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Calcium binding promotes conformational flexibility of the neuronal Ca(2+) sensor synaptotagmin.

ABSTRACT: Synaptotagmin 1 (Syt1) is a synaptic vesicle protein that serves as a calcium sensor of neuronal secretion. It is established that calcium binding to Syt1 triggers vesicle fusion and release of neuronal transmitters, however, the dynamics of this process is not fully understood. To investigate how Ca(2+) binding affects Syt1 conformational dynamics, we performed prolonged molecular dynamics (MD) simulations of Ca(2+)-unbound and Ca(2+)-bound forms of Syt1. MD simulations were performed at a microsecond scale and combined with Monte Carlo sampling. We found that in the absence of Ca(2+) Syt1 structure in the solution is represented by an ensemble of conformational states with tightly coupled domains. To investigate the effect of Ca(2+) binding, we used two different strategies to generate a molecular model of a Ca(2+)-bound form of Syt1. First, we employed subsequent replacements of monovalent cations transiently captured within Syt1 Ca(2+)-binding pockets by Ca(2+) ions. Second, we performed MD simulations of Syt1 at elevated Ca(2+) levels. All the simulations produced Syt1 structures bound to four Ca(2+) ions, two ions chelated at the binding pocket of each domain. MD simulations of the Ca(2+)-bound form of Syt1 revealed that Syt1 conformational flexibility drastically increased upon Ca(2+) binding. In the presence of Ca(2+), the separation between domains increased, and interdomain rotations became more frequent. These findings suggest that Ca(2+) binding to Syt1 may induce major changes in the Syt1 conformational state, which in turn may initiate the fusion process.

SUBMITTER: Bykhovskaia M

PROVIDER: S-EPMC4457270 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Calcium binding promotes conformational flexibility of the neuronal Ca(2+) sensor synaptotagmin.

Bykhovskaia Maria M

Biophysical journal 20150501 10

Synaptotagmin 1 (Syt1) is a synaptic vesicle protein that serves as a calcium sensor of neuronal secretion. It is established that calcium binding to Syt1 triggers vesicle fusion and release of neuronal transmitters, however, the dynamics of this process is not fully understood. To investigate how Ca(2+) binding affects Syt1 conformational dynamics, we performed prolonged molecular dynamics (MD) simulations of Ca(2+)-unbound and Ca(2+)-bound forms of Syt1. MD simulations were performed at a micr ...[more]

PMID: 25992729

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Project description:Neuronal calcium sensor-1 (NCS-1) protein is abundantly expressed in the central nervous system and retinal neurons, where it regulates many vital processes such as synaptic transmission. It coordinates three calcium ions by EF-hands 2-4, thereby transducing Ca2+ signals to a wide range of protein targets, including G protein-coupled receptors and their kinases. Here, we demonstrate that NCS-1 also has Zn2+-binding sites, which affect its structural and functional properties upon filling. Fluorescence and circular dichroism experiments reveal the impact of Zn2+ binding on NCS-1 secondary and tertiary structure. According to atomic absorption spectroscopy and isothermal titration calorimetry studies, apo-NCS-1 has two high-affinity (4 × 106 M-1) and one low-affinity (2 × 105 M-1) Zn2+-binding sites, whereas Mg2+-loaded and Ca2+-loaded forms (which dominate under physiological conditions) bind two zinc ions with submicromolar affinity. Metal competition analysis and circular dichroism studies suggest that Zn2+-binding sites of apo- and Mg2+-loaded NCS-1 overlap with functional EF-hands of the protein. Consistently, high Zn2+ concentrations displace Mg2+ from the EF-hands and decrease the stoichiometry of Ca2+ binding. Meanwhile, one of the EF-hands of Zn2+-saturated NCS-1 exhibits a 14-fold higher calcium affinity, which increases the overall calcium sensitivity of the protein. Based on QM/MM molecular dynamics simulations, Zn2+ binding to Ca2+-loaded NCS-1 could occur at EF-hands 2 and 4. The high-affinity zinc binding increases the thermal stability of Ca2+-free NCS-1 and favours the interaction of its Ca2+-loaded form with target proteins, such as dopamine receptor D2R and GRK1. In contrast, low-affinity zinc binding promotes NCS-1 aggregation accompanied by the formation of twisted rope-like structures. Altogether, our findings suggest a complex interplay between magnesium, calcium and zinc binding to NCS-1, leading to the appearance of multiple conformations of the protein, in turn modulating its functional status.

Project description:In neurons, entry of extracellular calcium (Ca(2+)) into synaptic terminals through Cav2.1 (P/Q-type) Ca(2+) channels is the driving force for exocytosis of neurotransmitter-containing synaptic vesicles. This class of Ca(2+) channel is, therefore, pivotal during normal neurotransmission in higher organisms. In response to channel opening and Ca(2+) influx, specific Ca(2+)-binding proteins associate with cytoplasmic regulatory domains of the P/Q channel to modulate subsequent channel opening. Channel modulation in this way influences synaptic plasticity with consequences for higher-level processes such as learning and memory acquisition. The ubiquitous Ca(2+)-sensing protein calmodulin (CaM) regulates the activity of all types of mammalian voltage-gated Ca(2+) channels, including the P/Q class, by direct binding to specific regulatory motifs. More recently, experimental evidence has highlighted a role for additional Ca(2+)-binding proteins, particularly of the CaBP and NCS families in the regulation of P/Q channels. NCS-1 is a protein found from yeast to humans and that regulates a diverse number of cellular functions. Physiological and genetic evidence indicates that NCS-1 regulates P/Q channel activity, including calcium-dependent facilitation, although a direct physical association between the proteins has yet to be demonstrated. In this study, we aimed to determine if there is a direct interaction between NCS-1 and the C-terminal cytoplasmic tail of the Cav2.1 ?-subunit. Using distinct but complementary approaches, including in vitro binding of bacterially expressed recombinant proteins, fluorescence spectrophotometry, isothermal titration calorimetry, nuclear magnetic resonance, and expression of fluorescently tagged proteins in mammalian cells, we show direct binding and demonstrate that CaM can compete for it. We speculate about how NCS-1/Cav2.1 association might add to the complexity of calcium channel regulation mediated by other known calcium-sensing proteins and how this might help to fine-tune neurotransmission in the mammalian central nervous system.

Dataset Information

Calcium binding promotes conformational flexibility of the neuronal Ca(2+) sensor synaptotagmin.

Publications

Calcium binding promotes conformational flexibility of the neuronal Ca(2+) sensor synaptotagmin.

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