Download The structural biology of the amyloid precursor protein

DOI 10.1515/hsz-2013-0280 Biol. Chem. 2014; 395(5): 485–498
Review
Ina Coburger, Sandra Hoefgen and Manuel E. Than*
The structural biology of the amyloid precursor
protein APP – a complex puzzle reveals its
multi-domain architecture
Abstract: The amyloid precursor protein (APP) and its
processing are widely believed to be central for the etiology of Alzheimer’s disease (AD) and appear essential for
neuronal development and cell homeostasis in mammals.
Many studies show the proteolysis of APP by the proteases
α-, β- and γ-secretase, functional aspects of the protein
and the structure of individual domains. It is, however,
largely unclear and currently also widely debated of how
the structures of individual domains and their interactions
determine the observed functionalities of APP and how
they are arranged within the three-dimensional architecture of the entire protein. Further unanswered questions
relate to the physiologic function of APP, the regulation
of its proteolytic processing and the structural and functional effect of its cellular trafficking and processing. In
this review, we summarize our current understanding of
the structure-function-relationship of the multi-domain
protein APP. This type-I transmembrane protein consists
of the two folded E1 and E2 segments that are connected
to one another and to the single transmembrane helix by
flexible segments and likely fulfills several independent
functions.
Keywords: 3D-Structure; Alzheimer’s disease; overall
topology; structure-function-relationship.
*Corresponding author: Manuel E. Than, Protein Crystallography
Group, Leibniz Institute for Age Research-Fritz Lipmann Institute
(FLI), Beutenbergstr. 11, D-07745 Jena, Germany,
e-mail: [email protected]
Ina Coburger and Sandra Hoefgen: Protein Crystallography Group,
Leibniz Institute for Age Research-Fritz Lipmann Institute (FLI),
Beutenbergstr. 11, D-07745 Jena, Germany
Introduction
Alzheimer’s disease (AD) is the most frequent form of progressive dementia, occurring predominantly in the elderly
population (Blennow et al., 2006; Huang and Mucke, 2012).
It currently affects about 1 million patients in Germany and
about 25 million patients worldwide. The type one transmembrane amyloid precursor protein (APP) and its processing by a series of proteolytic cleavages are generally believed
to be causative to the disease (Thinakaran and Koo, 2008).
APP is initially cleaved either by α- or β-secretase (Reiss
and Saftig, 2009; Willem et al., 2009), liberating its large
extracellular domain at slightly different cleavage sites to
the soluble fragments sAPPα and sAPPβ. The remaining
intramembrane stubs, C83 and C99, are then cleaved by
the transmembrane protease γ-secretase (Li et al., 2009)
by regulated intramembrane proteolysis (RIP), leading to
the release of the APP intracellular domain (AICD) that has
been implicated in signal transduction (McLoughlin and
Miller, 2008). As second cleavage products, the short ∼25
and the primarily 40/42 amino acid long peptides p3 and
Aβ, respectively, are formed (Figure 1). It is the Aβ-peptide
that is specifically found in the senile plaques, the diseasedefining depositions in the brains of AD-patients. Correspondingly, its proteolytic generation from APP as well
as its aggregation into highly neurotoxic small oligomeric
Aβ-aggregates (Walsh and Selkoe, 2007) are considered to
be central to the aetiology of AD. The tau-protein, a second
protein that has been found repeatedly associated with
the development of AD, is also involved in disease-causing
molecular effects and seems to function downstream of Aβ
and APP (Selkoe et al., 2012).
APP belongs to a family of three mammalian genes and
APP-homologues from other organisms that fulfill essential
functions in the nervous system and its development (Aydin
et al., 2012). Their exact physiological function remains to
be established. Genetic ablation of only APP in mice shows
a rather mild phenotype (Zheng et al., 1996). Severe phenotypes are generated, however, if more than one family
member is deleted. Some (but not all) of these phenotypes can be rescued by expression of the soluble ectodomain-fragment sAPPα (Ring et al., 2007). Accordingly,
APP-family proteins are essential for proper mammalian
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486 I. Coburger et al.: Multi-domain structure of APP
Figure 1 Processing of the amyloid precursor protein.
In the amyloidogenic pathway, APP is cleaved by β- and γ-secretase,
leading to the generation of the soluble fragment sAPPβ and the
short Aβ peptide. This peptide aggregates and builds up the Aβamyloid. Alternatively, α-secretase cleaves within the Aβ-sequence
and thereby prevents the generation of these toxic peptides. A
non-toxic peptide p3 and the C-terminally slightly longer sAPPα are
generated instead (non-amyloidogenic pathway).
development and the different homologues are responsible for both, redundant and specific physiologic functions
(e.g., reviewed in Anliker and Muller, 2006). All three mammalian homologues, APP, the amyloid β (A4) precursor-like
protein 1 (APLP1) and the amyloid β (A4) precursor-like
protein 2 (APLP2), share a highly similar domain architecture and proteolytic processing, but only APP contains the
Aβ-sequence important for the development of AD. Several
trophic and neuroprotective functions such as the regulation of neurite outgrowth and axon guidance are associated
with APP (e.g., reviewed in Thinakaran and Koo, 2008).
