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von Castelmur, Eleonore
Publications (10 of 11) Show all publications
Franke, B., Gasch, A., Rodriguez, D., Chami, M., Khan, M. M., Rudolf, R., . . . Mayans, O. (2014). Molecular basis for the fold organization and sarcomeric targeting of the muscle atrogin MuRF1. Open Biology, 4, Article ID 130172.
Open this publication in new window or tab >>Molecular basis for the fold organization and sarcomeric targeting of the muscle atrogin MuRF1
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2014 (English)In: Open Biology, ISSN 2046-2441, E-ISSN 2046-2441, Vol. 4, article id 130172Article in journal (Refereed) Published
Abstract [en]

MuRF1 is an E3 ubiquitin ligase central to muscle catabolism. It belongs to the TRIM protein family characterized by a tripartite fold of RING, B-box and coiled-coil (CC) motifs, followed by variable C-terminal domains. The CC motif is hypothesized to be responsible for domain organization in the fold as well as for high-order assembly into functional entities. But data on CC from this family that can clarify the structural significance of this motif are scarce. We have characterized the helical region from MuRF1 and show that, contrary to expectations, its CC domain assembles unproductively, being the B2- and COS-boxes in the fold (respectively flanking the CC) that promote a native quaternary structure. In particular, the C-terminal COS-box seemingly forms an α-hairpin that packs against the CC, influencing its dimerization. This shows that a C-terminal variable domain can be tightly integrated within the conserved TRIM fold to modulate its structure and function. Furthermore, data from transfected muscle show that in MuRF1 the COS-box mediates the in vivo targeting of sarcoskeletal structures and points to the pharmacological relevance of the COS domain for treating MuRF1-mediated muscle atrophy.

Place, publisher, year, edition, pages
London, United Kingdom: The Royal Society Publishing, 2014
Keywords
COS-box, RBCC/TRIM fold, X-ray crystallography, ab initio modelling, coiled-coil, electron microscopy
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-143661 (URN)10.1098/rsob.130172 (DOI)000333906500002 ()24671946 (PubMedID)2-s2.0-84900993019 (Scopus ID)
Available from: 2017-12-12 Created: 2017-12-12 Last updated: 2018-01-26Bibliographically approved
Nijenhuis, W., von Castelmur, E., Littler, D., De Marco, V., Tromer, E., Vleugel, M., . . . Kops, G. J. (2013). A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B. Journal of Cell Biology, 201(2), 217-231
Open this publication in new window or tab >>A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B
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2013 (English)In: Journal of Cell Biology, ISSN 0021-9525, E-ISSN 1540-8140, Vol. 201, no 2, p. 217-231Article in journal (Refereed) Published
Abstract [en]

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.

Place, publisher, year, edition, pages
Rockefeller University Press, 2013
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-143662 (URN)10.1083/jcb.201210033 (DOI)000317583500007 ()23569217 (PubMedID)2-s2.0-84876697755 (Scopus ID)
Available from: 2017-12-12 Created: 2017-12-12 Last updated: 2018-01-05Bibliographically approved
Heidebrecht, T., Fish, A., von Castelmur, E., Johnson, K. A., Zaccai, G., Borst, P. & Perrakis, A. (2012). Binding of the J-binding protein to DNA containing glucosylated hmU (base J) or 5-hmC: evidence for a rapid conformational change upon DNA binding. Journal of the American Chemical Society, 134(32), 13357-13365
Open this publication in new window or tab >>Binding of the J-binding protein to DNA containing glucosylated hmU (base J) or 5-hmC: evidence for a rapid conformational change upon DNA binding
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2012 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 32, p. 13357-13365Article in journal (Refereed) Published
Abstract [en]

