C2 Domains from Different Ca<sup>2+</sup> Signaling Pathways Display Functional and Mechanistic Diversity

Nalefski, Eric A.; Wisner, Mark A.; Chen, James Z.; Sprang, Stephen R.; Fukuda, Mitsunori; Mikoshiba, Katsuhiko; Falke, Joseph J.

doi:10.1021/bi001968a

Cited by 117 publications

(190 citation statements)

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“…The reduced magnitude of this electrostatic switch increases the relative importance of other activation mechanisms, particularly the direct or indirect coordination of the two Ca 2+ in the binding pocket by lipid headgroup oxygens upon membrane docking. Evidence supporting the essential roles of both the electrostatic switch activation mechanism and the coordinating lipid activation mechanism is now available for C2 domains, suggesting that both mechanisms contribute to Ca 2+ -activated membrane docking (5,6,14,(41)(42)(43)(44)(45).…”

Section: Discussionmentioning

confidence: 99%

“…Ca 2+ binding to these sites is known to trigger a local structural change that increases the intrinsic emission of Trp 71 by 15%. The resulting fluorescence increase has been used previously to monitor the titration of sites I and II with two Ca 2+ ions, yielding a [Ca 2+ ] 1/2 of 14 ± 2 μM and a Hill coefficient of 1.7 ± 0.2 (6,18). Since the Hill coefficient significantly exceeds 1.0, there is strong positive cooperativity between the two adjacent sites (18).…”

Section: Effects Of Coordinating Side Chain Substitutions On Ca 2+ Bimentioning

confidence: 99%

“…For three residues i, j and k, and for the probability that residue i is protonated before j and residue j before k, eq 5 reduces to a product of two terms (6) The probabilities that these three aspartates would be protonated in each of the six possible consecutive orders are listed in Table 2.…”

Section: Calculation Of Pseudo-pk a 'S For The Coordinating Aspartatesmentioning

confidence: 99%

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Ca²⁺ Activation of the cPLA₂ C2 Domain: Ordered Binding of Two Ca²⁺ Ions with Positive Cooperativity

et al. 2004

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During Ca 2+ activation, the Ca 2+ -binding sites of C2 domains typically bind multiple Ca 2+ ions in close proximity. These binding events exhibit positive cooperativity, despite the strong charge repulsion between the adjacent divalent cations. Using both experimental and computational approaches, the present study probes the detailed mechanisms of Ca 2+ activation and positive cooperativity for the C2 domain of cytosolic phospholipase A 2 , which binds two Ca 2+ ions in sites I and II, separated by only 4.1 Å. First, each of the five coordinating side chains in the Ca 2+ -binding cleft is individually mutated and the effect on Ca 2+ -binding affinity and cooperativity is measured. The results identify Asp 43 as the major contributor to Ca 2+ affinity, while the two coordinating side chains that provide bridging coordination to both Ca 2+ ions, Asp 43 and Asp 40, are observed to make the largest contributions to positive cooperativity. Electrostatic calculations reveal that Asp 43 possesses the highest pseudo-pK a of the coordinating acidic residues, as well as the highest general cation affinity, due to its relatively buried location within 3.5 Å of seven protein oxygens with full or partial negative charges. These calculations therefore explain the greater importance of Asp 43 in defining the Ca 2+ affinity. Overall, the experimental and computational results support an activation model in which the first Ca 2+ ion binds usually to site I, thereby preordering both bridging side chains Asp 40 and 43, and partially or fully deprotonating the three coordinating Asp residues. This initial binding event prepares the conformation and protonation state of the remaining site for Ca 2+ binding, enabling the second Ca 2+ ion to bind with higher affinity than the first as required for positive cooperativity.The C2 domain is a ubiquitous membrane-targeting protein module found in a diverse array of signal transducing proteins that regulate key cellular processes at membrane surfaces (reviewed in refs 1-10). These processes include the generation of lipid-derived second messengers, vesicular targetting and fusion, GTPase regulation, protein phosphorylation, pore formation by cytolytic T cells, and ubiquitin-mediated protein degradation. The majority of C2 domains are activated by cytoplasmic Ca 2+ signals and dock to specific membrane-associated targets such as phospholipids or, less commonly, to membrane-bound proteins.The structures of representative Ca 2+ -regulated C2 domains, including the C2A domain of synaptotagmin I (Syt-IA), 1 the C2 domain of cytosolic phospholipase A 2 (α-isoform, cPLA 2 α), and the C2 domain of protein kinase C (PKC), have been determined by X-ray † Support provided by NIH Grants GM R01-063235 (to J.J.F.) and GM R01-054651 (to H.L.S./E.J.), and by a DOE/GTL Grant (to E.J.).