Additionally, APP has been described to be involved in the
binding of metals (e.g., reviewed in Kepp, 2012) and of components of the extracellular matrix (ECM; see, for example,
Small et al., 1993), in cell adhesion (Thinakaran and Koo,
2008; Baumkotter et al., 2012) and to influence the synaptic function and long-term potentiation (e.g., Selkoe, 2008;
Wang et al., 2009; Aydin et al., 2012). Also tightly linked to
the physiologic function(s) of APP-family members is their
propensity to form homologous and heterologous dimers
that are involved in cell-cell-interactions in cis and trans.
Whereas Soba and coworkers described trans-dimerization
for all APP-family members (Soba et al., 2005), only APLP1
trans-dimerization was postulated by Kaden et al. (2009).
APP and its family members undergo alternative splicing.
Interestingly, the shortest isoform of APP (APP695) as well as
its homologue APLP1 show a neuron-specific distribution
(reviewed, for example, in Jacobsen and Iverfeldt, 2009),
whereas the longer isoforms of APP (APP751 and APP770) as
well as its homologue APLP2 are expressed ubiquitously.
As Aβ-deposits are specifically found in the brain of ADpatients and many data suggest a neuronal origin of this
peptide (see e.g., Sisodia et al., 1993), most research has
been concentrated on APP695. It is, however, not finally
clear from which APP-splice form the AD-associated Aβaggregates are derived.
Many investigations have targeted functional features of different segments of APP and its mammalian
paralogues and orthologues from other species as well as
of the respective overall proteins in the past. As for every
biomolecule, the function of APP is dependent on its threedimensional structures, of its domains and on the nature
of the linkages (flexible or fixed) between its structural
domains. Over the last few years, significant progress
has been made in our understanding of the structure of
the multi-domain protein APP and in the assignment of
its constituting domains to defined physiological functions. Several reviews and additional studies have also
placed this knowledge into a first overall picture of this
protein (see, for example, Reinhard et al., 2005; Gralle and
Ferreira, 2007). Recently, Coburger and coworkers (Coburger
et al., 2013) investigated how those domain-structures are
assembled within the entire APP-protein. Their study also
answers the question of whether they interact with one
another to form one defined overall, three-dimensional
structure of the entire protein or whether they form several
functional domains that are flexibly connected to one
another and hence act in part independently like balls on
a string. In addition we know only very little about the
structure-function-relationship of APP-homologues. From
a simple analysis of their sequences they cannot be of identical structure to APP, which is further underlined by their
largely homologous but not identical functionality observed
in various studies (Anliker and Muller, 2006). Given the
complex functional redundancy of the three mammalian
paralogues – APP, APLP1 and APLP2 – all homologues and
splicing isoforms must be considered together for any comprehensive understanding of the structure-function-relationship of this important family of proteins. In this review
we will summarize the current structural knowledge about
APP and its homologues and how this relates to the known
functional properties of this protein and its involvement in
the development of AD.
Overall structure
A large number of functional segments and isolated structural domains have been described in the last ∼20 years
for APP and its homologues (Figure 2A). Those include
crystallographic and NMR-structures of the growth-factorlike (GFLD; Rossjohn et al., 1999) and copper-bindingdomains (CuBD; Barnham et al., 2003; Kong et al., 2007)
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of APP, of its entire E1-domain (Dahms et al., 2010), of its
Kunitz type protease inhibitor domain (KPI; Hynes et al.,
1990; Scheidig et al., 1997), of its central APP domain (also
called CAPPD or E2 domain; Dulubova et al., 2004; Keil
et al., 2004; Wang and Ha, 2004; Dahms et al., 2012), of
its intracellular domain (AICD; Kroenke et al., 1997; Radzimanowski et al., 2008) and the recent elucidation of
the structure of its membrane-proximal part containing
the α-secretase cleavage site (Barrett et al., 2012), which
also showed that the protein’s very C-terminus is again
inserted into the lipid bilayer (see also Figure 2B). In addition, a larger number of other functional segments and
regions such as a heparin binding site within the GFLD
(Small et al., 1994), a zinc binding site next to the boundary between E1 and the acidic domain (AcD) (Bush et al.,
1993), phosphorylation sites within the extension domain
(ED) (Walter et al., 2000) as well as O-linked glycosylation sites within or next to the AcD (Perdivara et al., 2009;
Klatt et al., 2013) have been identified within its N-terminal half. Similarly, different domains are also specified for
the C-terminal half of the large APP-ectodomain, including, for example, the central CAPPD with an additional
N-linked glycosylation site (Pahlsson and Spitalnik, 1996),
a second heparin-binding domain (Clarris et al., 1997),
the RERMS-domain (Ninomiya et al., 1993), one collagenbinding domain (Beher et al., 1996), and the juxtamembrane region (JMR) that contains one additional O-linked
glycosylation site (Perdivara et al., 2009; Klatt et al., 2013).
The intracellular AICD contains further phosphorylation
sites (Chang et al., 2006). In addition, the structures of
domains of APP-homologues were resolved (Hoopes et al.,
2009; Lee et al., 2011; Xue et al., 2011a).
