Analysis of tripartite Synaptotagmin‐1‐SNARE‐complexin‐1 complexes in solution. Issue 1 (16th November 2022)

• Record Type: Journal Article
• Authors: Jaczynska, Klaudia; Esquivies, Luis; Pfuetzner, Richard A.; Alten, Baris; Brewer, Kyle D.; Zhou, Qiangjun; Kavalali, Ege T.; Brunger, Axel T.; Rizo, Josep
• Is Part Of: FEBS open bio. Volume 13:Issue 1(2023)
• Page Start: 26
• Page End: 50
• Publication Date: 2022-11-16
• Subjects: Ca2+ sensing -- complexin -- neurotransmitter release -- SNAREs -- synaptotagmin -- weak protein interactions; Molecular biology -- Periodicals; Cytology -- Periodicals; Life sciences -- Periodicals; Biological Science Disciplines -- Periodicals; Molecular Biology -- Periodicals; Cell Biology -- Periodicals; Cytology; Life sciences; Molecular biology; Periodicals; 572.805
• DOI: 10.1002/2211-5463.13503
• Languages: English
• ISSNs: 2211-5463

Abstract

Abstract : Characterizing interactions of Synaptotagmin‐1 with the SNARE complex is crucial to understand the mechanism of neurotransmitter release. X‐ray crystallography revealed how the Synaptotagmin‐1 C2 B domain binds to the SNARE complex through a so‐called primary interface and to a complexin‐1‐SNARE complex through a so‐called tripartite interface. Mutagenesis and electrophysiology supported the functional relevance of both interfaces, and extensive additional data validated the primary interface. However, ITC evidence suggesting that binding via the tripartite interface occurs in solution was called into question by subsequent NMR data. Here, we describe joint efforts to address this apparent contradiction. Using the same ITC approach with the same C2 B domain mutant used previously (C2 B KA‐Q ) but including ion exchange chromatography to purify it, which is crucial to remove polyacidic contaminants, we were unable to observe the substantial endothermic ITC signal that was previously attributed to binding of this mutant to the complexin‐1‐SNARE complex through the tripartite interface. We were also unable to detect substantial populations of the tripartite interface in NMR analyses of the ITC samples or in measurements of paramagnetic relaxation effects, despite the high sensitivity of this method to detect weak protein complexes. However, these experiments do not rule out the possibility of very low affinity ( K D > 1 mm ) binding through this interface. These results emphasize the need to develop methods to characterize the structure of synaptotagmin‐1‐SNARE complexes between two membranes and to perform further structure–function analyses to establish the physiological relevance of the tripartite interface. Abstract : Contradictory data were reported regarding the binding of the neuronal SNARE complex involved in neurotransmitter release to the Ca 2+ sensor Synaptotagmin‐1 through a so‐called tripartite interface. Using ITC and highly sensitive NMR methods, we were unable to detect this interaction in solution, indicating that establishing the physiological relevance of the tripartite interface will require further research.