nucleation complex

2019 ◽  
Author(s):  
Harish C. Thakur ◽  
Eric M. Lynch ◽  
Weronika E. Borek ◽  
Xun X. Bao ◽  
Sanju Ashraf ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Higher Order ◽  
Microtubule Nucleation ◽  
Cooperative Interactions ◽  
Dimerization Interface ◽  
Functional Version ◽  
Complex Architecture ◽  
Order Structures

ABSTRACTProteins that contain a Centrosomin Motif 1 (CM1) domain are key regulators ofγ-tubulin complex-dependent microtubule nucleation, but how they are organized in higher-order structures is largely unknown. Mto1[bonsai], a truncated functional version of theSchizosaccharomyces pombeCM1 protein Mto1, interacts with Mto2 to form an Mto1/2[bonsai] complexin vivo. Here we show that recombinant Mto1/2[bonsai] forms higher-order multimersin vitroand that Mto2 alone can also multimerize. We demonstrate that Mto2 multimerization involves two separate homodimerization domains, the near N-terminal domain (NND) and the twin-cysteine domain (TCD). The TCD crystal structure reveals a stable homodimer with a novel dimerization interface. While the NND homodimer is intrinsically less stable, using crosslinking mass spectrometry we show that within Mto1/2[bonsai] complexes, it can be reinforced by additional cooperative interactions involving both Mto2 and Mto1[bonsai]. We propose a model for Mto1/2[bonsai] complex architecture that is supported by functional analysis of mutantsin vivo.

scholarly journals LAP2alpha maintains a mobile and low assembly state of A-type lamins in the nuclear interior

2020 ◽  
Author(s):  
Nana Naetar ◽  
Konstantina Georgiou ◽  
Christian Knapp ◽  
Irena Bronshtein ◽  
Elisabeth Zier ◽  
...  
Keyword(s):  
Nuclear Periphery ◽  
Higher Order ◽  
Lamin A ◽  
Independent Manner ◽  
Nuclear Interior ◽  
Order Structures

AbstractLamins form stable filaments at the nuclear periphery in metazoans. Unlike B-type lamins, lamins A and C localize also in the nuclear interior, where they interact with lamin-associated polypeptide 2 alpha (LAP2α). We show that lamin A in the nuclear interior is formed from newly expressed pre-lamin A during processing and from soluble mitotic mature lamins in a LAP2α-independent manner. Binding of LAP2α to lamins A/C in the nuclear interior during interphase inhibits formation of higher order structures of lamin A/C in vitro and in vivo, keeping lamin A/C in a mobile low assembly state independent of lamin A/C S22 phosphorylation. Loss of LAP2α causes formation of larger, less mobile and biochemically stable lamin A/C structures in the nuclear interior, which reduce the mobility of chromatin. We propose that LAP2α is essential to maintain a mobile lamin A/C pool in the nuclear interior, which is required for proper nuclear functions.

Effects of dietary alteration of bicarbonate and magnesium on rat bone

1986 ◽  
Vol 250 (2) ◽  
pp. F302-F307 ◽  
Author(s):  
J. M. Burnell ◽  
C. Liu ◽  
A. G. Miller ◽  
E. Teubner
Keyword(s):  
Crystal Structure ◽  
Bone Formation ◽  
X Ray Diffraction ◽  
X Ray ◽  
Growing Rats ◽  
Final Composition ◽  
Rat Bone

To study the effects of bicarbonate and magnesium on bone, mild acidosis and/or hypermagnesemia were produced in growing rats by feeding ammonium chloride and/or magnesium sulfate. Bone composition, quantitative histomorphometry, and mineral x-ray diffraction (XRD) characteristics were measured after 6 wk of treatment. The results demonstrated that both acidosis (decreased HCO3) and hypermagnesemia inhibited periosteal bone formation, and, when combined, results were summative; and the previously observed in vitro role of HCO3- and Mg2+ as inhibitors of crystal growth were confirmed in vivo. XRD measurements demonstrated that decreased plasma HCO3 resulted in larger crystals and increased Mg resulted in smaller crystals. However, the combined XRD effects of acidosis and hypermagnesemia resembled acidosis alone. It is postulated that the final composition and crystal structure of bone are strongly influenced by HCO3- and Mg2+, and the effects are mediated by the combined influence on both osteoblastic bone formation and the growth of hydroxyapatite.

Ability to organize microtubules in taxol-treated mitotic PtK2 cells goes with the SPN antigen and not with the centrosome

1992 ◽  
Vol 102 (1) ◽  
pp. 91-102 ◽  
Author(s):  
M. Kallajoki ◽  
K. Weber ◽  
M. Osborn
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Nuclear Matrix ◽  
Essential Role ◽  
Soluble Form ◽  
Microtubule Nucleation ◽  
Brain Microtubules ◽  
Mitotic Cells

