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• W1981603102 abstract "During Ca2+ activation, the Ca2+-binding sites of C2 domains typically bind multiple Ca2+ 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 Ca2+ activation and positive cooperativity for the C2 domain of cytosolic phospholipase A2, which binds two Ca2+ ions in sites I and II, separated by only 4.1 Å. First, each of the five coordinating side chains in the Ca2+-binding cleft is individually mutated and the effect on Ca2+-binding affinity and cooperativity is measured. The results identify Asp 43 as the major contributor to Ca2+ affinity, while the two coordinating side chains that provide bridging coordination to both Ca2+ 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-pKa 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 Ca2+ affinity. Overall, the experimental and computational results support an activation model in which the first Ca2+ 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 Ca2+ binding, enabling the second Ca2+ ion to bind with higher affinity than the first as required for positive cooperativity." @default.
• W1981603102 created "2016-06-24" @default.
• W1981603102 creator A5004495836 @default.
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• W1981603102 date "2004-12-01" @default.
• W1981603102 modified "2023-09-26" @default.
• W1981603102 title "Ca²⁺ Activation of the cPLA₂ C2 Domain: Ordered Binding of Two Ca²⁺ Ions with Positive Cooperativity" @default.
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• W1981603102 doi "https://doi.org/10.1021/bi0482405" @default.
• W1981603102 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3657617" @default.
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• W1981603102 hasPublicationYear "2004" @default.
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