Figure 2 Overall structure and conservation of APP-family proteins.
(A) Domain architecture, functional segments and conservation of APP-family proteins. The transmembrane region of APP is assigned
according to Barrett et al. (2012) and the Kunitz type protease inhibitor domain (KPI)/OX2 is inserted few amino acid residues before
the beginning of E2. APP and its mammalian homologues APLP1 and APLP2 share on overall a similar domain architecture including the
E1-domain, the acidic region (AcD), the E2-domain (also called central APP domain, CAPPD), the juxtamembrane region (JMR), the transmembrane region (TM) and an intracellular domain [APP intracellular domain (AICD); APP-like intracellular domain (ALID1/ALID2)]. The
Aβ-peptide and the extension domain (ED) are only present in APP, the KPI/OX2-insertion only in APP and APLP2 and the E1-domain is less
conserved in APLP1 as indicated by its light green color. Additional functional units are indicated by brackets [heparin binding domain
(HBD); copper binding domain (CuBD); zinc binding domain (ZnBD); collagen binding domain (CBD); growth factor like domain (GFLD)].
Phosphorylation and glycosylation sites are shown as P and empty circles, respectively. (B) The two folded domains E1 and E2 of APP as
well as the transmembrane segment connecting the JMR with the AICD are shown as surface representation based on their respective
crystal or NMR structures [PDB-ID: 3KTM, (Dahms et al., 2010); 3NYL, (Wang and Ha, 2004); 2LP1, (Barrett et al., 2012)]. The KPI-domain,
only present in the longer splicing forms in between the AcD and the E2-domain, is likewise shown as surface representation (PDB-ID: 1AAP;
Hynes et al., 1990) and the surface-associated helix at the very C-terminus of APP (Barrett et al., 2012) is shown as idealized α-helix. Panel B
was prepared in part with PyMOL (http://www.pymol.org).
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488 I. Coburger et al.: Multi-domain structure of APP
Considering the function of the entire APP-protein
and its involvement in the aetiology of AD, it is essential to
know how these structural and functional segments form
and act within the context of the entire protein and within
the cellular environment: is it possible to link specific
functions to single sections of APP, which could hence act
in parallel in the context of the entire protein? Alternatively, do interactions between those subdomains couple
one functionality with the other? Do certain functionalities
depend on a previous processing step? In this regard the
structural and functional differences between the soluble
ectodomain fragments sAPPα and sAPPβ that are released
by the α- and β-secretase cleavages, respectively, are also
very intriguing. In addition to several shared features and
despite the fact that both shed ectodomain fragments
differ by only 16 most likely unstructured amino acids
residues at their C-terminus, largely different functions
were also found (Chasseigneaux and Allinquant, 2012).
Do those differences mainly result from different trafficking and from the different localization of the two ectodomain fragments within the cell? Are those few C-terminal
residues responsible for different interactions? Or do they
possibly give rise to different overall structures?
A first glimpse on the overall arrangement of the different domains of APP in space has been obtained by small
angle X-ray scattering (SAXS) (Gralle et al., 2006). These
results were contradicted in part, however, by a more recent
study employing the same method (Libeu et al., 2011).
Recently, Coburger and coworkers investigated the overall
structure of APP by a variety of methods, finding that in isolation it has a rather extended conformation without any
strong interactions between its constituting domains and
segments, which are arranged in space like balls on a string
(Coburger et al., 2013). Their data are in line with an earlier
analysis of the APP-sequence based on homology considerations (Wasco et al., 1992) and refined the definition of the
boundaries of the constituting domains. Correspondingly,
the large ectodomain of the neuronal isoform APP695 consists in sequence of an N-terminal folded domain, called
E1, that consists of its constituting subdomains GFLD and
CuBD (for their interaction within E1 see below), the small
ED, the highly flexible and negatively charged AcD, the
second folded domain of APP, called E2, that consist mainly
of flexible α-helices and a second highly flexible region,
called the JMR, which connects the entire ectodomain to
the single transmembrane helix (Figure 2B). At its C-terminus, APP features the short intracellular domain AICD. The
ubiquitously expressed isoforms of APP, APP751 and APP770
carry the additional KPI and the OX2-segment in-between
the AcD and E2. On one hand this overall domain architecture is in line with many experimental observations
obtained in the past and was more or less expected. There
are, however, also other domain and subdomain arrangements discussed in the literature, which clearly are not
compatible with this overall structure. For them to be of
physiological consequence, a structural re-arrangement or
processing step must occur to first produce the respective
alternative structures and/or peptide fragment(s). All more
complex structures and/or arrangements such as different
homo- and/or hetero-dimers between APP, parts of its ectodomain and its homologues (Kaden et al., 2012) are likely to
build on this overall structure of the momomeric protein.
