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. 2019 Oct 8;9:14408. doi: 10.1038/s41598-019-50684-1

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Molecular and phenotypic properties of Doc2b and its Ca²⁺-binding site mutants. (A) Cartoon showing C₂ domain structures of Doc2b based on crystallography⁵¹. Aspartates involved in Ca²⁺ binding are marked in red; poly-lysine sequences for SNARE complex and PIP₂ interaction are marked in orange⁶⁶. Note that the poly-lysine region is oriented opposite to the Ca²⁺-binding aspartates. Dashed lines represent linker sequences between domains. Dashed squares highlight Ca²⁺-binding pockets enlarged in C. (B) Linear representation of Doc2b^WT and two previously investigated mutants Doc2b^DN and Doc2b^6A (red lines indicate amino acid substitutions). (C) Aspartates substituted in Doc2b^DN (D218, 220N) or Doc2b^6A (D163, 218, 220, 303, 357, 359A). (D) Summary of functional effects of Doc2b^DN and Doc2b^6A mutations. Ca²⁺-binding capacity was assessed by tryptophan fluorescence measurements⁴⁹ for Doc2b^6A and isothermal titration calorimetry (ITC) measurement for Doc2b^DN (termed CLM mutant)⁴⁴. Synaptic release phenotypes were determined by electrophysiology in cultured neurons. Doc2b^WT supports spontaneous release (marked as ‘+’ in the table). Its role in asynchronous release is observed in some but not all systems^29,44,47 (marked as ‘~’). It does not function in synchronous release (‘none’). Doc2b^DN constitutively binds phosphatidylserine-containing membranes^44,48 and partially binds Ca²⁺ via its intact C₂B domain⁴⁴. It increases spontaneous release frequency (+++)^21,29 and is implicated in asynchronous release in some but not all studies (~)^21,44. No effect was noticed on synchronous release (none). Doc2b^6A was shown have an abrogated Ca²⁺-binding²⁹ capacity but its lipid association activity was not reported (n.a.). This mutant increased spontaneous release frequency²⁹ (+++) but its effect on evoked synchronous and asynchronous release were not investigated (n.a.). See^21,29,44 for more details.