Conformational variability and intramembrane recognition possibly associated with abnormal cleavage of APP transmembrane domain
Bitopic proteins having only one helical transmembrane (TM) domain are a class of biologically significant membrane proteins, including the type I receptors and amyloid precursor protein (APP), which are involved in regulating the homeostasis of human organism and recognized as substrate by c-secretase. Amyloid Ab-peptides forming plaques in brain during Alzheimer disease (AD) are the products of sequential intramembrane cleavage of APP. A lot of mutations associated with AD familial forms were found in the APP transmembrane (TM) domain and juxtamembrane (JM) regions. We designed highly productive systems of bacterial and cell-free expression and easy purification procedure for 13C/15N-isotope labeled APP JM-TM fragments of different length (corresponding to the sequential cleavage steps of APP) and with several familial AD mutations, as well as the TM fragments of c-secretase. The fragments were solubilized in membrane-mimicking complexes (detergent micelles and lipid bicelles), which allows to acquire proper high-resolution NMR spectra despite low sample
stability and to characterize their structural-dynamic properties. Molecular Dynamics relaxation of obtained NMR structures of the fragments in hydrated explicit lipid bilayers provided a detailed atomistic picture of the intra- and intermolecular interactions within membrane. The mutant APP JM-TM fragments are shown to be promising objects for elaboration the molecular aspects of the abnormal recognition and sequential proteolysis by c-secretase, revealing a straightforward mechanism of the pathogenesis associated with some familial AD mutation as well as with aging.