title: Molecular determinants for the recruitment of the ubiquitin-ligase MuRF-1 onto M-line titin.
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.
authors.affiliation: Division of Structural Biology, Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Mrosek
authors.forename: Dietmar
authors.initials: D
authors.lastname: Labeit
authors.forename: Stephanie
authors.initials: S
authors.lastname: Witt
authors.forename: Heiko
authors.initials: H
authors.lastname: Heerklotz
authors.forename: Eleonore
authors.initials: E
authors.lastname: von Castelmur
authors.forename: Siegfried
authors.initials: S
authors.lastname: Labeit
authors.forename: Olga
authors.initials: O
authors.lastname: Mayans
mesh_terms: D002151:Calorimetry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D064211:Connectin; D018360:Crystallography, X-Ray; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D000070584:Tripartite Motif Proteins; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D064211:Connectin; D009124:Muscle Proteins; C577285:TTN protein, human; D000070584:Tripartite Motif Proteins; C440908:TRIM63 protein, human; D044767:Ubiquitin-Protein Ligases; D011494:Protein Kinases