The function of BAP as a nucleotide exchange factor

stead of BAP. In the nucleotide free state, the percentage shifted from 5.2 % for BiP-167-519 with BAP to 17.4 % for BiP-∆lid with BAP and to 30.7 % for BiP-∆lid with BAP−∆N. The relatively high percentage of BiP molecules with a short distance between the two binding domains suggest that the lid keeps the two domains separated from each other.

An additional effect was the shift of a certain percentage of BiP-∆lid molecules to the high FRET conformation (d∼53 ˚A) either without a nucleotide or when ADP was present. From the measurements of BiP with BAP and ADP, we know that BiP is very inhomogeneous and has a broad distribution of conformations for the linker between the two binding domains.

Without the lid, the distribution of conformations gets narrower. By removing the N-terminal domain of BAP, the remaining molecules with low FRET efficiencies were also shifted to the two high FRET conformations (d ∼ 40 ˚A and d ∼ 53 ˚A). This supports the theory that the lid interacts with the N-terminal domain of BAP and pushes the two binding domains apart.

167 (NBD)

519 (SBD)

BiP-Δlid + BAP + ATP BiP-Δlid + BAP BiP-Δlid + BAP + ADP BiP (NBD & SBD) + BAP

BiP-Δlid + BAP-ΔN + ATP BiP-Δlid + BAP-ΔN BiP-Δlid + BAP-ΔN + ADP BiP (NBD & SBD) + BAP

A

B C

Figure 5.13: SpFRET analysis of BiP-∆lid with BAP and BAP-∆N. (A) A cartoon of BiP-∆lid indicating the labeling position is shown. (B) SpFRET distributions were measured for 25 pM BiP-167-519 with 10 µM BAP (grey line) or 25 pM BiP-∆lid with 10µM BAP alone (black line), in the presence of 10 µM BAP and 1 mM ATP (dark yellow line) or 10 µM BAP and 1 mM ADP (red line). (C) SpFRET distributions were measured for BiP-167-519 with 10 µM BAP (grey line), BiP-∆lid with 10µM BAP-∆N (black line), with 10 µM BAP-∆N and 1 mM ATP (dark yellow line) or with 10µM BAP and 1 mM ADP (red line).

In summary, the effect of deleting the lid of BiP or the N-terminal domain of BAP, suggest that the C-terminal lid of BiP interacts with the N-terminal domain of BAP. They push each other apart and, therefore, increase the distance between the NBD and SBD of BiP. When one or both interaction partners are missing, the two domains come close together.

the dissociation rate of ATP is to use commercial ADP, which contains a few percent of ATP contamination. For the other measurements, which are presented above, ADP was purified by an anion-exchange column in the Äkta (ÄKTAmicro, GE Healthcare, Uppsala, Sweden) and only the ADP fraction was used. For the determination of the K_d, we used the commercial ADP, because the measurements contains then two different populations, the ADP and the ATP conformation. Thus, we can only compare the measurements with and without BAP or BAP-∆N but cannot determine absolute values.

A + 1 mM ADP

+ 1 µM ADP + 10 µM ADP + 100 nM ADP + 10 nM ADP

+ BAP + 1 mM ADP + BAP + 10 µM ADP + BAP + 100 µM ADP + BAP + 1 µM ADP + BAP + 100 nM ADP

B

C

BiP-519-638 + ADP BiP-519-638 + BAP + ADP BiP-519-638 + BAP-ΔN + ADP + BAP-ΔN + 1 mM ADP

+ BAP-ΔN + 10 µM ADP + BAP-ΔN + 100 µM ADP + BAP-ΔN + 1 µM ADP + BAP-ΔN + 100 nM ADP + BAP-ΔN + 10 nM ADP

D

K_D=0.18 µM K_D=0.31 µM

KD=19.4 µM

Figure 5.14: Calculation of the K_d of ATP in the presence and absence of BAP by using spFRET. SpFRET distributions of 25 pM BiP-519-638 were measured with dif-ferent concentrations of ADP contaminated with a few percent of ATP (A) in the absence and (B) presence of 10 µM BAP and (C) presence of 10µM

BAP-∆N. (D) The normalized area under the FRET histograms up to a FRET value of 0.4 was summed up and plotted versus the ADP concentration. The K_d is given by the fit as 0.31 ±0.03µM for BiP alone, 19.4±4.8µM in the presence of BAP and 0.17± 0.04µM in the presence of BAP-∆N.

