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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 Unauthenticated Download Date | 8/12/17 6:51 AM 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) Unauthenticated Download Date | 8/12/17 6:51 AM I. Coburger et al.: Multi-domain structure of APP 487 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). Unauthenticated Download Date | 8/12/17 6:51 AM 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., Unauthenticated Download Date | 8/12/17 6:51 AM I. Coburger et al.: Multi-domain structure of APP 489 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 Unauthenticated Download Date | 8/12/17 6:51 AM 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. Unauthenticated Download Date | 8/12/17 6:51 AM I. Coburger et al.: Multi-domain structure of APP 491 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 Unauthenticated Download Date | 8/12/17 6:51 AM 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 Unauthenticated Download Date | 8/12/17 6:51 AM 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 Unauthenticated Download Date | 8/12/17 6:51 AM 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 Unauthenticated Download Date | 8/12/17 6:51 AM 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. 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Zheng, H., Jiang, M., Trumbauer, M.E., Hopkins, R., Sirinathsinghji, D.J., Stevens, K.A., Conner, M.W., Slunt, H.H., Sisodia, S.S., Chen, H.Y., et al. (1996). Mice deficient for the amyloid precursor protein gene. Ann. NY Acad. Sci. 777, 421–426. 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. Unauthenticated Download Date | 8/12/17 6:51 AM