What do we know about the overall structure of the
APP-homologues? The three mammalian APP-paralogues
are similar, but not identical in sequence and hence in
structure to APP and its different splicing forms (Figures 2A
and 3). In contrast to APP, its homologues APLP1 and
APLP2 show no conservation of the ED (Coburger et al.,
2013) and the purely neuronal APLP1 does, for example, not
contain the KPI domain whereas APLP2 does (Aydin et al.,
2012). Furthermore, the sequence conservation especially
between the E1-domains is more pronounced between
APP and APLP2 than between APP and APLP1 (Dahms
et al., 2010). This makes structural and hence functional
differences especially likely at the interface between its
constituting subdomains and with respect to a heparindependent dimerization of E1/APP (for details see below).
Comparing human APP to non-vertebrate homologues, an
even lower degree of conservation is observed. Whereas the
overall topology is likely to be similar, it becomes difficult
to predict details of the structural homology with certainty.
In addition, the multi-domain architecture of APP
and of its homologues as shown in Figure 2A gives also
rise to the parallel function of its independently folded
domains and segments and allows these proteins to fulfill
more than one physiologic function. Whereas E1 might be
responsible for a heparin-dependent dimerization of APP
(Soba et al., 2005; Kaden et al., 2008; Dahms et al., 2010)
and additionally might react by defined structural alterations to the cellular location of the molecule encoded
by the respective pH-values (Dahms et al., 2010), the
E2-domain might function as a conformational switch by
sensing the presence of the physiologically relevant metal
ions Cu2+ and Zn2+ (Dahms et al., 2012) and form a second
dimerization module (Wang and Ha, 2004; Xue et al.,
2011a). Further details will be discussed below.
The E1-domain
Initially, the two subdomains of E1, GFLD (Rossjohn
et al., 1999) and CuBD (Barnham et al., 2003; Kong et al.,
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Figure 3 Sequence alignment of APP-family members.
Human APP, APLP1 and APLP2 were aligned using Clustal X. Fully conserved amino acids are highlighted with (*). The (:) and (.) indicate a fully
conserved strong group and a conserved weaker group, respectively. Domain boundaries for APP are highlighted in colors according to Figure 2.
2007), were observed at the slightly basic pH of 7.5–8.0
as independent entities. No interaction between them
was found by SAXS studies (Gralle et al., 2006). A more
recent crystal structure obtained at slightly acidic pH
(Dahms et al., 2010) shows a distinct 3D-structure of the
entire E1-domain. Herein, the GFLD and the CuBD are connected by an ordered linker segment. They interact additionally over a tight interface consisting of a hydrophobic
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490 I. Coburger et al.: Multi-domain structure of APP
interaction core, one predominant salt bridge, several
hydrogen bonds and contacts mediated by water molecules (Figure 4A). At the N-terminus of APP, the GFLD is
made up by 7–9 β-strands, one α-helix, the intervening
segments of irregular structure and three disulfide bridges
that fold into a compact, globular domain. It is connected
by a linker of distinct fold but of no regular secondary
structure to the CuBD, which consists of a three-stranded
β-sheet that faces on one side a second α-helix, contains
three additional disulfide bridges and forms the second
compact subdomain of E1 with its somewhat elongated
overall shape. This structural arrangement spatially interferes with the binding of Cu2+-ions to His147, His151, Tyr168,
Met170 as observed for the isolated CuBD (Kong et al.,
2007). In line with this observation, no bound Cu2+-ions
were found in the crystals of the entire E1-domain (Dahms
et al., 2010). If one considers some structural flexibility,
the side chains of Asn89 and Glu87 of the GFLD could also
contribute to metal binding to the CuBD (Figure 4B).
Interestingly, the two E1-subdomains interact tightly
with one another only at an acidic pH value of around 5 as
observed in both, in the crystal lattice and also in solution.
Figure 4 Structure of the E1-domain.
(A) Cartoon representation of the entire E1-domain (PDB-ID: 3KTM;
Dahms et al., 2010). The growth factor-like domain (GFLD) is shown
in blue and the copper binding domain (CuBD) in green. The linker
in between is highlighted in red. Cysteine side chains are illustrated
as spheres. (B) Closeup of the copper binding site described for
the isolated CuBD within the context of the entire E1. Side chains
implicated in copper binding are shown as sticks. (C) Model of
the heparin-induced E1-dimer. The two interacting E1-domains
are shown as cartoon and surface representation, respectively.
The cross-connecting heparin-decamer is shown as sticks and the
cysteine residues 98 and 105 encompassing the loop, which is
involved in heparin binding, are illustrated as spheres. This figure
was prepared with PyMOL (http://www.pymol.org).
At pH 8.0 some accessibility of the linker region connecting the GFLD and the CuBD within E1 by V8 protease was
observed, suggesting a certain degree of plasticity of the
interaction at the higher pH (Dahms et al., 2010). As these
differences in interaction are caused by pH-changes characteristic for subcellular vesicles and extracellular space,
these pH-dependent structural alterations suggest a pHbased encoding of the subcellular location of APP resulting in a respectively altered functionality. This notion is
further supported by altered heparin binding properties
of E1 at these two pH-values (Dahms et al., 2010). It will be
interesting to see if there is a pH-dependency on binding
of Cu2+ to the E1-domain and/or if the binding of Cu2+ to the
CuBD affects its interaction with the GFLD.