Base J (β-D-glucosyl-hydroxymethyluracil) was discovered in the nuclear DNA of some pathogenic protozoa, such as trypanosomes and Leishmania, where it replaces a fraction of base T. We have found a J-Binding Protein 1 (JBP1) in these organisms, which contains a unique J-DNA binding domain (DB-JBP1) and a thymidine hydroxylase domain involved in the first step of J biosynthesis. This hydroxylase is related to the mammalian TET enzymes that hydroxylate 5-methylcytosine in DNA. We have now studied the binding of JBP1 and DB-JBP1 to oligonucleotides containing J or glucosylated 5-hydroxymethylcytosine (glu-5-hmC) using an equilibrium fluorescence polarization assay. We find that JBP1 binds glu-5-hmC-DNA with an affinity about 40-fold lower than J-DNA (~400 nM), which is still 200 times higher than the JBP1 affinity for T-DNA. The discrimination between glu-5-hmC-DNA and T-DNA by DB-JBP1 is about 2-fold less, but enough for DB-JBP1 to be useful as a tool to isolate 5-hmC-DNA. Pre-steady state kinetic data obtained in a stopped-flow device show that the initial binding of JBP1 to glucosylated DNA is very fast with a second order rate constant of 70 μM(-1) s(-1) and that JBP1 binds to J-DNA or glu-5-hmC-DNA in a two-step reaction, in contrast to DB-JBP1, which binds in a one-step reaction. As the second (slower) step in binding is concentration independent, we infer that JBP1 undergoes a conformational change upon binding to DNA. Global analysis of pre-steady state and equilibrium binding data supports such a two-step mechanism and allowed us to determine the kinetic parameters that describe it. This notion of a conformational change is supported by small-angle neutron scattering experiments, which show that the shape of JBP1 is more elongated in complex with DNA. The conformational change upon DNA binding may allow the hydroxylase domain of JBP1 to make contact with the DNA and hydroxylate T's in spatial proximity, resulting in regional introduction of base J into the DNA.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2012
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-143664 (URN)10.1021/ja303423t (DOI)000307487200040 ()22775585 (PubMedID)2-s2.0-84865111457 (Scopus ID)
Available from: 2017-12-12 Created: 2017-12-12 Last updated: 2017-12-20Bibliographically approved
von Castelmur, E., Strümpfer, J., Franke, B., Bogomolovas, J., Barbieri, S., Qadota, H., . . . Mayans, O. (2012). Identification of an N-terminal inhibitory extension as the primary mechanosensory regulator of twitchin kinase. Proceedings of the National Academy of Sciences of the United States of America, 109(34), 13608-13613
Open this publication in new window or tab >>Identification of an N-terminal inhibitory extension as the primary mechanosensory regulator of twitchin kinase
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2012 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 34, p. 13608-13613Article in journal (Refereed) Published
Abstract [en]

Titin-like kinases are an important class of cytoskeletal kinases that intervene in the response of muscle to mechanical stimulation, being central to myofibril homeostasis and development. These kinases exist in autoinhibited states and, allegedly, become activated during muscle activity by the elastic unfolding of a C-terminal regulatory segment (CRD). However, this mechano-activation model remains controversial. Here we explore the structural, catalytic, and tensile properties of the multidomain kinase region of Caenorhabditis elegans twitchin (Fn(31)-Nlinker-kinase-CRD-Ig(26)) using X-ray crystallography, small angle X-ray scattering, molecular dynamics simulations, and catalytic assays. This work uncovers the existence of an inhibitory segment that flanks the kinase N-terminally (N-linker) and that acts synergistically with the canonical CRD tail to silence catalysis. The N-linker region has high mechanical lability and acts as the primary stretch-sensor in twitchin kinase, while the CRD is poorly responsive to pulling forces. This poor response suggests that the CRD is not a generic mechanosensor in this kinase family. Instead, the CRD is shown here to be permissive to catalysis and might protect the kinase active site against mechanical damage. Thus, we put forward a regulatory model where kinase inhibition results from the combined action of both N- and C-terminal tails, but only the N-terminal extension undergoes mechanical removal, thereby affording partial activation. Further, we compare invertebrate and vertebrate titin-like kinases and identify variations in the regulatory segments that suggest a mechanical speciation of these kinase classes.