Different ADP concentrations and, thus, also different ATP concentrations, were measured in the absence of a NEF (Figure 5.14A), in the presence of BAP (Figure 5.14B) and in the presence of BAP-∆N (Figure 5.14C). The normalized area under the FRET efficiency histogram up to a FRET efficiency value of 0.4 was plotted versus the ADP concentration. The values were fitted by a sigmoid curve (Figure 5.14D). For BiP alone, aK_dof 0.31 ±0.03 µM was determined. By adding BAP to the measurements, the K_d increased to 19.4±4.8µM.

This is an increase in the dissociation constant of a factor of 65. Therefore, BAP increases the release of ATP. To analyze if this effect is due to the N-terminus of BAP, the same experiment was performed with BAP-∆N instead of BAP. The calculated value for the K_d

was 0.17 ±0.04 µM. This is in the same range as the value calculated for the measurements without a NEF and, therefore, the increase in the ATP release is due to the N-terminal domain of BAP.

To further address the effect of BAP on the nucleotide cycle, the hydrolysis process is analyzed in more details. When ATP hydrolyzes, an ADP and a pyrophosphate are bound to BiP. In the measurements before, only ADP was added instead of ADP and pyrophosphate. One effect could be that BAP speeds up the process of kicking off the pryophosphate and, therefore, speeding up the whole nucleotide cycle. To answer this question, pyrophosphate was added to the measurement of the lid-mutant with ADP and to the measurements with BAP and ADP. It was decided to simulate the hydrolyzed state because it is very hard to control the hydrolysis process and, therefore, it is unknown if an ATP or an ADP with pyrophosphate is present. However, this measurements are again performed with the non-purified ADP, which is the reason for the two FRET populations.

A _{+ ADP}

+ Pi (in measurement solution) + ADP (preincubated) + ADP + Pi (preincubated) + Pi (preincubated)

+ ADP (in measurement solution)

+ BAP + ADP

+ Pi (in measurement solution) + BAP + ADP (preincubated) + BAP + ADP + Pi (preincubated) + BAP + Pi (preincubated) + ADP (in measurement solution)

B

Figure 5.15: Conformational effect of pyrophosphate for BiP with ADP in the presence and absence of BAP. (A) SpFRET distributions of 25 pM BiP-519-638 were measured with different ways of incubation with 10 mM pyrophosphate and 1 mM ADP in the absence of BAP or (B) in the presence of 10µM BAP.

To figure out the effect of pyrophosphate, we measured the spFRET distribution of BiP-519-638 in the presence of ADP and a 10-fold Mol excess of pyrophosphate compared to ADP.

The incubation process was varied. When we first incubate with ADP at 37^◦C for 15 min and then add pyrophosphate to the measurement at 20^◦C (Figure 5.15A, red line) a small shift from low to high FRET efficiencies compared to the measurements without pyrophosphate was detected (Figure 5.15A, black line). By preincubating ADP and pyrophosphate at the same time at 37^◦C for 15 min, the lid is totally closed (Figure 5.15A, dark yellow line). This does not change anymore when we first incubate with pyrophosphate at 37^◦C for 15 min and then add ADP to the measurement at 20^◦C (Figure 5.15A, cyan line). The explanation for this effect can be given by the fact that pyrophoshate is known to increase the affinity of a Hsp70 to ADP. Therefore, as longer BiP is incubated with pyrophosphate as more likely it is that the affinity for ADP is increased.

By adding BAP to the different incubation processes, no changes in the conformation with a mixture of open and closed lid conformations were detected (Figure 5.15B). This means BAP keeps the lid conformation constant, independent of the presence of pyrophosphate. Thus, it is most likely that BAP has a opposite effect on the ADP affinity as the pyrophosphate and the two effects are canceled out.

In summary, we were not able to make statements about the hydrolyzed state and how BAP changes it. However, we detected that BAP can cancel out the increase in the ADP affinity induced by pyrophosphate.