Predominantly basic amino acid residues of the loop
encompassing cysteines 98 and 105 create a positively
charged surface patch that has been implicated in heparin-binding (Small et al., 1994). This region shows in part
elevated mobility of the constituting amino acid residues
and has been associated with the formation of APPdimers (Kaden et al., 2008; Dahms et al., 2010). Structural and biochemical data as well as a model developed
for the heparin-bridged E1-dimer (Dahms et al., 2010)
support that a moderate but significant interaction
between two opposing E1-molecules leads first to the formation of an elongated positively charged surface patch.
The resulting dimeric interaction is in turn stabilized
by the specific interaction with an elongated polyanion
such as heparin. Thus, two E1-entities of neighboring APP
molecules form a heparin (or heparansulfate proteoglycan, HSPG) cross-linked dimer (Figure 4C). Interestingly,
the binding of other negatively charged molecules to this
region was not described until now, but might be interesting to investigate in the future. This positively charged
surface patch is, in addition, well conserved between
APP and APLP2 but not in APLP1. Hence, the formation
of similar E1-based dimers is probably retained between
the paralogues APP and APLP2. APLP1, in contrast, has
been experimentally shown to dimerize differently in the
context of the entire ectodomain (Kaden et al., 2009).
This finding is very interesting, since dimerization of
APP has been implicated to play an important role for its
physiologic function. APP has been described to share
characteristics of a synaptic cell adhesion molecule,
connecting the pre- and postsynaptic neuron via dimerization (Baumkotter et al., 2012). As heterodimerization
between APP-family members was also shown (Soba
et al., 2005), this cell-cell-adhesion function could be
implemented differently in diverse cell-types depending
on expression levels and subcellular localization of the
proteins.
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The KPI domain
The KPI domain that is only present in the longer APPsplicing isoforms APP770 and APP751 was the first domain
of APP to be structurally characterized in isolation
(Hynes et al., 1990) and in complex with a protease target
(­Scheidig et al., 1997). Its structure is very similar to the
bovine pancreatic trypsin inhibitor (BPTI). Three conserved disulfide bridges dominate the fold consisting of
an α-helix that packs against one face of a twisted, twostranded β-sheet (Figure 5A). The positively charged side
chain of an arginine residue at position 15 of the inhibitor (Arg301 in APP770/APP751-numbering), which is part
of the reactive center loop (RCL), dominates its selectivity towards trypsin and other trypsin-like proteases
(Figure 5B).
As the neuronal isoform APP695 does not contain the
KPI domain, this domain was not in the main focus of ADresearch within recent years. Interestingly, recent findings
of Ben Khalifa and coworkers on the enhancement of the
APP-homodimerization caused by the presence of the KPI
(Ben Khalifa et al., 2012a) are contradicted by data of Isbert
and coworkers, who found the opposite effect (Isbert et al.,
2012). Using bimolecular f­luorescence complementation
(BiFC) Khalifa et al. could also show that APP-dimers containing mutations in the KPI were retained in the endoplasmatic reticulum, whereas wildtype APP751 was mostly
Figure 5 Structure of the KPI.
(A) Cartoon representation of the KPI (PDB-ID: 1AAP; Hynes et al.,
1990) in rainbow-coloring ranging from its N-terminus (blue) to its
C-terminus (red). Cysteines are shown as spheres. (B) Cartoon representation similar to panel A, highlighting the reactive center loop
(RCL) in darker brown. Arg301, which is important for the selectivity
towards trypsin and other trypsin-like proteases, is shown in stick
representation. This figure was prepared with PyMOL (http://www.
pymol.org).
localized in the Golgi region at steady state (Ben Khalifa
et al., 2012b). Together with the fact that the KPI-domain
binds to the LDL-receptor related protein, which in turn
is involved in the internalization of APP (Kounnas et al.,
1995), the KPI apparently influences the processing of the
ubiquitously expressed isoforms of APP. It will hence be
very interesting to obtain more information on the physiologic function of the KPI in the future and to see if it possibly relates to the genetic association between AD and the
ε4-allele of the apolipoprotein E (ApoE4; Verbeek et al.,
2000).
The E2-domain
The E2-domain represents the second region of defined
three-dimensional structure of the neuronal isoform
APP695. It consists exclusively of α-helical secondary structure (Dulubova et al., 2004; Keil et al., 2004; Wang and
Ha, 2004) and can be subdivided into an N-terminal coil
consisting of helices αB and roughly the first half of αC
as well as of a C-terminal four helix bundle consisting of
helices αC (second half) through αF (Figure 6). The two
motifs are connected by the ∼90 Å long, 15 turns encompassing helix αC that belongs to both regions. In some
structures an additional helix αA precedes the N-terminal
coiled coil. Interestingly, the helices constituting E2 show
in the uncomplexed state a high flexibility in their relative
orientation and elevated B-factors, especially for the N-terminal coiled coil. This is also reflected by different relative
orientations observed for independently solved structures of APP-E2 and of its homologues (Dulubova et al.,
2004; Wang and Ha, 2004; Hoopes et al., 2009; Lee et al.,
2011; Dahms et al., 2012). Within E2, several positively
charged amino acid residues that were initially identified
in different peptides to bind to heparin (Mok et al., 1997)
come together, forming one positively charged surface
patch that represents the heparin-binding surface of this
domain. For the homologous APLP1-E2, heparin binding
was also shown in the crystalline state and heparin was
suggested to have a dual role in the dimerization of the
E2-domain in APP and APLP1 (Lee et al., 2011; Xue et al.,
2011a). It currently remains to be finally established
how the E2-domain is involved in a functional heparindependent dimerization of APP and/or of its homologues.