Place, publisher, year, edition, pages
Washington, DC, United States: National Academy of Sciences, 2012
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-143663 (URN)10.1073/pnas.1200697109 (DOI)000308085200038 ()22869697 (PubMedID)2-s2.0-84865297039 (Scopus ID)
Available from: 2017-12-12 Created: 2017-12-12 Last updated: 2017-12-20Bibliographically approved
A. Strumpfer, J., von Castelmur, E., Franke, B., Barbieri, S., Bogomolovas, J., Qadota, H., . . . Mayans, O. (2012). Stretching of Twitchin Kinase. Biophysical Journal, 102(3 Supplement 1), 361a-362a
Open this publication in new window or tab >>Stretching of Twitchin Kinase
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2012 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 102, no 3 Supplement 1, p. 361a-362aArticle in journal (Refereed) Published
Abstract [en]

The giant proteins from the titin family, that form cytoskeletal filaments, have emerged as key mechanotransducers in the sarcomere. These proteins contain a conserved kinase region, which is auto-inhibited by a C-terminal tail domain. The inhibitory tail domain occludes the active sites of the kinases, thus preventing ATP from binding. It was proposed that through application of a force, such as that arising during muscle contraction, the inhibitory tail becomes detached, lifting inhibition. The force-sensing ability of titin kinase was demonstrated in AFM experiments and simulations [Puchner, et al., 2008, PNAS:105, 13385], which showed indeed that mechanical forces can remove the autoinhibitory tail of titin kinase. We report here steered molecular dynamics simulations (SMD) of the very recently resolved crystal structure of twitchin kinase, containing the kinase region and flanking fibronectin and immuniglobulin domains, that show a variant mechanism. Despite the significant structural and sequence similarity to titin kinase, the autoinhibitory tail of twitchin kinase remains in place upon stretching, while the N-terminal lobe of the kinase unfolds. The SMD simulations also show that the detachment and stretching of the linker between fibronectin and kinase regions, and the partial extension of the autoinhibitory tail, are the primary force-response. We postulate that this stretched state, where all structural elements are still intact, may represent the physiologically active state.

Place, publisher, year, edition, pages
St. Louis, MO, United States: Cell Press, 2012
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-143666 (URN)10.1016/j.bpj.2011.11.1973 (DOI)000321561202410 ()
Available from: 2017-12-12 Created: 2017-12-12 Last updated: 2017-12-21Bibliographically approved
Suijkerbuijk, S. J., van Dam, T. J., Karagöz, G. E., von Castelmur, E., Hubner, N. C., Duarte, A. M., . . . Kops, G. J. (2012). The vertebrate mitotic checkpoint protein BUBR1 is an unusual pseudokinase. Developmental Cell, 22(6), 1321-1329
Open this publication in new window or tab >>The vertebrate mitotic checkpoint protein BUBR1 is an unusual pseudokinase
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2012 (English)In: Developmental Cell, ISSN 1534-5807, E-ISSN 1878-1551, Vol. 22, no 6, p. 1321-1329Article in journal (Refereed) Published
Abstract [en]

Chromosomal stability is safeguarded by a mitotic checkpoint, of which BUB1 and Mad3/BUBR1 are core components. These paralogs have similar, but not identical, domain organization. We show that Mad3/BUBR1 and BUB1 paralogous pairs arose by nine independent gene duplications throughout evolution, followed by parallel subfunctionalization in which preservation of the ancestral, amino-terminal KEN box or kinase domain was mutually exclusive. In one exception, vertebrate BUBR1-defined by the KEN box-preserved the kinase domain but allowed nonconserved degeneration of catalytic motifs. Although BUBR1 evolved to a typical pseudokinase in some vertebrates, it retained the catalytic triad in humans. However, we show that putative catalysis by human BUBR1 is dispensable for error-free chromosome segregation. Instead, residues that interact with ATP in conventional kinases are essential for conformational stability in BUBR1. We propose that parallel evolution of BUBR1 orthologs rendered its kinase function dispensable in vertebrates, producing an unusual, triad-containing pseudokinase.

