Structural Biology of the Cytoskeleton

Structure and PIP2 regulation mechanism of human α-actinin

We determined the first complete structure of α-actinin from striated muscle and explored its functional implications on the biochemical and cellular level. The structure provides insight into the mechanism of α-actinin regulation by PIP₂ and the intramolecular pseudoligand autoinhibition mechanism. It also offers a foundation to study interactions with Z-disc partners and effects of pathogenic mutations with molecular resolution and mechanistic insight. (Ribeiro et al., Cell, 2014).

Read more: soft link to paper - https://doi.org/10.1016/j.cell.2014.10.056

Structure and calcium regulation mechanism of ancestral α-actinin

Actin is one of the most abundant proteins in eukaryotic cells. Actin filaments together with a large number of actin-binding proteins are critical players in many cellular functions, ranging from cell motility and muscle contraction to maintenance of cell shape and transcription regulation. α-Actinin—a member of the spectrin superfamily—is an archetypical F-actin–binding and –bundling protein. It is known that Ca²⁺ inhibits α-actinin capacity to bundle F-actin. We present a structure of a Ca²⁺ -regulated α-actinin and propose the mechanism for its regulation. We uncover that Ca²⁺ binding triggers an increase in protein rigidity, leading to reduced conformational flexibility and bundling activity. The proposed molecular mechanism is likely to be a blueprint for regulation of spectrin-like proteins (Pinotsis et al., PNAS, 2020).

Read more: soft link to paper - https://www.pnas.org/content/117/36/22101

Order from disorder in the sarcomere

In sarcomeres, α-actinin crosslinks actin filaments and anchors them to the Z-disk. FATZ proteins interact with α-actinin and five other core Z-disk proteins, contributing to myofibril assembly and maintenance as a protein interaction hub.

Here we report the first structure and its cellular validation of α-actinin-2 in complex with a Z-disk partner, FATZ-1, which is best described as a conformational ensemble. We show that FATZ-1 forms a tight fuzzy complex with α-actinin-2 and propose a molecular interaction mechanism via main molecular recognition elements and secondary binding sites. The obtained integrative model reveals a polar architecture of the complex which, in combination with FATZ-1 multivalent scaffold function, might organise interaction partners and stabilise α-actinin-2 preferential orientation in the Z-disk.

Finally, we uncover FATZ-1 ability to phase-separate and form biomolecular condensates with α-actinin-2, raising the intriguing question whether FATZ proteins can create an interaction hub for Z-disk proteins through membrane-less compartmentalization during myofibrillogenesis (Sponga et al., Sci Adv, 2021).

Read more: soft link to paper - https://www.science.org/doi/10.1126/sciadv.abg7653

Myotilin as a regulator of sarcomere assembly

The Z-disc is the boundary between adjacent sarcomeres, the basic contractile units of striated muscles. Myotilin is a Z-disc scaffold protein, providing structural integrity by multiple interactions, including F-actin and α-actinin-2. In our study, we provide the first integrative structural model of its complex with F-actin. We further show that myotilin displaces tropomyosin from F-actin, implying a novel role of myotilin in sarcomere biogenesis beyond an interaction hub for Z-disc partners (Kostan et al., PLOS Biol, 2021).

Read more: soft link to paper - https://doi.org/10.1371/journal.pbio.300114

Tailored Suits Fit Better

The major bottleneck in macromolecular crystallography is the production of well-diffracting crystals. To increase the crystallization success, we developed a crystallization strategy customizable for each protein. The underlying concept is to use in crystallization cocktails compounds that increase the thermal stability of the protein. This customized strategy yielded not only twice as many crystal hits as the standard approach but also crystal hits for two additional proteins compared to the benchmark workflow. The inherently simple design and the modular and flexible nature of the platform makes it easy to modify, further develop, and optimize individual steps. The information gained can also be used to increase the monodispersity and stability of proteins, improving in this way their amenability for biochemical studies and eventually derive a possible function of not yet annotated proteins (Mlynek et al., Cryst Growth Des, 2020).

Read more: soft link to paper - https://dx.doi.org/10.1021/acs.cgd.9b01328

Optical tweezers

Stable anchoring of titin in muscle Z-disc is essential for muscle integrity during stretching. α-Actinin provides attachment points through binding to Z-repeats of titin. In collaboration with M. Rief, we used optical tweezers to study the mechanics of this interaction at a single-molecule level. We suggest a model where multiple weak α-actinin/Z-repeat interactions co-operate to ensure a stable titin anchoring while allowing for dynamic exchange of components (Grison et al., PNAS, 2017).

Read more: soft link to paper - https://www.embopress.org/doi/full/10.15252/embr.201439267

FAP52

Cellular and organelle shape can remarkably modulate cellular function. Actin polymerization together with proteins that directly deform membranes, such as the BAR superfamily proteins have been implicated in regulation of membrane shape. Using a combination of structural (MX, cryo-EM), biochemical and cell biophysics approaches we showed that F-BAR domain of pacsin2 binds actin filaments using the same concave surface employed to also bind to membranes (Kostan et al., EMBO Rep, 2014).

Read more: soft link to paper - https://doi.org/10.15252/embr.201439267