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. 2011 Mar 4;286(17):15067–15072. doi: 10.1074/jbc.M111.224493

FIGURE 4.

FIGURE 4.

High affinity binding of LC/A to SNAP-25. a, schematic of SNAP-25 (SN25) bound to the plasma membrane. SNAP-25 is a peripheral, plasma membrane-binding protein that comprises two helixes that bind the membrane through four palmitoylated cysteine interactions. b and c, solid phase binding assays; SNAP-25 (SN) derivatives (0.5 μg) were coated in 96-well plates and incubated with the indicated amounts of 3xFLAG-LC/A (in nm). Bound LC/A was detected with mouse α-FLAG-HRP antibody and Ultra TMB. A450 was plotted versus [LC/A] to determine k50, the amount of LC/A needed to bind 50% SNAP-25. d, schematic of LC/A interactions with membrane-bound SNAP-25. (Panel 1) At the cell plasma membrane, syntaxin 1a (Syn) and SNAP-25 form a SNARE complex (31). (Panel 2) The N terminus of LC/A binds residues 80–110 of SNAP-25 (panel 3) which facilitates substrate binding in competition with syntaxin 1a. (Panel 4) After SNAP-25 cleavage, LC/A has a high affinity for membrane-bound SNAP-25(1–197).