Place, publisher, year, edition, pages
Cambridge, United States: Cell Press, 2012
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-143665 (URN)10.1016/j.devcel.2012.03.009 (DOI)000305498200020 ()22698286 (PubMedID)2-s2.0-84862131533 (Scopus ID)
Available from: 2017-12-12 Created: 2017-12-12 Last updated: 2017-12-20Bibliographically approved
Lee, E. H., Hsin, J., von Castelmur, E., Mayans, O. & Schulten, K. (2010). Tertiary and Secondary Structure Elasticity of a Six-Ig Titin Chain. Biophysical Journal, 98(6), 1085-1095
Open this publication in new window or tab >>Tertiary and Secondary Structure Elasticity of a Six-Ig Titin Chain
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2010 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 98, no 6, p. 1085-1095Article in journal (Refereed) Published
Abstract [en]

The protein titin functions as a mechanical spring conferring passive elasticity to muscle. Force spectroscopy studies have shown that titin exhibits several regimes of elasticity. Disordered segments bring about a soft, entropic spring-type elasticity; secondary structures of titin's immunoglobulin-like (Ig-) and fibronectin type III-like (FN-III) domains provide a stiff elasticity. In this study, we demonstrate a third type of elasticity due to tertiary structure and involving domain-domain interaction and reorganization along the titin chain. Through 870 ns of molecular dynamics simulations involving 29,000-635,000 atom systems, the mechanical properties of a six-Ig domain segment of titin (I65-I70), for which a crystallographic structure is available, are probed. The results reveal a soft tertiary structure elasticity. A remarkably accurate statistical mechanical description for this elasticity is derived and applied. Simulations also studied the stiff, secondary structure elasticity of the I65-I70 chain due to the unraveling of its domains and revealed how force propagates along the chain during the secondary structure elasticity response.

Place, publisher, year, edition, pages
St. Louis, MO, United States: Cell Press, 2010
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-143667 (URN)10.1016/j.bpj.2009.12.4192 (DOI)000275842200022 ()20303866 (PubMedID)2-s2.0-77949648622 (Scopus ID)
Available from: 2017-12-12 Created: 2017-12-12 Last updated: 2017-12-21Bibliographically approved
von Castelmur, E., Marino, M., Svergun, D. I., Kreplak, L., Ucurum-Fotiadis, Z., Konarev, P. V., . . . Mayans, O. (2008). A regular pattern of Ig super-motifs defines segmental flexibility as the elastic mechanism of the titin chain. Proceedings of the National Academy of Sciences of the United States of America, 105(4), 1186-1191
Open this publication in new window or tab >>A regular pattern of Ig super-motifs defines segmental flexibility as the elastic mechanism of the titin chain
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2008 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 4, p. 1186-1191Article in journal (Refereed) Published
Abstract [en]

Myofibril elasticity, critical to muscle function, is dictated by the intrasarcomeric filament titin, which acts as a molecular spring. To date, the molecular events underlying the mechanics of the folded titin chain remain largely unknown. We have elucidated the crystal structure of the 6-Ig fragment I65-I70 from the elastic I-band fraction of titin and validated its conformation in solution using small angle x-ray scattering. The long-range properties of the chain have been visualized by electron microscopy on a 19-Ig fragment and modeled for the full skeletal tandem. Results show that conserved Ig-Ig transition motifs generate high-order in the structure of the filament, where conformationally stiff segments interspersed with pliant hinges form a regular pattern of dynamic super-motifs leading to segmental flexibility in the chain. Pliant hinges support molecular shape rearrangements that dominate chain behavior at moderate stretch, whereas stiffer segments predictably oppose high stretch forces upon full chain extension. There, librational entropy can be expected to act as an energy barrier to prevent Ig unfolding while, instead, triggering the unraveling of flanking springs formed by proline, glutamate, valine, and lysine (PEVK) sequences. We propose a mechanistic model based on freely jointed rigid segments that rationalizes the response to stretch of titin Ig-tandems according to molecular features.

