Recoverin is a neuronal
calcium sensor involved in vision adaptation
that reversibly associates with cellular membranes via its calcium-activated
myristoyl switch. While experimental evidence shows that the myristoyl
group significantly enhances membrane affinity of this protein, molecular
details of the binding process are still under debate. Here, we present
results of extensive molecular dynamics simulations of recoverin in
the proximity of a phospholipid bilayer. We capture multiple events
of spontaneous membrane insertion of the myristoyl moiety and confirm
its critical role in the membrane binding. Moreover, we observe that
the binding strongly depends on the conformation of the N-terminal
domain. We propose that a suitable conformation of the N-terminal
domain can be stabilized by the disordered C-terminal segment or by
binding of the target enzyme, i.e., rhodopsin kinase. Finally, we
find that the presence of negatively charged lipids in the bilayer
stabilizes a physiologically functional orientation of the membrane-bound
recoverin.
Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein‐protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid‐liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA‐RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.
The detailed functional mechanism of recoverin, which acts as a myristoyl switch at the rod outer-segment disk membrane, is elucidated by direct and replica-exchange molecular dynamics. In accord with NMR structural evidence and calcium binding assays, simulations point to the key role of enhanced calcium binding to the EF3 loop of the semiopen state of recoverin as compared to the closed state. This 2-4-order decrease in calcium dissociation constant stabilizes the semiopen state in response to the increase of cytosolic calcium concentration in the vicinity of recoverin. A second calcium ion then binds to the EF2 loop and, consequently, the structure of the protein changes from the semiopen to the open state. The latter has the myristoyl chain extruded to the cytosol, ready to act as a membrane anchor of recoverin.
Multiconfiguration pair-density functional (MC-PDFT) theory provides an economical way to calculate the ground-state and excited-state energetics of strongly correlated systems. The energy is calculated from the kinetic energy, density, and on-top pair-density of a multiconfiguration wave function as the sum of kinetic energy, classical Coulomb energy, and on-top density functional energy. We have usually found good results with the translated Perdew-Burke-Ernzerhof (tPBE) on-top density functional, and in this article, we examine whether the results can be systematically improved by introducing scaling constants into the exchange and correlation terms. We find that only a small improvement is possible for electronic excitation energies and that no improvement is possible for bond energies.
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