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UK Dementia Research Institute@UCL and VIB@KuLeuven
Cysteine scanning as a tool for structure function analysis of presenilin
The new study by Sato et al.  utilizes cysteine scanning mutagenesis as a tool for the structure-function analysis of the catalytic site of presenilin. Earlier this year, we  showed by using a very similar approach and using a specific disulfide cross-linking strategy that the catalytic site of presenilin is accessible to a water-containing environment within the lipid bilayer, with the two catalytic aspartates facing each other with a maximal distance of 5.2Å. We also performed a “scanning” of the transmembrane domains 6 and 7 of presenilin and identified several important amino acid residues in this area.
It is important to stress that cysteine scanning mutagenesis is a biochemical technique which permits one to draw conclusions on the water accessibility of amino acid residues and their relative position versus each other in the tertiary conformation. It is, however, an extreme extrapolation to draw conclusions from such experiments on the global structure of a protein domain, like the funnel-like structure for the catalytic site of presenilin, as Sato et al. claim. Especially for presenilin this might be a dangerous exercise, since we have no hard structural information or even structures that can be used as models to interpret the data, and therefore caution with this type of conclusion is indicated. Sato et al. also did not take into account that presenilin might be present in the cell in distinct functional and therefore most probably structural conformations, depending on whether it is associated or not with the other γ-secretase complex components. The interpretation of the cysteine scanning data should take this into account as we actually did in our paper.
We feel somewhat surprised by Sato and colleagues’ aggressive criticism of our work. In contrast to Sato et al., we conclude that many of the data in our papers significantly overlap, suggesting that cysteine scanning mutagenesis is indeed a reliable methodology to probe structure function (within the indicated limits). For instance, Sato et al. confirm a differential labeling pattern for TM6 and TM7 which is in accordance with our conclusions and suggesting that TM6 and TM7 contribute in a different way to the activation and/or catalytic mechanism. In addition, Sato et al. show a similar labeling pattern in TM7 as we reported in our paper. Interestingly, this result suggests that the cytosol-oriented part of the TM7 exhibits a solvent accessible (flexible) non-regular secondary structure. A similar unstructured region was observed in the catalytic site-contributing TM4 in the crystal structure of rhomboid GlpG, another intramembrane cleaving protease from an entirely different functional class from presenilin. 
Sato et al. stress quite extensively the differences between their and our results. While they allude to methodological differences (for instance, the chemistry of the compounds used) and the absence of controls (which is, in fact, not the case), we believe that a more fundamental and interesting fact could explain at least part of the observed differences. Indeed, the basic construct (cysteine-less presenilin) used in the two studies is quite different, because our cysteine-less mutant is functionally similar to the wild-type presenilin, while Sato et al. used a cysteine-less presenilin that behaves as a clinical mutation, in fact, a loss-of-function form of wild-type presenilin. Indeed, Sato et al. (2006) introduced a Cys92Ser substitution, a known AD mutation, while we used a Cys92Ala form in order to create our cysteine-less template . We provide evidence that the latter mutant behaves like wt PS1 in the in vivo processing of at least three tested substrates. In contrast, the Sato et al. cysteine-less version of PS1 behaves clearly as an FAD mutation in the processing of APP (~threefold decrease in Aβ40 and ~twofold increase in Aβ42, resulting in a more than a fivefold increase of the ratio Aβ42/40). Thus, the few differences we see in both studies could reflect differences in the conformation of mutant versus wild-type presenilin.
Sato et al. also heavily criticized us about including inactive mutants in our accessibility study, such as G384C, D257C, and D385C, whereas they excluded them from their study arguing that they are liable to significant structural changes, again suggesting that we did not perform adequate controls. We would like to point out here briefly 1) that we demonstrated that none of our cysteine substitutions affected the incorporation of PS1 into the γ-secretase complex as assessed by blue native gel electrophoresis and 2) that this criticism implies that Sato et al. also do not accept any of the previous publications that investigated, for instance, the substitution of the catalytic aspartates. In contrast, we believe that one of the major findings in our manuscript was the close apposition of the two cysteines replacing the putative catalytic aspartates in TM6 and TM7, confirming for the first time that these residues, which are quite remote in the primary structure, are in close proximity to each other in the native conformation.
On the other hand, we feel that Sato et al. could have been a bit more critical when interpreting their own data with regard to the γ-secretase inhibitor binding experiments. Briefly, changes in the accessibility of certain amino acid residues in the complex upon inhibitor treatment does not prove that these residues are directly involved in the binding of the inhibitor. The alternative interpretation is, indeed, that the inhibitor induces a conformational change in the catalytic site, like transition state analogues do, and that this actually changes the accessibility of the residue. This possibility must also be taken into account when “modulators” of enzyme activity are studied, such as the non-transition state inhibitor DAPT.
In conclusion, we welcome the work of Sato et al. as an interesting study on the structure function of the catalytic site of presenilin, and we are very pleased with the partial confirmation and the extensions of our previous work. We would like to stress, however, that it is important to take the limitations and considerations discussed above into account when interpreting the data in these two first studies. We also anticipate that further studies around the lines of ours  and others  will shed further light into the complex structure of γ-secretase. The real value of this work, as with other site-directed mutagenesis work, will, however, be fully realized when it is combined with other approaches.
Sato C, Morohashi Y, Tomita T, Iwatsubo T. Structure of the catalytic pore of gamma-secretase probed by the accessibility of substituted cysteines. J Neurosci. 2006 Nov 15;26(46):12081-8. PubMed.
Tolia A, Chávez-Gutiérrez L, De Strooper B. Contribution of presenilin transmembrane domains 6 and 7 to a water-containing cavity in the gamma-secretase complex. J Biol Chem. 2006 Sep 15;281(37):27633-42. PubMed.
Wang Y, Zhang Y, Ha Y. Crystal structure of a rhomboid family intramembrane protease. Nature. 2006 Nov 9;444(7116):179-80. PubMed.