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Section: Discussionmentioning

confidence: 99%

Section: Effects Of Coordinating Side Chain Substitutions On Ca 2+ Bimentioning

confidence: 99%

Section: Calculation Of Pseudo-pk a 'S For The Coordinating Aspartatesmentioning

confidence: 99%

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Ca²⁺ Activation of the cPLA₂ C2 Domain: Ordered Binding of Two Ca²⁺ Ions with Positive Cooperativity

et al. 2004

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“…C2 domain is a unique conserved membrane docking module that targets numerous signaling proteins to membrane surfaces where they regulate diverse process critical for cell signaling (Nalefski et al, 2001;Rizo and Sudhof, 1998). C2 domain contains approximately 120 aa residues that were first identified in protein kinase C (Nishizuka, 1988).…”

Section: Introductionmentioning

confidence: 99%

“…The first category contains proteins involved in membrane protein phosphorylation, in generation or inactivation of lipid-derived second messengers, in activation of GTPase, in ubiquitin ligation, in vesicle targeting and fusion, and in formation of transmembrane pores. The second category includes synaptotagmins, rabphilin-3, RIM, and Munc (Ponting and Parker, 1996;Nalefski and False, 1996;Rizo and Sudhof, 1998;Nalefski et al, 2001). Besides the well characterized proteins, several open reading frames with C2 domains reported in GenBank (for example, proteins with three or four C2 domains in C. elegans and yeast, the putative proteins with just one C2 domain in zebrafish) have not been identified for their biological roles, suggesting a broad range of functional specialization in the C2 domain superfamily and additional interesting functions for C2 domain proteins remain to be discovered (Nalefski et al, 2001;Rizo and Sudhof, 1998).…”

Section: Introductionmentioning

confidence: 99%

Identification of a novel C2 domain factor in ovaries of orange-spotted grouper (Epinephelus coioides)

Ji¹,

Zhou²,

Wang³

et al. 2006

Comparative Biochemistry and Physiology Part B: Biochemistry an

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Membrane BindingC2‐Like Domains

Verdaguer¹,

Corbal��n-Garc��a²,

Ochoa³

et al. 2004

Handbook of Metalloproteins

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C2‐domains are independently folded modules, of about 130 residues, found in a large and diverse set of eukaryotic proteins. Many of the best‐characterized C2‐domains are found in proteins involved in membrane trafficking and fusion, such as synaptotagmins or rabphilin, and in signal transduction, such as phospholipases A2 (cPLA2) and C (PLC) or the protein kinase C isoforms (PKCs). All C2‐domains share a common overall fold: a single compact Greek‐key motif organized as an eight‐stranded antiparallel β‐sandwich consisting of a pair of four‐stranded β‐sheets – the A and B sheets – with a long axis of about 50 Å. Connections between β‐strands emerge from the top and bottom of the domain according to the two distinct topologies of C2‐domains. In topology I (the S family), the first β‐strand occupies the same structural position as the eighth β‐strand of topology II (the P family), which shifts in a circular permutation the order of homologous strands in the primary structure. The structural information defines adjacent calcium binding sites within a broad acidic cleft that contains highly conserved acidic residues. These provide most of the oxygen atoms that coordinate with the calcium ions. The present work will focus on the wealth of functional and structural information, which is being obtained at an exponential pace, on the membrane‐binding C2‐domains that require calcium.

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C2 Domains from Different Ca²⁺ Signaling Pathways Display Functional and Mechanistic Diversity

Cited by 117 publications

References 64 publications

Ca²⁺ Activation of the cPLA₂ C2 Domain: Ordered Binding of Two Ca²⁺ Ions with Positive Cooperativity

Ca²⁺ Activation of the cPLA₂ C2 Domain: Ordered Binding of Two Ca²⁺ Ions with Positive Cooperativity

Identification of a novel C2 domain factor in ovaries of orange-spotted grouper (Epinephelus coioides)

Membrane BindingC2‐Like Domains

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C2 Domains from Different Ca2+ Signaling Pathways Display Functional and Mechanistic Diversity

Cited by 117 publications

References 64 publications

Ca2+ Activation of the cPLA2 C2 Domain: Ordered Binding of Two Ca2+ Ions with Positive Cooperativity

Ca2+ Activation of the cPLA2 C2 Domain: Ordered Binding of Two Ca2+ Ions with Positive Cooperativity

Identification of a novel C2 domain factor in ovaries of orange-spotted grouper (Epinephelus coioides)

Membrane BindingC2‐Like Domains

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C2 Domains from Different Ca²⁺ Signaling Pathways Display Functional and Mechanistic Diversity

Ca²⁺ Activation of the cPLA₂ C2 Domain: Ordered Binding of Two Ca²⁺ Ions with Positive Cooperativity

Ca²⁺ Activation of the cPLA₂ C2 Domain: Ordered Binding of Two Ca²⁺ Ions with Positive Cooperativity