Conformational plasticity and evolutionary analysis of the myotilin tandem Ig domains

Abstract

Myotilin is a component of the sarcomere where it plays an important role in organisation and maintenance of Z-disk integrity. This involves direct binding to F-actin and filamin C, a function mediated by its Ig domain pair. While the structures of these two individual domains are known, information about their relative orientation and flexibility remains limited. We set on to characterise the Ig domain pair of myotilin with emphasis on its molecular structure, dynamics and phylogeny. First, sequence conservation analysis of myotilin shed light on the molecular basis of myotilinopathies and revealed several motifs in Ig domains found also in I-band proteins. In particular, a highly conserved Glu344 mapping to Ig domain linker, was identified as a critical component of the inter-domain hinge mechanism. Next, SAXS and molecular dynamics revealed that Ig domain pair exists as a multi-conformation species with dynamic exchange between extended and compact orientations. Mutation of AKE motif to AAA further confirmed its impact on inter-domain flexibility. We hypothesise that the conformational plasticity of the Ig domain pair in its unbound form is part of the binding partner recognition mechanism.

Molecular basis of F-actin regulation and sarcomere assembly via myotilin

“…Overall, the EOM-selected models tended to be more compact than those in the random pool. This is visible from the shift to lower values of D max and R g derived from the selected models compared to the distribution derived from the original pool, as the most extended models were not selected (Fig 1E,S1C and S1D Fig) . Based on this analysis, Trx-MYOT displays a tendency to occupy more compact conformations compared to a chain with random coil linkers, in line with the predicted compactness (Fig 1B ) and the disorder tendency analysis [23].…”

“…The previously performed disorder tendency analysis of full-length myotilin revealed that the N-terminal region of myotilin displays characteristics of an intrinsically disordered protein (IDP) [23]. The meta-structure analysis, which reflects how likely a specific residue is to be located within a compact 3D structure, was carried out as described previously [6], and indicated a tendency for local compactions in the N-terminal ID region (Fig 1B).…”

“…The muscular dystrophy-causing mutant R405K (Ig1Ig2 R405K ) [27] was also tested, but did not show any notable difference in F-actin affinity with respect to the wild type (Fig 2A Since binding data are available for other actin-binding Ig domains, e.g., for Ig domains of palladin and filamin A [20], we conducted a comparative structural analysis of the actin-binding Ig domains of myotilin, the Ig3 domain of palladin, and Ig10 of filamin A, which revealed [23]. Surfaces are colored on a red white blue gradient, as calculated by Adaptive Poisson-Boltzmann Solver [28].…”

The Role of Z-disc Proteins in Myopathy and Cardiomyopathy

“…Almost all MYOT variants have been located in exon 2 in the amino-terminal region, an intrinsically unstructured region [ 135 ]. Two separate studies described MYOT missense variants affecting the same residue (p.Arg6Gly and p.Arg6His), both causing severe forms of skeletal myopathy, suggesting this residue is a mutational hotspot [ 136 , 137 ].…”

Myotilin, a New Topotecan Resistant Protein in Ovarian Cancer Cell Lines

“…This mechanism is not restricted only to collagens but was also observed for pancreatic cancer cells growing on the surface composed of fibronectin and laminin, where cancer cells became resistant to DOX, CIS and 5-fluorouracil (5-FU) 44 and for breast cancer cell line MDA-MB-231 growing on the surface of fibronectin and type I collagen that became resistant to PAC 45 . MYOT is a structural protein that interacts with many other proteins in cells 46 . IgG-like domains of MYOT are responsible for dimerization and binding of other proteins like F- and G-actin 31 , 47 or filamin C 32 .…”