Both, monomeric (Hoopes et al., 2009; Dahms et al., 2012)
and dimeric forms of this domain (Wang and Ha, 2004;
Lee et al., 2011) have been described for APP and its homo­
logues. Although one would instinctively anticipate the
same functionality of the E2-domain of APP and APLP1
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492 I. Coburger et al.: Multi-domain structure of APP
interaction of the entire RERMS-sequence with its receptor
is thus structurally only feasible if this part of E2 becomes
either unfolded or proteolytically cleaved out. In addition, metal-ion specific structures were also implicated to
induce such conformational changes of APP-E2, and with
that of the entire APP-protein, which alter its interaction
with ligands, its subcellular trafficking and its proteolytic
processing as a consequence of binding to the respective
metal ions (Dahms et al., 2012).
Another structurally very interesting observation is
the closeness in space of the SOS (Xue et al., 2011b) and
heparin (Xue et al., 2011a) binding sites within APLP1-E2
and this M1-site within APP-E2 (Dahms et al., 2012). This
might hint towards an explanation for the functionally
observed effect of, for example, the binding of Zn2+ on the
binding of heparin (Multhaup et al., 1995). Details of this
interaction remain, however, to be established.
Figure 6 Structure of the E2-domain and its metal binding site M1.
On the left the entire E2-domain is shown in cartoon representation
(PDB-ID: 3UMK; Dahms et al., 2012). The termini and helices αB
through αF are labeled and the M1 metal binding site is shown with
its four coordinating histidines in stick representation. On the right,
two close ups of M1 coordinating Cu2+- (PDB-ID: 3UMK; Dahms et al.,
2012) and Zn2+-ions (PDB-ID: 3UMI; Dahms et al., 2012) with different geometries are shown. This figure was prepared with PyMOL
(http://www.pymol.org).
given their sequence homology, studies show that the two
paralogues can dimerize via different domains (Kaden
et al., 2009).
Recently, Dahms and coworkers identified a novel
metal binding site within the E2-domain of APP (Dahms
et al., 2012). Crystallizing this domain in the presence of
Cd2+ ions, the authors found that E2 features several intraand intermolecular binding sites for this ion, which stabilize this domain within the crystal lattice. In addition, the
intramolecular metal binding site M1 sits like a welding
point in the center of E2 (Figure 6). As a consequence,
metal binding stabilizes E2. The M1-site shows a metal-specific coordination for the two ions Zn2+ and Cu2+ and hence
represents a metal-ion driven molecular switch or sensor,
which binds to these two physiologically interacting ions
with different geometries and affinities. Interestingly,
the E2-domain contains, for example, the pentapeptide
RERMS, a sequence that was implicated with growth
promoting functions of APP (Ninomiya et al., 1993). This
sequence is part of the N-terminal coiled coil of E2 and
hence shows an α-helical conformation. A peptide-like
The flexible linkers AcD and JMR
As already described above, the E1- and the E2-domains
are connected to one another by the flexible segment AcD.
E2 is connected to the single transmembrane helix of APP
by the likewise flexible JMR (Figure 2B). Between E1 and
AcD lies the small ED (Coburger et al., 2013), that is conserved among mammalian APP. Within the ED four phosphorylation sites were previously described at Ser193,
Ser198, Ser206 and Ser221 (Walter et al., 1997, 2000), but it
is currently undetermined what molecular and functional
property of APP this phosphorylation event influences.
Although not as widely studied as regulation by intracellular phosphorylation, the existence of extracellular
phosphorylation is well established (Ehrlich et al., 1990).
Similarly, the conservation of the AcD within vertebrates
and its existence in APP-homologues from lower organisms makes the presence of such a flexible connection of
strong negative charge density probably essential for the
functionality of APP. Hence both regions, the ED and the
AcD, seem to be of evolutionary value. Their exact function remains, however, to be established.
Likewise the JMR is mostly disordered. Its presence is
also conserved among the different APP-homologues. As
the sequence conservation is much lower in this region
of the molecule, it might function as a “plain linker”. The
sites of β- and α-secretase cleavage are within or at the
C-terminus of this segment, and the 16 amino acids that
are contained in sAPPα, but not in sAPPβ, belong to the
JMR. It will thus be very interesting to find out if there is
some residual structure within the JMR that possibly might
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I. Coburger et al.: Multi-domain structure of APP 493
explain the functional differences observed between
sAPPα and sAPPβ and might also influence the cleavage
partition between α- and β-secretase and with that also
the formation of the neurotoxic Aβ and the neutral p3.
The three-dimensional structure of the very C-terminus
of the JMR and the subsequent transmembrane helix
has recently been determined (Barrett et al., 2012) and is
described below.
