RESULTADO DE LA EVALUACIÓN DE LA
2. Se diseñaron los indicadores de eficacia teniendo en cuenta los objetivos previstos para la planificación estratégica.
The SPR/IAC result obtained demonstrated a substantial reduction in binding – half the response of the WT epsin 1 (highest binding response) from the epsin clathrin box mutants. This reduction was equal in all the three mutants (257, 480 and DKO) (Figure 5.9.3 (A)), with no significant difference between them in their ability to bind clathrin TD. These results are representative of three independent repeats (n=3) for each WT and mutant with the mean of
WT
257
480
Figure 5.9.2: Diagram illustrating the ‘clathrin binding box’ motif mutants (257, 480 and DKO) on a linear representation of the epsin 1 structure. The
mutants were designed in line with Drake et al., 2000 and Holkar et al., 2015. The
WT form of the epsin 1 structure is represented at the top of the diagram. The 257 mutant has one of the ‘clathrin binding boxes’ starting at residue 257 mutated to alanines (LMDLA-> AAAAA). The 480 mutant has one of the ‘clathrin binding boxes’ starting at residue 480 mutated to alanines (LVDLD-> AAAAA). The DKO mutant has both ‘clathrin binding boxes’ mutated to alanines (LMDLA->AAAAA and LVDLD-> AAAAA).
Mutant Names Mutations
257 LMDLA -> AAAAA 257 LMDLA -> AAAAA 480 LVDLD -> AAAAA - 480 LVDLD -> AAAAA DKO
the highest response unit of each sample and standard deviation plotted. The WT epsin 1 has the highest response units of 1171 ± 287.5 RU and the 257, 480 and DKO having significantly lower responses of 586.7 ± 147.2 RU, 578.10 ± 94.43 RU and 550 ± 69.37 RU respectively (Figure 5.9.3 (B)). Overall, these results are consistent with the hypothesis of Drake et al. 2000, where reduction of clathrin binding was observed when one clathrin box motif- 257 is deleted and that the unstructured region/DPW between the two clathrin box motifs was suggested to bind to clathrin non- specifically/independently (Drake et al., 2000).
On the other hand, these results are partially consistent with ultracentrifugation assays conducted by Dr. Michael Baker with clathrin cages as described above, with the exception that the SPR data with clathrin TD indicates an equivalent reduction in binding between all three mutants, whereas a greater reduction for the DKO mutant for clathrin TD binding was observed from GST- pulldown assays, as conducted by Dr. Michael Baker (Baker, 2016). It is important to note that pulldown assays were carried out with whole clathrin cages, whereas SPR experiments were carried out using clathrin TD (residue 1-363). An overall reduction in binding is observed between the WT and the three epsin 1 mutants both in cases when clathrin cages (binding pulldown assays) or clathrin TD (SPR data) used.
The two epsin clathrin box motifs function were hypothesized to function cooperatively (Drake et al., 2000; Drake and Traub, 2001), and promote clathrin assembly, a result which was confirmed with evidence from Holkar et al. 2015 (Holkar et al., 2015). However, the SPR data in this section indicate a similar binding affinity of the clathrin TD for each of the two clathrin box motifs and the unstructured region/DPW motifs suggesting that they bind in a cooperative manner to clathrin. This was shown from the SPR data, which revealed no significant difference in response between the three mutants (257, 480, DKO). This has not been previously suggested or observed and therefore adds an important insight into how the epsin 1 structure effects the interaction with clathrin.
Preliminary investigations from Dr. Michael Baker confirmed that mutating the epsin 1 clathrin box motifs (257 and 480 mutants) still had a subtle effect on altering the cage size distribution from an average radius of ~ 45 nm to ~ 35 nm, in a manner similar to WT epsin as determined by DLS and EM (Baker, 2016). Even though there are different biodynamic interactions between clathrin cages and clathrin TD, the DLS/EM results could relate to the clathrin-TD SPR data obtained in this section, suggesting that even when the reduction in clathrin TD binding between the epsin 1 clathrin box mutants is equal there is more complex behavior of epsin 1:clathrin interaction is likely much more complex.
A strong epsin-clathrin interaction is suggested due to the slow dissociation of the complex observed in WT and three epsin 1 mutants, with the response curve not reaching the baseline. This could possibility demonstrate the strong and cooperative behavior characteristic of multiple site interaction of epsin 1’s two clathrin box motifs and the unstructured/DPW motif region binding to clathrin TD- a much longer dissociation time will be required to disrupt many existing interactions.
(A)
(B)
Figure 5.9.3: Binding of epsin 1 full-length (residues 1-575) WT and ‘clathrin binding box’ mutants (10 μM) to GST-TD (1-363) (1 μM), to investigate epsin:clathrin interactions. (A) Binding of purified epsin 1 of WT and 3 mutants (257, 480 and DKO) to GST-TD. WT binds the strongest, with an unusual binding curve, which is hypothesized not to be a 1:1 stoichiometric binding with TD. Whereas all the 3 mutants have a very similar binding capacity to the GST-TD with a hypothesised 1:1 stoichiometric ratio, due to the shape of the binding curve. Each experiment was carried out in a series of three repeats in randomised order. Overall, no saturation was observed in the SPR experiments, even in 1:10 molar ratio of clathrin to epsin 1, which was the recommended ratio with excess epsin 1
concentration (B) The results from (A) are plotted on a bar chart representing the
mean from the highest response value of three independent repeats (n=3) and the standard deviation. The WT epsin 1 has the highest response of 1171.0 and the 257, 480 and DKO having significantly equal lower responses of 586.7, 578.10 and 550.00 respectively. 100 200 300 400 500 0 500 1000 1500 Time (s) Response Diff. WT (10 µM) 257 (10 µM) 480 (10 µM) DKO (10 µM) WT 257 480 DKO 0 500 1000 1500 2000 Hi g h e s t re s p o n s e v a lu e s (RU)
5.9.4 Mutagenesis studies of epsin 1 unstructured/DPW
region
Using the SPR/IAC technique, the observation that mutating single clathrin box motifs or both in epsin 1 resulted in a substantial reduction in its ability to bind to clathrin TD was confirmed. In addition, I demonstrated how the clathrin box motifs and the unstructured/DPW region could possibly have an equal contribution to epsin 1 binding to clathrin TD. Thus, both epsin 1 structure components could be equivalently strong in their affinity to bind clathrin TD. A number of interesting questions arose from the above initial observations as to how the two clathrin box motifs and the unstructured/DPW motif function could promote clathrin assembly. Hence, after investigating the two clathrin box motifs; epsin’s unstructured/DPW motif region via mutagenesis studies and SPR/IAC technique was aimed to be investigated.
In order to investigate this unstructured/DPW region of epsin 1, we designed and obtained mutants in which the unstructured/DPW region was deleted or shortened, hence reducing the distance between the two ‘clathrin box motifs. The mutants are illustrated in Figure 5.9.4 and are abbreviated as follows: ½ DPW, ¼ DPW and ΔDPW. Briefly, in the ½ DPW, 93 amino acids were cut (approx. half of the region) from the original 216 amino acid sequence of the unstructured region. No DPW motifs of the region were deleted in this mutant. In the ¼ DPW mutant, 153 amino acids were deleted (approx. quarter of the region), with four DPW motifs deleted. In the ΔDPW, 209 amino acids were deleted together with all eight DPW motifs in this region. Thus the whole unstructured/DPW region has been completely deleted in this mutant.