The SPN antigen plays an essential role in mitosis, since microinjection of antibodies causes mitotic arrest. Here we show, by examination of the relative locations of SPN antigen, the centrosomal 5051 antigen and tubulin in normal mitotic, and in taxol-treated mitotic cells, that the SPN antigen is involved in organizing the microtubules of the spindle. The 210 kDa protein defined as SPN antigen relocates from the nuclear matrix to the centrosome at prophase, remains associated with the poles at metaphase and anaphase, and dissociates from the centrosomes in telophase. In taxol-treated mitotic cells, SPN staining shows a striking redistribution while 5051 antigen remains associated with centrosomes. SPN antigen is seen at the plasma membrane end of the rearranged microtubules. SPN antigen is always at the center of the multiple microtubule asters (5 to 20 per cell) induced by taxol, whereas 5051 again remains associated with the centrosomal complex (1 to 2 foci per cell). Microtubule nucleation is associated with the SPN antigen rather than with the 5051 antigen. Microinjection of SPN-3 antibody into taxol-treated mitotic PtK2 cells causes disruption of the asters as judged by tubulin staining of the same cells. Finally, SPN antigen extracted in soluble form from synchronized mitotic HeLa cells binds to, and sediments with, pig brain microtubules stabilized by taxol. This association of SPN antigen with microtubules is partially dissociated by 0.5 M NaCl but not by 5 mM ATP. Thus SPN antigen binds to microtubules in vitro and seems to act as a microtubular minus-end organizer in mitotic cells in vivo.

scholarly journals Crystal Structure of a Variant PAM2 Motif of LARP4B Bound to the MLLE Domain of PABPC1

Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 872 ◽  
Author(s):  
Clemens Grimm ◽  
Jann-Patrick Pelz ◽  
Cornelius Schneider ◽  
Katrin Schäffler ◽  
Utz Fischer
Keyword(s):  
Crystal Structure ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Turnover Rates ◽  
Mrna Transcription ◽  
Terminal Part ◽  
Protein Output

Eukaryotic cells determine the protein output of their genetic program by regulating mRNA transcription, localization, translation and turnover rates. This regulation is accomplished by an ensemble of RNA-binding proteins (RBPs) that bind to any given mRNA, thus forming mRNPs. Poly(A) binding proteins (PABPs) are prominent members of virtually all mRNPs that possess poly(A) tails. They serve as multifunctional scaffolds, allowing the recruitment of diverse factors containing a poly(A)-interacting motif (PAM) into mRNPs. We present the crystal structure of the variant PAM motif (termed PAM2w) in the N-terminal part of the positive translation factor LARP4B, which binds to the MLLE domain of the poly(A) binding protein C1 cytoplasmic 1 (PABPC1). The structural analysis, along with mutational studies in vitro and in vivo, uncovered a new mode of interaction between PAM2 motifs and MLLE domains.

scholarly journals Crystallizing the 6S and 8S spliceosomal assembly intermediates: a complex project

2015 ◽  
Vol 71 (10) ◽  
pp. 2040-2053 ◽  
Author(s):  
Jann-Patrick Pelz ◽  
Hermann Schindelin ◽  
Katharina van Pee ◽  
Jochen Kuper ◽  
Caroline Kisker ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Protein Complexes ◽  
Sm Proteins ◽  
Entropy Reduction ◽  
Multimeric Protein ◽  
Complex Project ◽  
Key Steps ◽  
Small Nuclear Ribonucleoproteins

The small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/6 and U5 are major constituents of the pre-mRNA processing spliceosome. They contain a common RNP core that is formed by the ordered binding of Sm proteins onto the single-stranded Sm site of the snRNA. Although spontaneousin vitro, assembly of the Sm core requires assistance from the PRMT5 and SMN complexesin vivo. To gain insight into the key steps of the assembly process, the crystal structures of two assembly intermediates of U snRNPs termed the 6S and 8S complexes have recently been reported. These multimeric protein complexes could only be crystallized after the application of various rescue strategies. The developed strategy leading to the crystallization and solution of the 8S crystal structure was subsequently used to guide a combination of rational crystal-contact optimization with surface-entropy reduction of crystals of the related 6S complex. Conversely, the resulting high-resolution 6S crystal structure was used during the restrained refinement of the 8S crystal structure.

scholarly journals Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells

2003 ◽  
Vol 162 (5) ◽  
pp. 757-764 ◽  
Author(s):  
Yasuhiko Terada ◽  
Yumi Uetake ◽  
Ryoko Kuriyama
Keyword(s):  
Mammalian Cells ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Mitotic Spindles ◽  
Microtubule Nucleation ◽  
Microtubule Organization ◽  
Centrosomal Protein ◽  
Spindle Poles

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of γ-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with γ-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring γ-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

scholarly journals Natural chromatin is heterogeneous and self-associates in vitro

2018 ◽  
Vol 29 (13) ◽  
pp. 1652-1663 ◽  
Author(s):  
Shujun Cai ◽  
Yajiao Song ◽  
Chen Chen ◽  
Jian Shi ◽  
Lu Gan
Keyword(s):  
Divalent Cations ◽  
Large Mass ◽  
Higher Order ◽  
Linker Histone ◽  
Order Structure ◽  
Face To Face ◽  
Chromatin Packing ◽  
Electron Cryotomography

