Evidence for a Robust Target Recognition of the Disassembly

Inhibiting SNARE disassembly with the use of ternary complex-antibodies A different approach to investigate whether SNARE disassembly can be blocked on the target level, is not to delete parts of the complex but rather attach a factor, which either hides one or more interaction sites or sterically hinders conformational changes required for disassembly. Good candidates for proteins, whose attachment might influence function, are antibodies.

Figure 4.12: Two antibodies against the ternary SNARE complex act as synergis-tic inhibitors of SNARE disassembly. Approximately 70nM FRET-SNARE complex (Sb^28OG/SNAP25^{130T R}/H3) were incubated with the respective antibodies in disassem-bly buffer. Subsequently, NSF (1,5nM) andαSNAP (1,2µM) were added and the reaction started with MgCl₂ at t=-40s.

Three different antibodies raised against the purified SNARE ’mini’-complex (Dirk Fasshauer & Michaela Hellwig) had already been mapped to recognize dif-ferent regions of the SNARE complex by Tabrez Siddiqui.¹ Each of them alone as well as combinations of the antibodies were tested for their ability to inhibit SNARE disassembly using the FRET assay. As is shown in figure 4.12, all three antibodies partially impair disassembly, clones 131.1 and 131.2 having a slightly more pronounced effect than clone 131.5. Interestingly, if clones 131.1 and 131.2 are combined, they completely block disassembly. Considering that only half of the amounts used in the previous single-antibody experiments was applied in order to

1Tabrez Jamal Siddiqui, SNARE assembly and regulation on membranes, PhD thesis

exclude a dosage dependent cause of the block, this finding indicates that the anti-bodies act synergistically, probably by interfering with the disassembly mechanism at two different sites of the complex.

The combination of two non-inhibitory mutations can lead to inhibition of disassembly

The last finding suggests that the NSF machinery might indeed be robust enough to function even though one of the interaction sites between substrate and machinery is weakened or blocked. Considering that the antibodies have been mapped to different regions of the complex, it is not likely that the two synergistic clones 131.1 and 131.2 interfere with the same site of interaction. This might indicate that even if one site is blocked, other sites might compensate for the defect and suffice to mediate disassembly. Similar to the observation made for the anti-complex antibodies, other structural alterations of the neuronal SNARE complex might also lead to inhibitions of function and maybe even show synergistic effects if they interfere with different sites of interaction. As will be shown in the next section, this synergy indeed turned out to be the case for two alterations, both of which did not exhibit any defect in disassembly on their own.

Attachment of fluorescent dyes to Sb⁶¹ and Sx²²⁵ alone does not impair disassembly

As pointed out in section 4.1.4, the FRET pair used throughout all experiments up to now (Sb^28OG/SNAP25^{130T R}) is equally prone to disassembly as wildtype ternary complex. This does not necessarily need to be true for all FRET pairs, since the attachment of labels at different positions might have a negative effect caused by steric hindrance or ablation of an important interface in any way. Hence, for every new FRET pair used, this possibility needs to be excluded first. Figure 4.13 therefore compares the speed of SNARE disassembly of a FRET pair which will be used in some of the following experiments (Sb^61OG/H3^{225T R}), to that of the one used hitherto. As can be seen, there is no significant difference between the disassembly-kinetics of the two different FRET pairs.

The FRET pair Sb⁶¹/Syntaxin²²⁵ combined with C-terminal SNAP25 deletions severely impairs disassembly

Notwithstanding the fact that each of the two alterations alone discussed in sec-tions 4.2.3 and 4.2.2 did not influence disassembly kinetics, the combination of both severely impairs disassembly as can be witnessed in figure 4.14. Here, the kinet-ics of SNAP25 C-terminally deleted SNARE complexes carrying the FRET pair Sb^61OG/H3^{225T R} are significantly slower than the wildtype reaction. Furthermore, of the SNAP25^layer5-, SNAP25¹⁻¹⁹⁷- and SNAP25¹⁻¹⁸⁸-complexes no more than 10%, 20% or 30%, respectively, are successfully disassembled, compared to 75% in the wildtype reaction.

- 1 0 0 0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 - 0 , 1

0 , 0 0 , 1 0 , 2 0 , 3 0 , 4 0 , 5 0 , 6 0 , 7 0 , 8 0 , 9

parts of full disassembly

t i m e / s

S N A P 2 5 ( 1 3 0 O G ) / S b ( 2 8 T R ) S x ( 2 2 5 T R ) / S b ( 6 1 O G )

Figure 4.13: The two different FRET complexes Sb^28OG/SNAP25^{130T R}/H3 and Sb^61OG/H3^{225T R}/SNAP25) were disassembled in presence of 1,2µMαSNAP, 1,5nM NSF and 2mM ATP, triggered by addition of MgCl2 at t=160s. The parts of full disassembly are defined as the fraction of signal increase at a given point divided by the change of signal observed during full assembly of the complex (F-Fasscomplete)/(Fdiscomplete-Fasscomplete), Fdis_complete and Fass_complete representing the fluorescence at complete disassembly or as-sembly, respectively.

To exclude the possibility that the reactions with the SNAP25-mutations show a pronounced re-assembly, possibly due to slight inaccuracies with respect to the pro-tein determination, the impact of an enhanced assembly and subsequent re-assembly was tested in further experiments, where the concentration of the respective SNAP25 was raised by a factor of four to promote faster assembly. As shown in figure 4.15, even though the acceleration has a subtle influence on disassembly, the differences between the mutant and the wildtype reaction remain much more prominent than those between the wildtype and the defective mutants.

-200 0 200 400 600 800

Figure 4.14: Two structural alterations of the SNARE complex show a pronounced synergy. Different SNAP25 mutants (BotA mutant: residues 1-197, layer-‘5’ mutant:

M71A/I192A) or wildtype SNAP were assembled with their cognate SNAREs to form complexes carrying the FRET pair (Sb^61OG/H3^{225T R} introduced in section 4.2.3 (A) and subsequently disassembled in the presence of 1,2µMαSNAP, 1,5nM NSF and 2mM ATP by addition of MgCl₂ at t=0s (B). The ‘parts of disassembly’ are defined as the fraction of signal increase at a given point divided by the change of signal observed during full assem-bly of the complex (F-Fass_complete)/(Fdis_complete-Fass_complete), Fdis_completeand Fass_complete representing the fluorescence at complete disassembly or assembly, respectively.

A

B

Figure 4.15: SNARE-complex assembly (A) and disassembly (B) were performed as in figure 4.14 except that a four-fold excess of SNAP25 (800nM) was used in two of the reactions to exclude ostensive differences in disassembly caused by slightly different re-assembly kinetics.