Phosphatidylinositol 4,5-Bisphosphate Increases Ca2+ Affinity of Synaptotagmin-1 by 40-fold

Bogaart, Geert van den; Meyenberg, Karsten; Diederichsen, Ulf; Jahn, Reinhard

doi:10.1074/jbc.m112.343418

Cited by 119 publications

(159 citation statements)

References 30 publications

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“…S3), showing that Rasal possesses a similar affinity for both lipids. The resultant apparent dissociation constants (K d s) were 35 ± 23 and 29 ± 18 μM for PI3P and PS, respectively, which are in a similar range of other C2 domain affinities for lipids (21)(22)(23)(24).…”

Section: Resultsmentioning

confidence: 72%

Ras GTPase activating (RasGAP) activity of the dual specificity GAP protein Rasal requires colocalization and C2 domain binding to lipid membranes

Sot

Behrmann

Raunser

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Rasal, belonging to the GAP1 subfamily of Ras GTPase-activating proteins (RasGAPs) with dual RasGAP/RapGAP specificity, is epigenetically silenced in several tumor types. Surprisingly, the isolated protein has GAP activity on Rap but not on Ras. Its membrane recruitment is regulated by interaction with calcium and lipids, which simultaneously induces its RasGAP activity through a yet unknown mechanism. Here we show that the interaction of Rasal with membranes induces Rasal RasGAP activity by spatial and conformational regulation, although it does not have any effect on its RapGAP activity. Not only is colocalization of Rasal and Ras in the membrane essential for RasGAP activation, but direct and Ca-dependent interaction between the tandem C2 domains of Rasal and lipids of the membrane is also required. Whereas the C2A domain binds specifically phosphatidylserine, the C2B domain interacts with several phosphoinositol lipids. Finally we show, that similar to the C2 domains of synaptotagmins, the Rasal tandem C2 domains are able to sense and induce membrane curvature by the insertion of hydrophobic loops into the membrane.

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Section: Resultsmentioning

confidence: 72%

Ras GTPase activating (RasGAP) activity of the dual specificity GAP protein Rasal requires colocalization and C2 domain binding to lipid membranes

Sot

Behrmann

Raunser

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Ca 2+ binds to the Ca 2+ -binding region, comprising two inter-strand loops, via five conserved acidic residues [4,5]. Many C 2 domains also bind soluble NSF attachment receptor (SNARE) proteins, key proteins in vesicle fusion, and anionic phospholipids (PLs), including phosphatidylserine and phosphatidylinositides, in a Ca 2+ -dependent manner [6][7][8][9][10][11]. In turn, the binding of PLs increases the affinity of the C 2 domains for Ca 2+ [8,[12][13][14][15][16].…”

Section: Introductionmentioning

confidence: 99%

The C2B Domain Is the Primary Ca2+ Sensor in DOC2B: A Structural and Functional Analysis

Giladi

Michaeli

Almagor

et al. 2013

Journal of Molecular Biology

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DOC2B (double-C 2 domain) protein is thought to be a high-affinity Ca 2+ sensor for spontaneous and asynchronous neurotransmitter release. To elucidate the molecular features underlying its physiological role, we determined the crystal structures of its isolated C2A and C2B domains and examined their Ca 2+ -binding properties. We further characterized the solution structure of the tandem domains (C2AB) using small-angle X-ray scattering. In parallel, we tested structure-function correlates with live cell imaging tools. We found that, despite striking structural similarity, C2B binds Ca 2+ with considerably higher affinity than C2A. The C2AB solution structure is best modeled as two domains with a highly flexible orientation and no difference in the presence or absence of Ca 2+ . In addition, kinetic studies of C2AB demonstrate that, in the presence of unilamellar vesicles, Ca 2+ binding is stabilized, as reflected by the~10-fold slower rate of Ca 2+ dissociation than in the absence of vesicles. In cells, isolated C2B translocates to the plasma membrane (PM) with an EC 50 of 400 nM while the C2A does not translocate at submicromolar Ca 2+ concentrations, supporting the biochemical observations. Nevertheless, C2AB translocates to the PM with an~2-fold lower EC 50 and to a greater extent than C2B. Our results, together with previous studies, reveal that the C2B is the primary Ca 2+ sensing unit in DOC2B, whereas C2A enhances the interaction of C2AB with the PM.

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“…This site is also conserved in a wide variety of C2 domains of topology I, for example synaptotagmins, rabphilin 3A, DOC2, and PI3KC2α (10,(16)(17)(18)(19). Given the importance of PI(4,5)P 2 for bringing the vesicle and plasma membranes together before exocytosis to ensure rapid and efficient fusion upon calcium influx (20-23), it is crucial to understand the molecular mechanisms beneath this event.Many studies have reported different and contradictory results about the membrane binding properties of C2A and C2B domains of synaptotagmin 1 and rabphilin 3A providing an unclear picture about how Ca 2+ and PI(4,5)P 2 combine to orchestrate the vesicle fusion and repriming processes by acting through the two C2 domains existing in each of these proteins (16,20,22,(24)(25)(26)(27)(28). A myriad of works have explored the 3D structure of the individual C2 domains of both synaptotagmins and rabphilin 3A (5,26,27,29,30).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

