C2 domain conformational changes in phospholipase C-δ1

Grobler, Jay A.; Essen, Lars‐Oliver; Williams, Roger L.; Hurley, James H.

doi:10.1038/nsb0996-788

Cited by 111 publications

(115 citation statements)

References 42 publications

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“…The C2A domain exhibits a similar conformational change in limited proteolysis experiments (Davletov and Südhof 1994). These changes may reflect the movement of charged residues within or near the Ca 2+ -binding loops (Ubach et al 1998b; Chae et al 1998), as indicated by structural studies of the C2 domain of phospholipase C-δ1 (Grobler et al 1996). Given the diffusion-limited kinetics of the C2 domains of synaptotagmin, these conformational changes are unlikely to involve large-scale structural rearrangements (Davis et al 1999).…”

Section: Discussionmentioning

confidence: 99%

“…(A) Schematic diagram of putative Ca 2+ ligands within the C2B domain of synaptotagmin I. This model is based on structural studies of a variety of C2 domains (Sutton and Sprang 1995; Sutton et al 1995; Grobler et al 1996; Perisic et al 1998; Ubach et al 1998a,Ubach et al 1998b) and alignments between the C2A and C2B domains of synaptotagmin. (B) Wild-type (WT) and the indicated synaptotagmin Ib point mutants were analyzed for Ca 2+ -triggered oligomerization activity as described in Fig.…”

Section: Figurementioning

confidence: 99%

“…Three flexible loops protrude from the tip of the domain. In some cases, two of these loops form a pocket that mediates the binding of Ca 2 + and other divalent cations (Sutton and Sprang 1995; Sutton et al 1995, Sutton et al 1999; Grobler et al 1996; Perisic et al 1998; Ubach et al 1998a,Ubach et al 1998b; Pratt et al 1999; Macedo-Ribeiro et al 1999). Ca 2 + binding regulates the interaction of some C2 domains with target molecules including lipids and proteins, while other C2 domains do not appear to bind Ca 2 +.…”

Section: Introductionmentioning

confidence: 99%

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The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

Desai

Vyas

Earles

et al. 2000

The Journal of Cell Biology

115

165

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The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca2+ sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca2+ ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca2+-sensing module. Here, we report that Ca2+ drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca2+ are mediated by a set of conserved acidic Ca2+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca2+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

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Section: Discussionmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

Desai

Vyas

Earles

et al. 2000

The Journal of Cell Biology

115

165

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“…The crystal structures of PI-PLC␦1 (35)(36)(37) show the active site to be in a ␤␣-barrel similar to B. thuringiensis PI-PLC. The rim of this barrel, like the bacterial enzyme, has a number of hydrophobic residues (Trp, Leu, and Phe).…”

Section: Discussionmentioning

confidence: 99%

Role of Tryptophan Residues in Interfacial Binding of Phosphatidylinositol-specific Phospholipase C

Feng

Wehbi

Roberts

2002

Journal of Biological Chemistry

156

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The phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis exhibits several types of interfacial activation. In the crystal structure of the closely related Bacillus cereus PI-PLC, the rim of the active site is flanked by a short helix B and a loop that show an unusual clustering of hydrophobic amino acids. Two of the seven tryptophans in PI-PLC are among the exposed residues. To test the importance of these residues in substrate and activator binding, we prepared several mutants of Trp-47 (in helix B) and Trp-242 (in the loop). Two other tryptophans, Trp-178 and Trp-280, which are not near the rim, were mutated as controls. Kinetic (both phosphotransferase and cyclic phosphodiesterase activities), fluorescence, and vesicle binding analyses showed that both Trp-47 and Trp-242 residues are important for the enzyme to bind to interfaces, both activating zwitterionic and substrate anionic surfaces. Partitioning of the enzyme to vesicles is decreased more than 10-fold for either W47A or W242A, and removal of both tryptophans (W47A/W242A) yields enzyme with virtually no affinity for phospholipid surfaces. Replacement of either tryptophan with phenylalanine or isoleucine has moderate effects on enzyme affinity for surfaces but yields a fully active enzyme. These results are used to describe how the enzyme is activated by interfaces. Phosphatidylinositol-specific phospholipase C (PI-PLC)1 catalyzes the hydrolysis of PI in two steps: (i) an intramolecular phosphotransferase reaction to form inositol 1,2-cyclic-phosphate (cIP), followed by (ii) a cyclic phosphodiesterase activity that converts cIP to inositol 1-phosphate (I1P). Although the enzymes in eukaryotes play key roles in generating membraneassociated second messengers and in some case water-soluble second messengers (1, 2), PI-PLC enzymes in bacteria are secreted and play critical roles in cell infectivity (3, 4). The PI-PLC from Bacillus thuringiensis exhibits several types of kinetic interfacial activation by interfaces. Micellar PI is a better substrate than monomeric PI (5, 6), and interfaces of phosphatidylcholine, a nonsubstrate that does not bind at the active site, activate the enzyme for both PI cleavage (7) and cIP hydrolysis (8). In the available crystal structure (9) of the closely related Bacillus cereus PI-PLC, bound myo-inositol localized the active site inside the ␤␣ barrel. However, the orientation of PI substrate side chains and any other sites for interfacial PC were not defined.The rim of the active site of bacterial PI-PLC (9) has a short helix B and one particular loop (residues 237-243) that show an unusual clustering of hydrophobic amino acids that are fully exposed to solvent (Fig. 1). This structural characteristic could contribute to the binding of substrate fatty acyl chains (for PI) but also to the binding of the PC activator. Tryptophan residues are often elements inserted into bilayers when a peripheral protein binds (e.g. annexin V (10) or phospholipase A 2 (11)). Two of the seven tryptophans (Trp-47 an...

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“…2A). However, with POPA in the vesicles, the amount of the free rEF decreased proportional to the amount of SUVs present ( ( No calcium ion is found associated with the EF-hand domain that is visible in crystal structures of PLC ␦1, even though the enzyme is soaked in calcium or its analogs (7,(25)(26)(27). The lack of requirement for calcium, taken together with the selective affinity for anionic phospholipids, suggests that basic residues of the EF-hand domain, rather than its acidic residues in coordination with calcium, are likely to contribute to the binding of the EF-hand domain to the membrane surfaces.…”

Section: Isolated Ef-hand Domain Of Plc ␦1 Binds To Anionic Phospholimentioning

confidence: 99%

Ca2+-independent Binding of Anionic Phospholipids by Phospholipase C δ1 EF-hand Domain

Cai

Guo

Lomasney

et al. 2013

Journal of Biological Chemistry

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Background:The conserved EF-hand (EF) domain is necessary for active phospholipase. Results: EF binds to anionic phospholipid-containing vesicles; EF mutations introduced into PLC ␦1 reduce activity not recoverable with added PIP 2 . Conclusion: EF-hand domain aids substrate binding in the active site when the protein is membrane-anchored. Significance: This may be the function of the EF-hand domain in other PLC enzymes as well.

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C2 domain conformational changes in phospholipase C-δ1

Cited by 111 publications

References 42 publications

The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

Role of Tryptophan Residues in Interfacial Binding of Phosphatidylinositol-specific Phospholipase C

Ca2+-independent Binding of Anionic Phospholipids by Phospholipase C δ1 EF-hand Domain

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