| 1. |
- A. Strumpfer, Johan, et al.
(författare)
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Stretching of Twitchin Kinase
- 2012
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Ingår i: Biophysical Journal. - St. Louis, MO, United States : Cell Press. - 0006-3495 .- 1542-0086. ; 102:3 Supplement 1, s. 361a-362a
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Franke, Barbara, et al.
(författare)
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Molecular basis for the fold organization and sarcomeric targeting of the muscle atrogin MuRF1
- 2014
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Ingår i: Open Biology. - London, United Kingdom : The Royal Society Publishing. - 2046-2441. ; 4
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Lee, Eric H, et al.
(författare)
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Tertiary and Secondary Structure Elasticity of a Six-Ig Titin Chain
- 2010
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Ingår i: Biophysical Journal. - St. Louis, MO, United States : Cell Press. - 0006-3495 .- 1542-0086. ; 98:6, s. 1085-1095
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Mrosek, Michael, et al.
(författare)
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Molecular determinants for the recruitment of the ubiquitin-ligase MuRF-1 onto M-line titin.
- 2007
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Ingår i: The FASEB Journal. - : Wiley. - 0892-6638 .- 1530-6860. ; 21:7, s. 1383-1392
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Tidskriftsartikel (refereegranskat)abstract
- Titin forms an intrasarcomeric filament system in vertebrate striated muscle, which has elastic and signaling properties and is thereby central to mechanotransduction. Near its C-terminus and directly preceding a kinase domain, titin contains a conserved pattern of Ig and FnIII modules (Ig(A168)-Ig(A169)-FnIII(A170), hereby A168-A170) that recruits the E3 ubiquitin-ligase MuRF-1 to the filament. This interaction is thought to regulate myofibril turnover and the trophic state of muscle. We have elucidated the crystal structure of A168-A170, characterized MuRF-1 variants by circular dichroism (CD) and SEC-MALS, and studied the interaction of both components by isothermal calorimetry, SPOTS blots, and pull-down assays. This has led to the identification of the molecular determinants of the binding. A168-A170 shows an extended, rigid architecture, which is characterized by a shallow surface groove that spans its full length and a distinct loop protrusion in its middle point. In MuRF-1, a C-terminal helical domain is sufficient to bind A168-A170 with high affinity. This helical region predictably docks into the surface groove of A168-A170. Furthermore, pull-down assays demonstrate that the loop protrusion in A168-A170 is a key mediator of MuRF-1 recognition. Our findings indicate that this region of titin could serve as a target to attempt therapeutic inhibition of MuRF-1-mediated muscle turnover, where binding of small molecules to its distinctive structural features could block MuRF-1 access.
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| 5. |
- Mrosek, Michael, et al.
(författare)
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Structural analysis of B-Box 2 from MuRF1 : identification of a novel self-association pattern in a RING-like fold.
- 2008
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 47:40, s. 10722-10730
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
- Urzhumtsev, Alexandre, et al.
(författare)
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Ultralow-resolution ab initio phasing of filamentous proteins : crystals from a six-Ig fragment of titin as a case study.
- 2008
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Ingår i: Acta Crystallographica Section D. - : Wiley-Blackwell Publishing Inc.. - 0907-4449 .- 1399-0047. ; 64:Pt 5, s. 478-486
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- von Castelmur, Eleonore, et al.
(författare)
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A regular pattern of Ig super-motifs defines segmental flexibility as the elastic mechanism of the titin chain
- 2008
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - Washington, DC, United States : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 105:4, s. 1186-1191
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 8. |
- von Castelmur, Eleonore, et al.
(författare)
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Identification of an N-terminal inhibitory extension as the primary mechanosensory regulator of twitchin kinase
- 2012
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - Washington, DC, United States : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 109:34, s. 13608-13613
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 9. |
- Zacharchenko, Thomas, et al.
(författare)
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Structural advances on titin : towards an atomic understanding of multi-domain functions in myofilament mechanics and scaffolding.
- 2015
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Ingår i: Biochemical Society Transactions. - : Portland Press Ltd. - 0300-5127 .- 1470-8752. ; 43:5, s. 850-5
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Tidskriftsartikel (refereegranskat)abstract
- Titin is a gigantic filamentous protein of the muscle sarcomere that plays roles in myofibril mechanics and homoeostasis. 3D-structures of multi-domain fragments of titin are now available that start revealing the molecular mechanisms governing its mechanical and scaffolding functions. This knowledge is now being translated into the fabrication of self-assembling biopolymers. Here we review the structural advances on titin, the novel concepts derived from these and the emerging translational avenues.
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