Effect of PIP<sub>2</sub> Binding on the Membrane Docking Geometry of PKCα C2 Domain: An EPR Site-Directed Spin-Labeling and Relaxation Study

Landgraf, Kyle E.; Malmberg, Nathan J.; Falke, Joseph J.

doi:10.1021/bi800711t

Cited by 43 publications

(102 citation statements)

References 53 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: 79%

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: 79%

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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“…Noteworthy, the Ca 2ϩ binding affinity determined here is close to the concentrations of Ca 2ϩ microdomains required for the vesicle fusion process (49). For several other C2 domains it was also shown that the formation of localized pools of PIP2 plays an important role for enhancing Ca 2ϩ binding affinity (26,50). This may also be the case for the C2 domains of rabphilin-3A.…”

Section: Discussionsupporting

confidence: 72%

Structural Determinants for Ca2+ and Phosphatidylinositol 4,5-Bisphosphate Binding by the C2A Domain of Rabphilin-3A

Coudevylle

Montaville

Leonov

et al. 2008

Journal of Biological Chemistry

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Rabphilin-3A is a neuronal C2 domain tandem containing protein involved in vesicle trafficking. Both its C2 domains (C2A and C2B) are able to bind phosphatidylinositol 4,5-bisphosphate, a key player in the neurotransmitter release process. The rabphilin-3A C2A domain has previously been shown to bind inositol-1,4,5-trisphosphate (IP3; phosphatidylinositol 4,5-bisphosphate headgroup) in a Ca 2؉ -dependent manner with a relatively high affinity (50 M 2؉ binding enables the interaction with IP3 and vice versa, in line with a target-activated messenger affinity mechanism. Our data provide detailed structural insight into the functional properties of the rabphilin-3A C2A domain and reveal for the first time the structural determinants of a target-activated messenger affinity mechanism.)C2 domains are ubiquitous protein modules initially identified in protein kinase C (1). They all consist of ϳ130 residues (2) and share a common fold composed of two four-stranded ␤-sheets arranged in a compact ␤-sandwich and surrounded by variable loops and helices. C2 domain-containing proteins such as phospholipase C, cytosolic phospholipase A2, or protein kinase C are generally involved in signal transduction or membrane trafficking. The main function of the C2 domains in these proteins appears to be the targeting to biological membranes via their Ca 2ϩ -dependent phospholipid binding properties (3).Primary lipid targets are phosphatidylcholine (for cytosolic phospholipase A2 (4)) and phosphatidylserine (for phospholipase C␦ (5), synaptotagmins (6), and protein kinase C␣ (7)), the most abundant phospholipids in the inner membrane leaflet. Although not known to be classical phosphoinositide binding modules, some C2 domains have been shown to interact rather specifically with phosphoinositides (phospholipase C, protein kinase C␣), especially phosphatidylinositol 4,5-bisphosphate (PIP2) 3 (8). PIP2 represents less than 1% of the lipids in the plasma membrane and forms microdomains to regulate several key signaling events (9, 10). The interaction between the C2 domain of protein kinase C␣ and PIP2 is essential for the proper subcellular localization of protein kinase C␣ (11, 12). Several C2 domains of proteins involved in synaptic vesicle fusion have been described to bind PIP2, e.g. C2 domains of some synaptotagmins (6), CAPS-1 (13), and rabphilin-3A (14). The binding of the C2B domain of synaptotagmin 1 to PIP2 prelocalizes the protein to PIP2-enriched membranes (15). Recently a role for the CAPS-1 PIP2 interaction has been shown in the prefusion step of dense core vesicle fusion (13).PIP2 is a key player in the different stages of the neurotransmitter release process. It is required for clathrin-mediated endocytosis (16) or for specific stages of exocytosis and synaptic vesicle trafficking (10). PIP2 recruits proteins to specific locations in the plasma membrane to modulate spatially localized events involved in membrane trafficking (17).Rabphilin-3A was originally identified as a Rab3 effector in neuronal cells (18). It was found t...

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“…Thus, Ca 2ϩ serves a specific structural role in allowing the C2 domain to bind anionic membranes. In addition to the Ca 2ϩ -binding site, a lysine-rich cluster within the ␤3-and ␤4-sheets of the C2 domain binds phosphatidylinositol 4,5-bisphosphate (27,(32)(33)(34)(35)(36). It is this interaction with phosphatidylinositol 4,5-bisphosphate that directs conventional PKC isozymes to the plasma membrane, which is enriched in this lipid (38 -41).…”

mentioning

confidence: 99%

Electrostatic and Hydrophobic Interactions Differentially Tune Membrane Binding Kinetics of the C2 Domain of Protein Kinase Cα

Scott

Antal

Newton³

2013

Journal of Biological Chemistry

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Background:The C2 domain is a Ca 2ϩ sensor that drives the first step in the activation of conventional PKC isozymes. Results: Hydrophobic interactions drive membrane association and electrostatic interactions drive membrane retention of the C2 domain. Conclusion:The amplitude and location of conventional PKC signaling is controlled by residues in the C2 domain. Significance: Point mutations in the C2 domain are associated with disease.

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Effect of PIP₂ Binding on the Membrane Docking Geometry of PKCα C2 Domain: An EPR Site-Directed Spin-Labeling and Relaxation Study

Cited by 43 publications

References 53 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

Structural Determinants for Ca2+ and Phosphatidylinositol 4,5-Bisphosphate Binding by the C2A Domain of Rabphilin-3A

Electrostatic and Hydrophobic Interactions Differentially Tune Membrane Binding Kinetics of the C2 Domain of Protein Kinase Cα

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Effect of PIP2 Binding on the Membrane Docking Geometry of PKCα C2 Domain: An EPR Site-Directed Spin-Labeling and Relaxation Study

Cited by 43 publications

References 53 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

Structural Determinants for Ca2+ and Phosphatidylinositol 4,5-Bisphosphate Binding by the C2A Domain of Rabphilin-3A

Electrostatic and Hydrophobic Interactions Differentially Tune Membrane Binding Kinetics of the C2 Domain of Protein Kinase Cα

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Effect of PIP₂ Binding on the Membrane Docking Geometry of PKCα C2 Domain: An EPR Site-Directed Spin-Labeling and Relaxation Study

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