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Structural investigation of the molecular mechanisms underlying titin elasticity and signaling

Castelmur, Eleonore von. Structural investigation of the molecular mechanisms underlying titin elasticity and signaling. 2010, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_8995

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Abstract

Titin is a giant protein that spans >1µm from the Z-disc to the M-line, forming an intrasarcomeric filament system in vertebrate striated muscle, which is not only essential for the assembly of the sarcomere, but also critical for myofibril signaling and metabolism. Furthermore, it provides the sarcomere with resting tension, elasticity and restoring forces upon stretch, ensuring the correct positioning of the actin-myosin motors during muscle function. Titin is composed of ~300 immunoglobulin (Ig) and fibronectin-III (FnIII) domains, arranged in linear tandems. They are interspersed by an auto-inhibited Ser kinase (TK) close to its C-terminus as well as several unique sequences, most prominently a differentially spliced stretch rich in PEVK residues which localizes to the I-band part of titin where its elastic properties reside. There, the PEVK segment is flanked by a long Ig tandem, which together act as serial molecular springs that determine titin elastic response.
The focus of this work lay in the elucidation of the molecular mechanisms governing titin I-band elasticity and the recruitment of the M-line signalosome around TK involved in the control of myofibril turnover and the trophic state of muscle. To that effect, we have elucidated the crystal structure of a six-Ig fragment representative of the elastic Ig-tandem at 3.3Å resolution. The model reveals the molecular principles of Ig-arraying at the skeletal I-band of titin as mediated by conserved Ig-Ig transition motifs. Regular domain arrangements within this fragment point at the existence of a high-order in the fine structure of the filament, which is confirmed by EM data on a 19-mer poly-Ig segment. Our findings indicate a long-range, supra-order in the skeletal I-band of titin, where assembly of Ig domains into dynamical super-motifs is essential for the elastic function of the filament. We propose a novel model of spring mechanism for poly-Ig elasticity in titin based on a “carpenter ruler” model of skeletal I-band architecture. Furthermore, we have focused on the recruitment of the ubiquitin ligase MURF1 to the M-line signalosome through its specific interaction with titin domains A168 A170. MuRF1 contains several oligomerization motifs in succession, which indicates a possible need for tight regulation. We have therefore analyzed their influence on the oligomeric state of the protein. Our SEC-MALS data showed that the a-helical region of MuRF1 is dimeric in isolation, while in combination with the preceding B-Box domain, itself a dimerization motif, higher-order assembly is induced, which might be of physiological importance. We could also show that higher-order assembly of MuRF1 did not disrupt binding to A168-A170 in pull-down assays. Further biophysical or structural characterization of the complex of A168-A170 with MuRF1 constructs was hindered by the severely compromised solubility of the complex. Finally, we have successfully solved the crystal structure of the FnIII-Kin-Ig region of twitchin, which corresponds to titin A170-TK-M1. The N-terminal linker wraps around the kinase domain and positions the preceding FnIII domain in such a way that it blocks the autoregulatory tail in its inhibitory positon. Thus, from the structure we could conclude that stretch-activation of Twc kinase seems unlikely and instead propose phosphorylation of Y 104 as a possible activation mechanism.
Our findings illustrate how the structural and functional diversity in titin’s modular architecture has evolved not only on the basis of individual domains. Rather, functionality often involves adaptation of several neighboring domains or even whole Ig tandems/super-repeats. This is reflected in variations in mechanical and dynamic properties observed in different parts of the chain and highlights the necessity of working with representative multi-domain fragments to gain a comprehensive understanding of the titin chain
Advisors:Hennig, Michael
Committee Members:Mayans, Olga and Schirmer, Tilman
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics
Item Type:Thesis
Thesis no:8995
Bibsysno:Link to catalogue
Number of Pages:151 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:41
Deposited On:21 May 2010 08:06

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