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J. Biol. Chem., Vol. 281, Issue 37, 27633-27642, September 15, 2006

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Contribution of Presenilin Transmembrane Domains 6 and 7 to a Water-containing Cavity in the -Secretase Complex^*

From the Neuronal Cell Biology and Gene Transfer Laboratory, Center for Human Genetics, VIB4 and K. U. Leuven, Herestraat 49, 3000 Leuven, Belgium

-Secretase is a multiprotein complex responsible for the intramembranous cleavage of the amyloid precursor protein and other type I transmembrane proteins. Mutations in Presenilin, the catalytic core of this complex, cause Alzheimer disease. Little is known about the structure of the protein and even less about the catalytic mechanism, which involves proteolytic cleavage in the hydrophobic environment of the cell membrane. It is basically unclear how water, needed to perform hydrolysis, is provided to this reaction. Presenilin transmembrane domains 6 and 7 seem critical in this regard, as each bears a critical aspartate contributing to catalytic activity. Current models imply that both aspartyl groups should closely oppose each other and have access to water. This is, however, still to be experimentally verified. Here, we have performed cysteine-scanning mutagenesis of both domains and have demonstrated that several of the introduced residues are exposed to water, providing experimental evidence for the existence of a water-filled cavity in the catalytic core of Presenilin. In addition, we have demonstrated that the two aspartates reside within this cavity and are opposed to each other in the native complex. We have also identified the conserved tyrosine 389 as a critical partner in the catalytic mechanism. Several additional amino acid substitutions affect differentially the processing of -secretase substrates, implying that they contribute to enzyme specificity. Our data suggest the possibility that more selective -secretase inhibitors could be designed.

Received for publication, May 24, 2006 , and in revised form, July 12, 2006.

^* This work was supported by a Freedom to Discover grant from Bristol-Myers-Squibb; a Pioneer award from the Alzheimer's Association; the Fund for Scientific Research (Flanders); K. U. Leuven (GOA); European Union (APOPIS, LSHM-CT-2003-503330 and NeuroNe); and the Federal Office for Scientific Affairs (Belgium) (IUAP P5/19). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3.

¹ To whom correspondence should be addressed. Tel.: 32-16346227; Fax: 32-16347181; E-mail: Bart.destrooper{at}med.kuleuven.be.

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