Trans-membrane-helix
Besides its mostly helical structure, little is known about
the exact conformation of the single transmembrane helix
that connects the JMR with the AICD. It is often also referred
to as the transmembrane domain (TMD) or transmembrane segment (TMS) of APP. During the last 20–30 years
many NMR-structures of Aβ (or of its truncated forms) have
been determined in different solvents (e.g., Luhrs et al.,
2005; Tomaselli et al., 2006; Vivekanandan et al., 2011).
Those studies also show the mostly helical nature of the
transmembrane segment. Recently, Barrett and coworkers
reported the structure of the entire C99 peptide (Barrett
et al., 2012), which represents the β-secretase product and
γ-secretase substrate. Hence it also contains the transmembrane segment of APP (Figure 7). Beside the mostly
α-helical structure of the transmembrane segment, the
authors found that the β-secretase cleavage site is situated
roughly 16 amino acid residues away from the membrane
surface and hence belongs to the unstructured JMR. The
α-secretase cleavage site, in contrast, sits directly at the
membrane surface. The few amino acid residues between
this site and the beginning of the transmembrane helix
form an additional helical patch, the N-helix, basically
folding back onto the membrane (Barrett et al., 2012). In
addition, the flexibly curved nature of the transmembrane
helix of APP/C99 might be central to its recognition and
proteolysis by γ-secretase. The authors also describe a
cholesterol binding site that is composed of amino acid
residues belonging to the N-helix, the transmembrane
helix and the N-loop connecting the two and hence is
ideally located to influence the proteolytic processing of
APP.
Another interesting feature of the transmembrane
segment is the presence of a paired GxxxG (or GxxxGxxxG)
motif that was shown to be involved in the dimerization
of the transmembrane region and to affect the formation
of Aβ (Munter et al., 2007). Whereas a recent NMR-study
of the transmembrane segment of APP revealed a dimeric
arrangement of this region under micellar conditions
(Nadezhdin et al., 2012) another study suggested a rather
monomeric state under physiologic conditions, as binding
to cholesterol in a 1:1 stoichiometry might be favored in the
cell membrane (Song et al., 2013). Thus, it will be exciting
to see future experiments on the dimerization of APP, its
consequence for its proteolytic processing and the effect
of cholesterol binding.
The intracellular domain AICD
Figure 7 NMR-structure of C99 in the micellar state.
The transmembrane helix (TM) and the N-helix are shown as cartoon
representation with rainbow-coloring ranging from its N-terminus
(blue) to its C-terminus (red) as based on PDB-ID: 2LP1 (Barrett
et al., 2012). The cleavage sites of the α- and γ-secretase are indicated by a horizontal arrow. Glycines Gly700, Gly704 and Gly708
that are part of the GxxxGxxxG motif are illustrated as spheres. This
figure was prepared with PyMOL (http://www.pymol.org).
The AICD consists of 49 amino acid residues that in isolation form no defined or rather a transient three-dimensional structure (Kroenke et al., 1997; Ramelot et al.,
2000). It has been shown to interact with a large number
of effector and adaptor proteins (Cao and Sudhof, 2001,
2004; McLoughlin and Miller, 2008; Muller et al., 2008;
Chakrabarti and Mukhopadhyay, 2012; Pardossi-Piquard
and Checler, 2012). This interaction has been implicated
to the conserved YENPTY sequence patch within the AICD
(Borg et al., 1996) and the anticipated signal transduction
function of APP (Pardossi-Piquard and Checler, 2012). In
this scenario, a so-far unidentified extracellular ligand or
signal would influence the proteolytic processing of APP
and hence the release of AICD into the cytosol, which in
turn would trigger the intracellular response in a typical
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494 I. Coburger et al.: Multi-domain structure of APP
RIP based signaling. In fact, several potential target proteins have been described in the past, including APP
itself, BACE, Tip60, GSK3b and KAI1 (von Rotz et al., 2004)
and genes affecting the dynamics of the actin cytoskeleton (Muller et al., 2007). Also, the functional concept of
a receptor function of APP similar to the well understood
notch-signaling (Nichols et al., 2007) is quite appealing.
The final proof of this has, however, been elusive so far
and it is also discussed that APP does not have such a
receptor function (reviewed, for example, in Thinakaran
and Koo, 2008; Jacobsen and Iverfeldt, 2009).
Radzimanowski and coworkers solved the crystal
structure of the AICD in complex with its interacting
domain of Fe65 (Radzimanowski et al., 2008). In this structure (Figure 8), large parts of the AICD become structured
upon its interaction with Fe65-PTB2. This non-typical
complex between a PTB-family protein and its interacting YENPTY motif of APP shows a more intricate interaction between both partners compared to other structural
studies employing AICD-derived peptides (Zhang et al.,
1997; Yun et al., 2003; Li et al., 2008). Fe65-PTB2 stabilizes the two α-helices αN (Pro669-Gln679, APP695 numbering) and αC (Pro685-Gln692) of AICD. The conserved
interaction motif YENPTY lies mostly in-between these
two helices and makes strong contacts to Fe65-PTB2.
