Analysis of the Overall Structure of the Multi-Domain Amyloid Precursor Protein (APP)

Abstract

The amyloid precursor protein (APP) and its processing by the α-, β- and γ-secretases is widely believed to play a central role during the development of Alzheimer´s disease. The three-dimensional structure of the entire protein, its physiologic function and the regulation of its proteolytic processing remain, however, largely unclear to date. To gain a deeper understanding of the structure of APP that underlies all of its functions, we first cloned and recombinantly expressed different constructs in E. coli. Using limited proteolysis followed by mass spectrometry and Edman degradation as well as analytical gel permeation chromatography coupled static light scattering, we experimentally analyzed the structural domain boundaries and determined that the large ectodomain of APP consists of exactly two rigidly folded domains – the E1-domain (Leu18-Ala190) and the E2-domain (Ser295-Asp500). Both, the acidic domain (AcD) connecting E1 and E2 as well as the juxtamembrane region (JMR) connecting E2 to the single transmembrane helix are highly flexible and extended. We identified in-between the E1-domain and the AcD an additional domain of conservation and partial flexibility that we termed extension domain (ED, Glu191-Glu227). Using Bio-layer interferometry, pull-down assays and analytical gel filtration experiments we demonstrated that the E1-domain does not tightly interact with the E2-domain, both in the presence and in the absence of heparin. APP hence forms an extended molecule that is flexibly tethered to the membrane. Its multi-domain architecture enables together with the many known functionalities the concomitant performance of several, independent functions, which might be regulated by cellular, compartment specific pH-changes.

The crystal structure of DR6 in complex with the amyloid precursor protein provides insight into death receptor activation

“…Thus, the studies described here suggest that DR6 activation could be triggered by dimerization induced by APP binding. Interestingly, it was also suggested that the dimeric state of APP can be stabilized by heparin and, specifically, that heparin induces APP dimerization via interactions with either the E1 or E2 domain (Hoopes et al 2010;Lee et al 2011;Xue et al 2011a,b;Coburger et al 2013;Hoefgen et al 2014). Based on the experiments reported here and the previously published data, we propose a simple model for DR6 activation by APP-induced dimerization that ultimately leads to axon pruning and synapse elimination (Fig.…”

“…The extracellular domain of APP is comprised of an N-terminal domain termed E1 and a more C-terminal domain termed E2 that are linked via an acidic unstructured sequence and followed by a transmembrane helix and a cytoplasmic region. Previous studies have elucidated the structures of the unbound APP extracellular domains (Wang and Ha 2004;Dahms et al 2010Dahms et al , 2012Hoopes et al 2010;Lee et al 2011; Xue et al 2011a,b;Coburger et al 2013Coburger et al , 2014. High-affinity binding of APP to death receptor 6 (DR6, also known as TNFRSF21), a death domain-containing member of the extended TNF receptor superfamily (Pan et al 1998), was recently documented (Nikolaev et al 2009) and shown to be mediated by the E2 domain of APP (Olsen et al 2014).…”

Novel Zinc-binding Site in the E2 Domain Regulates Amyloid Precursor-like Protein 1 (APLP1) Oligomerization

“…In the absence of zinc, we conclude that recombinantly purified APLP1 E2 is monomeric at the employed experimental conditions. Similarly to APLP1 E2, the E2 domain of APP was also shown to exist in a monomeric form in solution (40). In the presence of 10 M ZnCl 2 , a significant peak broadening was observed with APP E2 and APLP1 E2, which corresponded to the theoretical mass of dimers (Fig.…”

The Amyloid Precursor Protein Shows a pH-Dependent Conformational Switch in Its E1 Domain

“…[18]). Its large ectodomain consists of the two rigidly folded segments E1 [19] and E2 [6,20,21] that are connected to one another and to the transmembrane helix of APP by flexible segments called the acidic domain and the juxtamembrane region, respectively [22]. In turn, E1 consists of the subdomains growth factor like (GFLD) and copper binding domain (CuBD).…”