“…Many studies have reported different and contradictory results about the membrane binding properties of C2A and C2B domains of synaptotagmin 1 and rabphilin 3A providing an unclear picture about how Ca 2+ and PI(4,5)P 2 combine to orchestrate the vesicle fusion and repriming processes by acting through the two C2 domains existing in each of these proteins (16,20,22,(24)(25)(26)(27)(28). A myriad of works have explored the 3D structure of the individual C2 domains of both synaptotagmins and rabphilin 3A (5,26,27,29,30).…”

mentioning

confidence: 99%

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Structural insights into the Ca ²⁺ and PI(4,5)P ₂ binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

Guillén

Ferrer-Orta

Buxaderas

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Proteins containing C2 domains are the sensors for Ca 2+ and PI (4,5)P 2 in a myriad of secretory pathways. Here, the use of a freemounting system has enabled us to capture an intermediate state of Ca 2+ binding to the C2A domain of rabphilin 3A that suggests a different mechanism of ion interaction. We have also determined the structure of this domain in complex with PI(4,5)P 2 and IP 3 at resolutions of 1.75 and 1.9 Å, respectively, unveiling that the polybasic cluster formed by strands β3-β4 is involved in the interaction with the phosphoinositides. A comparative study demonstrates that the C2A domain is highly specific for PI(4,5)P 2 /PI (3,4,5)P 3 , whereas the C2B domain cannot discriminate among any of the diphosphorylated forms. Structural comparisons between C2A domains of rabphilin 3A and synaptotagmin 1 indicated the presence of a key glutamic residue in the polybasic cluster of synaptotagmin 1 that abolishes the interaction with PI (4,5)P 2 . Together, these results provide a structural explanation for the ability of different C2 domains to pull plasma and vesicle membranes close together in a Ca 2+ -dependent manner and reveal how this family of proteins can use subtle structural changes to modulate their sensitivity and specificity to various cellular signals.PIP2 | calcium | vesicle fusion C 2 modules are most commonly found in enzymes involved in lipid modifications and signal transduction and in proteins involved in membrane trafficking. They consist of 130 residues and share a common fold composed of two four-stranded β-sheets arranged in a compact β-sandwich connected by surface loops and helices (1-4). Many of these C2 domains have been demonstrated to function in a Ca 2+ -dependent membrane-binding manner and hence act as cellular Ca 2+ sensors. Calcium ions bind in a cupshaped invagination formed by three loops at one tip of the β-sandwich where the coordination spheres for the Ca 2+ ions are incomplete (5-7). This incomplete coordination sphere can be occupied by neutral and anionic (7-9) phospholipids, enabling the C2 domain to dock at the membrane.Previous work in our laboratory has shed light on the 3D structure of the C2 domain of PKCα in complex with both PS and PI(4,5)P 2 simultaneously (10). This revealed an additional lipidbinding site located in the polybasic region formed by β3-β4 strands that preferentially binds to PI(4,5)P 2 (11-15). This site is also conserved in a wide variety of C2 domains of topology I, for example synaptotagmins, rabphilin 3A, DOC2, and PI3KC2α (10,(16)(17)(18)(19). Given the importance of PI(4,5)P 2 for bringing the vesicle and plasma membranes together before exocytosis to ensure rapid and efficient fusion upon calcium influx (20-23), it is crucial to understand the molecular mechanisms beneath this event.Many studies have reported different and contradictory results about the membrane binding properties of C2A and C2B domains of synaptotagmin 1 and rabphilin 3A providing an unclear picture about how Ca 2+ and PI(4,5)P 2 combine to orchestrate the vesi...

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Phosphatidylinositol 4,5-Bisphosphate Increases Ca2+ Affinity of Synaptotagmin-1 by 40-fold

Cited by 119 publications

References 30 publications

Ras GTPase activating (RasGAP) activity of the dual specificity GAP protein Rasal requires colocalization and C2 domain binding to lipid membranes

Ras GTPase activating (RasGAP) activity of the dual specificity GAP protein Rasal requires colocalization and C2 domain binding to lipid membranes

The C2B Domain Is the Primary Ca2+ Sensor in DOC2B: A Structural and Functional Analysis

Structural insights into the Ca ²⁺ and PI(4,5)P ₂ binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

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Phosphatidylinositol 4,5-Bisphosphate Increases Ca2+ Affinity of Synaptotagmin-1 by 40-fold

Cited by 119 publications

References 30 publications

Ras GTPase activating (RasGAP) activity of the dual specificity GAP protein Rasal requires colocalization and C2 domain binding to lipid membranes

Ras GTPase activating (RasGAP) activity of the dual specificity GAP protein Rasal requires colocalization and C2 domain binding to lipid membranes

The C2B Domain Is the Primary Ca2+ Sensor in DOC2B: A Structural and Functional Analysis

Structural insights into the Ca 2+ and PI(4,5)P 2 binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1

Contact Info

Product

Resources

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Structural insights into the Ca ²⁺ and PI(4,5)P ₂ binding modes of the C2 domains of rabphilin 3A and synaptotagmin 1