5 BiP’s interaction with BAP
5.9 Discussion and Outlook
higher oligomers disappeared in the presence of CH1, which was in line with [Blond-Elguindi et al., 1993]. Adding BAP to BiP (Figure 5.17 (red line)) resulted in a larger sedimentation coefficient because of the larger complex size. Surprisingly, the complex peak of BiP with BAP and CH1 shifted towards a smaller sedimentation coefficient than the BiP-BAP complex and was in between the peak of BiP with CH1 and the peak of BiP with BAP (Figure 5.17 (dark yellow line)).
3 4 5 6 7 8
0 400 800 1200 1600
c(s)
s20,w (S)
BiP BiP + BAP BiP + CH1 BiP + BAP + CH1
Figure 5.17: Analytical ultracentrifugation measurements of BiP with BAP and CH1. Com-plex formation by sedimentation velocity (SV) runs of 0.37µM ATTO 488-labeled BiP (gray line), 0.37 µM ATTO 488-labeled BiP and 37 µM CH1 (black line), 0.37 µM ATTO 488-labeled BiP and 3.7 µM BAP (red line) or 0.37 µM ATTO 488-labeled BiP, 3.7 µM BAP and 37 µM CH1 (dark yellow line). The measurements were performed by Mathias Rosam in the group of Prof. Buchner.
Hence, we can conclude that BAP and CH1 cannot bind both at the same time. This result is in line with the experimental spFRET data, where the conformation of BiP with BAP and CH1 is either similar to the conformation of BiP with CH1 alone or to the conformation of BiP with BAP.
In addition, the effect of BAP on the nucleotide cycle was investigated. The dissociation con-stant of ATP from BiP and from BiP with BAP was indirectly determined. BAP increases the Kd by a factor of 65 and, thus, speeds up the release of ATP. One possible explanation would be that BAP changes the nucleotide binding pocket to a more open conformation to facilitate the release of the nucleotides. This is in line with the comparison of the crystal structures of the NBD with BAP and the NBD with BAP and ADP [Yan et al., 2011].
Furthermore, by testing to use of non-hydrolyzable ATP analogues, differences in the FRET histograms were identified. For BiP alone the FRET efficiencies measured with ATP and the ATP analogues are similar. By adding BAP, the detected conformations are similar to the ADP conformations. The differences between ATP and the ATP analogues are due to small chemical changes which are recognized by BAP. One possible explanation for the different behavior with and without BAP is that the binding domain of BiP changes from a pocket to a more tunnel-like structure, when BAP binds and, therefore, BAP itself interacts with the nucleotide. Thus, we have to use ATP instead of non-hydrolyzable ATP analogues for studying the ATP conformation of BiP. Nevertheless, the ATP analogues help to understand that the binding pocket in some case changes when BAP binds.
In the presence of a substrate, the intrinsically disordered domain CH1 of an antibody, the conformation upon binding of ADP changes. The lid has to be open, when CH1 is bound to the substrate binding pocket. Upon binding of a substrate and BAP, a mixed conformation of the BAP-bound and the CH1-bound state was detected. This result is also supported by AUC measurements that showed that either BAP or CH1 can bind. This is in line with the idea that BAP should assist the chaperone cycle and help a substrate free BiP to accelerate the ADP/ATP exchange and, therefore, makes BiP again able to bind a new substrate [Cyr, 2008].
The overall nucleotide cycle of BiP is given in Figure 5.18. In the ATP state, the lid is in an open conformation and the two binding domains are close together. In this conformation, a substrate, in our case CH1, can bind (Figure 5.18, outer cycle) [Marcinowski et al., 2011], but the affinity for the binding is low. When ATP hydrolyzes to ADP, the affinity of BiP for binding a substrate increases. The substrate stays bound to BiP, but the overall structure is very similar to the ATP bound conformation. Upon release of the ADP, the lid closes and catches the substrate. The two binding domains are well separated. By binding new ATP, the substrate is released and the whole nucleotide cycle starts again. Alternatively, BAP can interact transiently with BiP in the ATP conformation (Figure 5.18, inner cycle). When ATP hydrolyzes to ADP, BAP stable binds to BiP and separates the two binding domains and closes the lid. This conformation does not change when ADP is released. Upon binding of a new ATP, BAP is released from the complex. Whenever both interaction partners, BAP and CH1, are present, only one can interact with BiP at a time.
The increase in the chaperone cycle due to BAP that was identified in the cooperation with the group of Prof. Buchner was in total much lower than for other NEFs. Other NEFs, for example Bag-1, are known to stimulate the dissociation of ADP from Hsc70 and Hsp70 by up to 100-fold [Mayer and Bukau, 2005]. Thus, BAP may have an additional function in the chaperone cycle. To further analyze the binding and dissociation rates in the presence and absence of BAP, TIRF measurements could be performed. We have already performed first experiments with immobilize BiP in vesicles but the protein seems to interact with the lipids.
Direct binding of BiP to the surface was also tried but this results so far in the loss of the functionality of BiP. One idea to overcome this problem would be to use a flow system as described in [Tyagi et al., 2014].
ATP
Phosphate ADP
BAP
CH1
Figure 5.18: Model for the BAP-regulated chaperone cycle of BiP. The different domains of BiP and BAP are color-coded as in Figure 5.1 and in Figure 5.4. For BiP, the NBD is shown in red, the SBD in green and the lid in blue, BAP is depicted in purple. For all combinations of BiP, BAP and CH1, which are depicted, the most populated conformation is drawn.
Furthermore, it would be of interest to study the chaperone cycle of BiP in the presence of BAP and other co-chaperones, for example, a J-protein. J-proteins are known to influence the chaperone cycle and are assumed to interact between the two binding domains of BiP [Kampinga and Craig, 2010], [Mapa et al., 2010]. Due to the fact that BAP binds to the NBD and, therefore, is in close proximity to the linker between the NBD and SBD, it would be important to know if both proteins can interact at the same time with BiP or if they are competing with each other.
One of the major open questions is how mutations in BAP, like observed for patients with the disease Marinesco-Sjögren syndrome [Howes et al., 2012], are changing the interaction of BAP with BiP and how they influence the chaperone cycle. BAP was identified as a NEF, which increases the chaperone cycle much less than other NEFs. Furthermore, BiP is known to be functional without BAP. Therefore, the important question is, what is the effect of the mutations that causes the autosomal recessive form of ataxia, cataract and myopathy [Anttonen et al., 2005], [Senderek et al., 2005], [Krieger et al., 2013].