Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment

Honigmann, Alf; Bogaart, Geert van den; Iraheta, Emilio; Risselada, Herre Jelger; Milovanović, Dragomir; Mueller, Veronika; Müllar, Stefan; Diederichsen, Ulf; Fasshauer, Dirk; Grubmüller, Helmut; Hell, Stefan W.; Eggeling, Christian; Kühnel, Karin; Jahn, Reinhard

doi:10.1038/nsmb.2570

Cited by 263 publications

(341 citation statements)

References 57 publications

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“…Given the close proximity of the lysine-rich cluster and the CBR, these results are compatible with a parallel orientation model (Fig. 6F) in which these two motifs would interact with a vesicle representing the plasma membrane, leaving the bottom face free to interact in trans with another lipid vesicle (19,(47)(48)(49)(50)(51). How these two C2 domains act in tandem and in cooperation with other SNARE proteins to regulate vesicle fusion is still under debate and will need further exploration.…”

Section: Resultssupporting

confidence: 68%

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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Section: Resultssupporting

confidence: 68%

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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“…EPR studies also yielded strong support for the direct bridging model for lnC 2 AB (24), and no oligomerization of lnC 2 AB under membrane bridging conditions was observed by EPR (24) or fluorescence spectroscopy (44). The study that proposed the oligomerization model using lnC 2 AB (29) did not account for the EPR data or the results that were already available for C 2 AB (20); relied on the cryo-EM images of a separate study (25); and supported the model with a series of indirect experiments, without providing direct evidence for the existence of proteinprotein interactions that help to bridge the membranes.…”

Section: Discussionmentioning

confidence: 99%

Prevalent mechanism of membrane bridging by synaptotagmin-1

Seven¹,

Brewer²,

Shi

et al. 2013

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

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Synaptotagmin-1 functions as a Ca 2+ sensor in neurotransmitter release through its two C 2 domains (the C 2 A and C 2 B domain). The ability of synaptotagmin-1 to bridge two membranes is likely crucial for its function, enabling cooperation with the soluble Nethylmaleimide sensitive factor adaptor protein receptors (SNAREs) in membrane fusion, but two bridging mechanisms have been proposed. A highly soluble synaptotagmin-1 fragment containing both domains (C 2 AB) was shown to bind simultaneously to two membranes via the Ca 2+ -binding loops at the top of both domains and basic residues at the bottom of the C 2 B domain (direct bridging mechanism). In contrast, a longer fragment including a linker sequence (lnC 2 AB) was found to aggregate in solution and was proposed to bridge membranes through trans interactions between lnC 2 AB oligomers bound to each membrane via the Ca 2+ -binding loops, with no contact of the bottom of the C 2 B domain with the membranes. We now show that lnC 2 AB containing impurities indeed aggregates in solution, but properly purified lnC 2 AB is highly soluble. Moreover, cryo-EM images reveal that a majority of lnC 2 AB molecules bridge membranes directly. Fluorescence spectroscopy indicates that the bottom of the C 2 B domain contacts the membrane in a sizeable population of molecules of both membranebound C 2 AB and membrane-bound lnC 2 AB. NMR data on nanodiscs show that a fraction of C 2 AB molecules bind to membranes with antiparallel orientations of the C 2 domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by both C 2 AB and lnC 2 AB, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release.membrane bending | Ca2+ sensing | exocytosis | synaptic transmission N eurotransmitter release is a key event in interneuronal communication that is acutely triggered by Ca 2+ influx into a presynaptic terminal (1). The synaptic vesicle protein synaptotagmin-1 acts as a Ca 2+ sensor in fast release through the two C 2 domains that form most of its cytoplasmic region (the C 2 A and C 2 B domains) (2-5). This function is coupled to membrane fusion through the neuronal soluble N-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) proteins (6, 7), which bring the synaptic vesicle and plasma membranes together by forming SNARE complexes (2, 3). Synaptotagmin-1 function also depends on a tight interplay with complexins (8-10) and other key proteins of the release machinery (11, 12). The synaptotagmin-1 C 2 domains bind three or two Ca 2+ ions through loops at the top of β-sandwich structures (13-15) (Fig. 1A). These top loops also mediate Ca 2+ -dependent phospholipid binding (15-17), which is crucial for synaptotagmin-1 function (5, 18). Moreover, the synaptotagmin-1 cytoplasmic region clusters chromaffin granules and liposomes (19), and a fragment containing its two C 2 domains was shown by cryo-EM to bind simultaneously to two membranes, bringing them i...

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“…Thus, it appears to be necessary for Ca 2ϩ sensor Syt1 to gain intimate access to the SNAREpin (32). In fact, it is shown that Syt1 has the capacity to bind the SNARE core and negatively charged lipids clustered in the immediate vicinity of the SNARE complex (38,54).…”

Section: Discussionmentioning

confidence: 99%

Synaptotagmin-1 Is an Antagonist for Munc18-1 in SNARE Zippering

Lou

Shin

Yang

et al. 2015

Journal of Biological Chemistry

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Background: The molecular mechanisms of the critical necessity of Munc18-1 protein for neurotransmitter release remain unclear. Results: Synaptotagmin-1 competes with Munc18-1 in SNARE zippering and fusion pore opening. Conclusion: Synaptotagmin-1 wins the tug-of-war in gaining control of the SNAREpin at the moment of membrane fusion. Significance: This work clarifies an ambiguity concerning the Munc18-1 function in neuroexocytosis.

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Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment

Cited by 263 publications

References 57 publications

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

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

Prevalent mechanism of membrane bridging by synaptotagmin-1

Synaptotagmin-1 Is an Antagonist for Munc18-1 in SNARE Zippering

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Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment

Cited by 263 publications

References 57 publications

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

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

Prevalent mechanism of membrane bridging by synaptotagmin-1

Synaptotagmin-1 Is an Antagonist for Munc18-1 in SNARE Zippering

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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

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