Place, publisher, year, edition, pages
Washington, DC, United States: National Academy of Sciences, 2008
Keywords
electron microscopy, poly-Ig tandem structure, small angle x-ray scattering, titin elasticity, x-ray crystallography
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-143657 (URN)10.1073/pnas.0707163105 (DOI)000252873900020 ()18212128 (PubMedID)2-s2.0-39549086109 (Scopus ID)
Available from: 2017-12-12 Created: 2017-12-12 Last updated: 2017-12-19Bibliographically approved
Mrosek, M., Meier, S., Ucurum-Fotiadis, Z., von Castelmur, E., Hedbom, E., Lustig, A., . . . Mayans, O. (2008). Structural analysis of B-Box 2 from MuRF1: identification of a novel self-association pattern in a RING-like fold.. Biochemistry, 47(40), 10722-10730
Open this publication in new window or tab >>Structural analysis of B-Box 2 from MuRF1: identification of a novel self-association pattern in a RING-like fold.
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2008 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 40, p. 10722-10730Article in journal (Refereed) Published
Abstract [en]

The B-box motif is the defining feature of the TRIM family of proteins, characterized by a RING finger-B-box-coiled coil tripartite fold. We have elucidated the crystal structure of B-box 2 (B2) from MuRF1, a TRIM protein that supports a wide variety of protein interactions in the sarcomere and regulates the trophic state of striated muscle tissue. MuRF1 B2 coordinates two zinc ions through a cross-brace alpha/beta-topology typical of members of the RING finger superfamily. However, it self-associates into dimers with high affinity. The dimerization pattern is mediated by the helical component of this fold and is unique among RING-like folds. This B2 reveals a long shallow groove that encircles the C-terminal metal binding site ZnII and appears as the defining protein-protein interaction feature of this domain. A cluster of conserved hydrophobic residues in this groove and, in particular, a highly conserved aromatic residue (Y133 in MuRF1 B2) is likely to be central to this role. We expect these findings to aid the future exploration of the cellular function and therapeutic potential of MuRF1.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2008
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-143668 (URN)10.1021/bi800733z (DOI)000259603600021 ()18795805 (PubMedID)2-s2.0-53249130853 (Scopus ID)
Available from: 2017-12-12 Created: 2017-12-12 Last updated: 2017-12-21Bibliographically approved
Urzhumtsev, A., von Castelmur, E. & Mayans, O. (2008). Ultralow-resolution ab initio phasing of filamentous proteins: crystals from a six-Ig fragment of titin as a case study.. Acta Crystallographica Section D: Biological Crystallography, 64(Pt 5), 478-486
Open this publication in new window or tab >>Ultralow-resolution ab initio phasing of filamentous proteins: crystals from a six-Ig fragment of titin as a case study.
2008 (English)In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 64, no Pt 5, p. 478-486Article in journal (Refereed) Published
Abstract [en]

Low-resolution diffraction data (resolution below 12 angstroms) from crystals of a filamentous six-Ig fragment of titin, I65-I70, were used in ab initio phasing with the aim of calculating its lattice packing and molecular envelope. Filamentous molecules, characterized by marked anisometry and idiosyncratic crystal lattices, have not been addressed before using this methodology. In this study, low-resolution phasing (19-122 angstroms) successfully identified the region of the unit cell occupied by the molecule. Phase extension to a higher resolution (12 angstroms) yielded regions of high density that corresponded either to the positions of individual Ig domains or to zones of dense intermolecular contacts, hindering the identification of individual domains and the interpretation of electron-density maps in terms of a molecular model. This problem resulted from the acutely uneven packing of the molecules in the crystal and it was further accentuated by the presence of partially disordered regions in the molecule. Addition of low-resolution reflections with phases computed ab initio to those obtained experimentally using MIRAS improved the initial electron-density maps of the atomic model, demonstrating the generic utility of low-resolution phases for the structure-elucidation process, even when individual molecules cannot be resolved in the lattice.

Place, publisher, year, edition, pages
Wiley-Blackwell Publishing Inc., 2008
Keywords
ultra-low resolution ab initio phasing, filamentous proteins, titin, crystal packing
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-143669 (URN)10.1107/S0907444908003442 (DOI)000255077900002 ()18453683 (PubMedID)2-s2.0-43249092361 (Scopus ID)
Available from: 2017-12-12 Created: 2017-12-12 Last updated: 2017-12-21Bibliographically approved
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