| Abstract
| - Accumulation of the cytotoxic 40- to 42-residue β-amyloid peptide represents the primarypathological process in Alzheimer's disease (AD). BACE1 (β-site APP cleaving enzyme 1) is responsiblefor the initial required step in the neuronal amyloidogenic processing of β-amyloid precursor protein andis a major drug target for the therapeutic intervention of AD. In the present study, BACE1 is initiallysynthesized as an immature precursor protein containing part of the pre domain and the entire pro domain,and undergoes autocatalytic conversion to yield the well-folded mature BACE1 enzyme. To understandthe mechanism of the conversion and the role of the pro domain, we monitored the autocatalytic conversionof BACE1 by heteronuclear NMR spectroscopy and used chemical shift perturbations as a probe to studythe structural changes accompanying the autocatalytic conversion. NMR data revealed local conformationalchanges from a partially disordered to a well-folded conformation associated with the conversion. Theconformational changes are largely concentrated in the NH2-terminal lobe. Conversely, the active siteconformations are conserved during the autocatalytic conversion. The precursor and mature BACE1 proteinswere further characterized for their ability to interact with a substrate-based transition state BACE1 peptideinhibitor. The precursor BACE1 rapidly adopted the bound conformation in the presence of the inhibitor,which is identical to the bound conformation of the mature protein. The interaction of the inhibitor withboth the precursor BACE1 and the fully processed BACE1 is in slow exchange on the NMR time scale,indicating a tight binding interaction. Overall, the NMR data demonstrated that the pro domain does nothinder inhibitor binding and may assist in the proper folding of the protein. The fully processed BACE1represents a high quality well-folded protein which is highly stable over a long period of time, and issuitable for evaluation of inhibitor binding by NMR for drug intervention.
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