Abstract
The activity of several cytosolic proteins critically depends on the concentration of calcium ions. One important intracellular calcium-sensing protein is α-actinin-1, the major actin crosslinking protein in focal adhesions and stress fibers. The actin crosslinking activity of α-actinin-1 has been proposed to be negatively regulated by calcium, but the underlying molecular mechanisms are poorly understood. To address this, we determined the first high-resolution NMR structure of its functional calmodulin-like domain (CaMD) in calcium-bound and calcium-free form. These structures reveal that in the absence of calcium, CaMD displays a conformationally flexible ensemble that undergoes a structural change upon calcium binding, leading to limited rotation of the N- and C-terminal lobes around the connecting linker and consequent stabilization of the calcium-loaded structure. Mutagenesis experiments, coupled with mass-spectrometry and isothermal calorimetry data designed to validate the calcium binding stoichiometry and binding site, showed that human non-muscle α-actinin-1 binds a single calcium ion within the N-terminal lobe. Finally, based on our structural data and analogy with other α-actinins, we provide a structural model of regulation of the actin crosslinking activity of α-actinin-1 where calcium induced structural stabilisation causes fastening of the juxtaposed actin binding domain, leading to impaired capacity to crosslink actin.
Highlights
Are located on opposite ends of the antiparallel dimer, stabilized by extensive subunit interactions between the central rod domain, which is composed of four spectrin repeats (SR)[7,8,9]
These results suggest that calcium binding to EF1-2 is necessary for the interaction of EF3-4 with the neck region
To assess the calcium-binding potential of the four putative calcium sites of α-actinin-1 (EF1 to EF4), we compared their sequences to sequences of EF hands from several other calcium-binding proteins, in which the Ca2+ ion is coordinated in a canonical pentagonal bipyramidal configuration (Fig. 2a,b)
Summary
Are located on opposite ends of the antiparallel dimer, stabilized by extensive subunit interactions between the central rod domain, which is composed of four spectrin repeats (SR)[7,8,9]. The calmodulin-like domain (CaMD) of the opposite subunit is located in close proximity, where it regulates the actin-crosslinking activity of α-actinin[9]. Partner helices within each EF hand are re-oriented from nearly antiparallel to an almost perpendicular orientation This results in exposure of large hydrophobic patches on the surface of each lobe (closed-to-open transition) and increased affinity toward its interaction partners[12]. Some insight into the calcium regulation mechanism of non-muscle α-actinin is provided by a model of the ternary complex between ABD, adjacent helical neck region, and the CaMD of α-actinin-4 Mutagenesis experiments showed that complex disruption enhances binding of α-actinin to actin[13] These results suggest that calcium binding to EF1-2 is necessary for the interaction of EF3-4 with the neck region. We constructed a model of Ca2+-induced structural changes of α-actinin-1, which are central to its actin-crosslinking activity
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