The 30-nm fiber is commonly formed by oligonucleosome arrays in vitro but rarely found inside cells. To determine how chromatin higher-order structure is controlled, we used electron cryotomography (cryo-ET) to study the undigested natural chromatin released from two single-celled organisms in which 30-nm fibers have not been observed in vivo: picoplankton and yeast. In the presence of divalent cations, most of the chromatin from both organisms is condensed into a large mass in vitro. Rare irregular 30-nm fibers, some of which include face-to-face nucleosome interactions, do form at the periphery of this mass. In the absence of divalent cations, picoplankton chromatin decondenses into open zigzags. By contrast, yeast chromatin mostly remains condensed, with very few open motifs. Yeast chromatin packing is largely unchanged in the absence of linker histone and mildly decondensed when histones are more acetylated. Natural chromatin is therefore generally nonpermissive of regular motifs, even at the level of oligonucleosomes.

scholarly journals Structural analysis reveals features of the spindle checkpoint kinase Bub1–kinetochore subunit Knl1 interaction

2012 ◽  
Vol 196 (4) ◽  
pp. 451-467 ◽  
Author(s):  
Veronica Krenn ◽  
Annemarie Wehenkel ◽  
Xiaozheng Li ◽  
Stefano Santaguida ◽  
Andrea Musacchio
Keyword(s):  
Crystal Structure ◽  
Structural Analysis ◽  
Convex Surface ◽  
Point Mutations ◽  
Tetratricopeptide Repeats ◽  
Checkpoint Protein ◽  
Checkpoint Kinases ◽  
Shed Light

The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR–KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1.

scholarly journals Analysis of the cross-reactivity and of the 1.5 Å crystal structure of the Malassezia sympodialis Mala s 6 allergen, a member of the cyclophilin pan-allergen family

2006 ◽  
Vol 396 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Andreas G. Glaser ◽  
Andreas Limacher ◽  
Sabine Flückiger ◽  
Annika Scheynius ◽  
Leonardo Scapozza ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Three Dimensional ◽  
Cross Reactivity ◽  
Dimensional Structure ◽  
Cellular Functions ◽  
Human Proteins ◽  
Ige Mediated ◽  
Unit Cell Dimensions

Cyclophilins constitute a family of proteins involved in many essential cellular functions. They have also been identified as a panallergen family able to elicit IgE-mediated hypersensitivity reactions. Moreover, it has been shown that human cyclophilins are recognized by serum IgE from patients sensitized to environmental cyclophilins. IgE-mediated autoreactivity to self-antigens that have similarity to environmental allergens is often observed in atopic disorders. Therefore comparison of the crystal structure of human proteins with similarity to allergens should allow the identification of structural similarities to rationally explain autoreactivity. A new cyclophilin from Aspergillus fumigatus (Asp f 27) has been cloned, expressed and showed to exhibit cross-reactivity in vitro and in vivo. The three-dimensional structure of cyclophilin from the yeast Malassezia sympodialis (Mala s 6) has been determined at 1.5 Å (1 Å=0.1 nm) by X-ray diffraction. Crystals belong to space group P41212 with unit cell dimensions of a=b=71.99 Å and c=106.18 Å. The structure was solved by molecular replacement using the structure of human cyclophilin A as the search model. The refined structure includes all 162 amino acids of Mala s 6, an active-site-bound Ala-Pro dipeptide and 173 water molecules, with a crystallographic R- and free R-factor of 14.3% and 14.9% respectively. The overall structure consists of an eight-stranded antiparallel β-barrel and two α-helices covering the top and bottom of the barrel, typical for cyclophilins. We identified conserved solvent-exposed residues in the fungal and human structures that are potentially involved in the IgE-mediated cross-reactivity.

scholarly journals Virus Particle Core Defects Caused by Mutations in the Human Immunodeficiency Virus Capsid N-Terminal Domain

2005 ◽  
Vol 79 (3) ◽  
pp. 1470-1479 ◽  
Author(s):  
Isabel Scholz ◽  
Brian Arvidson ◽  
Doug Huseby ◽  
Eric Barklis
Keyword(s):  
Human Immunodeficiency Virus ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Order Structures ◽  
Binding Loop ◽  
Hiv 1 ◽  
Cypa Binding

ABSTRACT The N-terminal domains (NTDs) of the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein have been modeled to form hexamer rings in the mature cores of virions. In vitro, hexamer ring units organize into either tubes or spheres, in a pH-dependent fashion. To probe factors which might govern hexamer assembly preferences in vivo, we examined the effects of mutations at CA histidine residue 84 (H84), modeled at the outer edges of NTD hexamers, as well as a nearby histidine (H87) in the cyclophilin A (CypA) binding loop. Although mutations at H87 yielded infectious virions, mutations at H84 produced assembly-competent but poorly infectious virions. The H84 mutant viruses incorporated wild-type levels of CypA and viral RNAs and showed nearly normal signals in virus entry assays. However, mutant CA proteins assembled aberrant virus cores, and mutant core fractions retained abnormally high levels of CA but reduced reverse transcriptase activities. Our results suggest that HIV-1 CA residue 84 contributes to a structure which helps control either NTD hexamer assembly or the organization of hexamers into higher-order structures.

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