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Order from disorder in the sarcomeric Z-disks

In sarcomeres, α-actinin cross-links actin filaments and anchors them to the Z-disk. The structure and cellular validation of α-actinin-2 in complex with its Z-disk partner, FATZ-1, shows that FATZ-1 forms a tight fuzzy complex with α-actinin-2 and suggests an interaction mechanism via main molecular recognition elements and secondary binding sites.

Approximately 40% of the human body consists of skeletal muscle, the contraction of which leads to locomotion. The contractile actin and myosin filaments are integrated in a paracrystalline lattice by the action of accessory cytoskeletal proteins and anchored at the boundaries of the sarcomeres, constituting the Z-disk and M-band, respectively. The protein α-actinin-2 crosslinks actin filaments from neighbouring sarcomeres at the Z-disks, which are composed of proteins important for its structural integrity and function. α-actinin-2 is the major Z-disk component, which binds not only to actin filaments from adjacent sarcomeres but also serves as an interaction platform for a number of Z-disk proteins, among which titin and FATZ-1. In muscle, α-actinin-2 interacts with F-actin and titin in a phosphoinositide-regulated manner [1], while bundling of actin filaments by non-muscle isoforms of α-actinin is regulated by calcium [2].

FATZ-1 is an intrinsically disordered scaffold protein that interacts with α-actinin-2 and other four core Z-disk proteins, contributing to myofibril assembly and maintenance as a protein interaction hub (Figure 43a).

This work focuses on α-actinin-2/FATZ-1 interaction and structure to elucidate the molecular mechanism of FATZ-1 scaffolding function.

X-ray diffraction data collected at beamline ID29 and Petra III were used to determine the crystal structure of the C-terminal portions of FATZ-1 in complex with α-actinin-2 rod domain or half-dimer to resolutions between 2.7 and 3.8 Å, but the electron density for bound FATZ-1 could not be unambiguously interpreted. Diffraction data at the Se-absorption edge were therefore collected on beamline ID30B, combined with complementary experiments that corroborated the FATZ-1 amino-acid sequence assignments (Figure 43b). FATZ-1 binds to α-actinin-2 via two short linear motifs (LM1, LM2) (Figure 43c). Combining X-ray diffraction and small-angle X-ray scattering data collected at beamline BM29 and PETRA III, an integrative model of fuzzy α-actinin-2/FATZ-1 complex was generated, which revealed that FATZ-1 stems radially from the α-actinin-2 rod anchoring points, resulting in a polar architecture of the complex (Figure 44a), as also observed in the α-actinin-2/ FATZ-1 complex placed into a cryo-electron tomography model of the Z-disk (Figure 44b) [3]. In combination with the FATZ-1 multivalent scaffolding function, this assembly is likely to organise interaction partners, and stabilise the preferential orientation of α-actinin-2 in the Z-disk, thus advancing understanding of the role of scaffold proteins in muscle ultrastructure.

The work further uncovers the ability of FATZ-1 to phase-separate and form biomolecular condensates with α-actinin-2 (Figure 44c), and shows that FATZ-1 condensates are prevented and dissolved by increasing

Fig. 43: a) Schematics of the FATZ-1 interactome and binding sites. b) Schematics of the main FATZ-1 constructs used in this work, along with their amino acid boundaries. c) Crystal structure of rod-α-actinin-2/mini-FATZ-1 (in green/magenta), with linear motifs LM1 and LM2 shown in magenta. Cross-linked residues are indicated by blue, red, and grey balls on the structure. Identified Se-Mets are shown in yellow.