Helix αN is capped at its N-terminus by Thr668. Whereas
this residue and its phosphorylation-dependent properties have been repeatedly implicated with the regulatory function of the AICD (see e.g., Chang et al., 2006) no
contact to the Fe65-PTB2 is seen in the crystal. Based on
their findings and previous data (Ramelot and Nicholson,
2001), Radzimanowski et al. propose a molecular switch
model in which the phosphorylation of Thr668 influences the formation of the capping box at the N-terminus
of αN that in turn affects the interaction between AICD
and Fe65 (Radzimanowski et al., 2008). Taken together,
the AICD interacts differently with the various identified
binding partners and the structural details of those interactions possibly influence their functional fate. In several
or all cases an interaction-dependent folding of the other­
wise only transiently structured AICD seems to be part of
contact formation.
Another very interesting structural observation on the
AICD comes from the recent work of Barrett and coworkers. In their NMR and EPR studies regarding the structure
of the entire C99 peptide (Barrett et al., 2012), they found
that the protein’s very C-terminus was associated with the
membrane, suggesting an important role of this section
in, for example, the phosphorylation of the AICD or its
interaction with binding partners.
Conclusions and outlook
Figure 8 Structure of the APP intracellular domain (AICD) in
complex with Fe65-PTB2.
Cartoon representation of the AICD highlighted in reddish colors
(αN in orange and αC in red) bound to Fe65-PTB2 shown in gray as
based on PDB-ID: 3DXC, (Radzimanowski et al., 2008). The YENPTY
motif important for the interaction with effector/adaptor proteins,
and Thr668 that can be phosphorylated are illustrated as sticks.
This figure was prepared with PyMOL (http://www.pymol.org).
The central position of the APP within the development of
AD strongly calls for a better understanding of its physio­
logical function and its malfunction in AD. Currently the
proteolytic processing of APP is quite well understood and
a large number of potential physiologic functions have
been described. Compared to this knowledge, our structural insight in APP and that of its structure-functionrelationship is still quite limited. Over the last few years,
significant progress has been made in our understanding
of the three-dimensional structure of the multi-domain
protein APP and in the assignment of its constituting
domains to defined physiological functions, as discussed
in this review.
The next steps towards a better understanding of the
structure-function-relationship of APP and the molecular
basis of AD include a better comprehension of the structure of the entire APP-family. Given their complex redundancy, all three homologues, APP, APLP1 and APLP2 as
well as all splicing isoforms must be considered together
and we must understand what physiological function
and which structural properties are provided by which
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I. Coburger et al.: Multi-domain structure of APP 495
domain, homologue and isoform. Another central question relates to the dimerization of APP and its homologues. The functional and structural dissection of the
involved domains and contact types will lead to the development of specific interacting molecules. Those, in turn,
will drastically improve our understanding of the underlying molecular principles and might also represent novel
compounds of pharmaceutical interest for a future treatment of AD. In addition, we must strive to further link the
observed structural and functional properties of the APPfamily proteins in order to gain a better understanding of
the structure-function-relationship of this important class
of proteins and to rationally develop new strategies and
compounds to effectively treat the currently uncurable AD
in the future.
Acknowledgments: The work of M. E. Than was supported
by a grant from the Deutsche Forschungsgemeinschaft
(SFBs 596/604) and the work of S. Hoefgen from the Graduate School “Leibniz Graduate School on Ageing and AgeRelated Diseases, LGSA” of the FLI. The authors apologize
for the many interesting publications that could not be
cited in this review because of space restrictions.
Received November 15, 2013; accepted February 4, 2014; previously
published online February 7, 2014
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Ina Coburger studied biochemistry at the Friedrich-Schiller-University in Jena, Germany and obtained her diploma in 2010. Since then
she has worked on her PhD in the laboratory of Manuel E. Than at
the Leibniz Institute for Age Research-Fritz Lipmann Institute (FLI).
Her thesis is focused on structure-function relationships of APP.
Manuel E. Than is currently independent junior group leader and
head of the protein crystallography group at the Leibniz Institute
for Age Research – Fritz Lipmann Institute (FLI), Jena, Germany. He
studied Chemistry and Biochemistry in Bayreuth, Germany and
Delaware, USA. In 2000 he received his PhD from the TU Munich,
Germany, for structural biology work in the department of Robert
Huber at the Max-Planck-Institute of Biochemistry on transmembrane and soluble proteins of the energy metabolism. During his
postdoctoral time with Wolfram Bode and his habilitation at the
Gene Center of the Ludwig Maximilians University Munich he predominantly worked on the structural biology of proteases and methodological developments in protein crystallography. His group that
he founded in 2006 focuses on X-ray crystallographic, biochemical
and biophysical investigations of proteins involved in the development of Alzheimer’s disease and other aging-related processes.
Sandra Hoefgen studied biology at the Friedrich-Schiller-University
in Jena, Germany and obtained her diploma in 2009. Since then she
has worked on her PhD in the laboratory of Manuel E. Than at the
Leibniz Institute for Age Research-Fritz Lipmann Institute (FLI). Her
thesis is focused on the dimerization behavior of APP.
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