Exploring the potential role of Q75 in modulating the pK a of C71

C71

6.5.1. Sequence alignment of PDI a and PDIp a domains

Studies of C71 identified two pKa dependent events (see figure 6.12) suggesting that

the reactivity of the N-terminal cysteine of PDIp a was being modulated by nearby residues. In this way the N-terminal cysteine may be slightly less reactive than would be expected for a reduced cysteine. To identify amino acids in PDIp a that could potentially contribute to this result, a sequence alignment was performed for PDIp a

and PDI a (see figure 6.14).

It is clear from figure 6.14 that there are 21 differences in charge for PDI a and PDIp

a. In total there are 13 charged residues in PDI that are not conserved in PDIp and 8 charged residues in PDIp that are not conserved in PDI. However R120, the residue implicated in modulation of PDI a, is conserved in PDIp a (Karala et al. 2010). It remains uncertain which residues are involved in maintaining/modulating the high pKa of the N-terminal cysteine of PDIp a but PDIp Q75 seems like a particularly

probable candidate because it is immediately adjacent to the active site in PDIp a

Figure 6.14: Sequence alignment of PDI a and PDIp a (CLUSTAL). Charged

residues in PDI that are not conserved in PDIp are shown in green and charged residues in PDIp that are not conserved in PDI are shown in red.

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(WCGHCQ). Furthermore figure 6.15 shows that in PDI, ERp57 and ERp72, a lysine residue is conserved at this position (WCGHCK).

In PDI the positive net charge of K57 interacts with the thiolate ion at the N- terminal cysteine stabilising it by electrostatics but for PDIp this is not the case because glutamine is uncharged. However it is possible that the the side group of glutamine could be acting as a hydrogen bond acceptor (see figure 6.16). If a hydrogen bond between Q75 and the unreactive thiol group this may explain why PDIp a exhibits some reactivity despite the N-terminal cysteine being in the predominantly unreactive thiol state. Overall this may explain the low oxido- reductase activity seen for wild-type full length PDIp.

Figure 6.15: Sequence alignment of PDI, PDIp, ERp57 and ERp72. PDIp Q75 is highlighted in red, typically a lysine residue is conserved at this position (highlighted in green).

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6.6. Concluding Remarks

Previously we showed that PDIp has ~50% of the oxido-reductase activity of PDI. This was expected to be caused by differences in substrate specificity conferred by the b’ domain and also the unusual a’ domain active site sequence (CTHC). We have since shown that while the b’ and a’ do have a slight negative effect on the activity of PDIp , the effect of PDIp a is far more significant.

Stopped flow studies of PDIp a that measured the reactivity of the active site with either GSH or GSSG confirmed that it was much less reactive than originally postulated. As PDIp a has a normal active site sequence (CGHC) this was unexpected. Additional stopped flow studies to measure the pKa of the active site

cysteines of PDIp a showed that the N-terminal cysteine had an exceptionally high pKa. This means that at physiological pH the N-terminal cysteine is in the unreactive

Figure 6.16: PyMOL images of the active site regions of PDI a and PDIp a. The structure of PDI Leu44-Tyr63 was observed using PyMOL and PDB: 4EKZ because this region is conserved in PDI and PDIp (with the exception of Q75 adjacent to active site. For PDIp K57 is mutated to Q using PyMOL mutagenesis function. N-terminal cysteine is shown in red, histidine in blue and either lysine or glutamine in orange (PDI and PDIp respectively). A) Interaction between positive charge of lysine and the negative charge of the thiolate ion on N-terminal cysteine (shown by white line). B) Hydrogen bond between SH group and oxygen on the side chain of glutamine leading to slight negative charge on the donating sulphur atom (shown by white line).

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thiol state and explains the lack of activity seen for PDIp a and also the reduced activity of full length PDIp.

Further study identified Q75, an amino acid found immediately adjacent to the PDIp

a domain active site that is not conserved in other PDI family members (which typically have a lysine residue at this position). Additional work is required to confirm our hypothesis, but we believe that the presence of a glutamine residue at position 75 in PDIp a instead of a lysine residue (seen for other PDI family members), leads to overall stabilisation of the thiol state at the N-terminal cysteine. Consequently the N-terminal cysteine of PDIp a is not as reactive as that of PDI a

which is stabilised in the thiolate form by electrostatic interactions with lysine. A PDIp a Q75K mutation should therefore stabilise the thiolate state of the N-terminal cysteine and therefore increase the overall reactivity of the active site.

In regards to structure, an unreactive N-terminal cysteine could explain why only minor redox-mediated conformational changes were observed. For example if the N- terminal cysteine is highly unreactive this would mean that it would be very difficult to oxidise PDIp a. This means that in our studies of redox structure oxidised PDIp could have a reduced a active site and an oxidised a’ active site and explains the polymorphisms seen by native-PAGE (chapter 5). This does however indicate a role for the a domain in the redox conformations of PDIp. This is not the case for PDI for which b’xa’ is the minimal requirement for redox conformational change. Therefore PDI and PDIp must be different in terms of redox structure. Further work is required to confirm this.

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