Solution Conformation and Heparin-induced Dimerization of the Full-length Extracellular Domain of the Human Amyloid Precursor Protein

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  • Hg

    Matthias Gralle , Cris4

    ie

    biological signaling are discussed in terms of the structural models

    most widespread form of dementia and one of accumulate in the brains of AD patients, originatesfrom proteolytic cleavage of APP.2 Second, the

    doi:10.1016/j.jmb.2005.12.053Parkville, Vic. 3010, Australia

    6The Mental Health ResearchInstitute of Victoria, ParkvilleVic. 3010, Australia

    proposed.q 2005 Elsevier Ltd. All rights reserved.

    Keywords: analytical ultracentrifugation; modeling; signal transduction;small-angle X-ray scattering; structural domains*Corresponding author

    Introduction

    The amyloid precursor protein (APP) is linkedin two ways to Alzheimers disease (AD), the

    the leading causes of death among the elderly. Thefirst link came from the discovery that theb-amyloid peptide (Ab), the main component ofthe extracellular plaques that characteristically

    1William J. McKinstryRoberto Cappai5,6, MIris Torriani2,3 and S

    1Instituto de Bioqumica MedicaPrograma de Bioqumica eBiofsica Celular, UniversidadeFederal do Rio de JaneiroRio de Janeiro, RJ 21944-590Brazil

    2Instituto de Fsica GlebWataghin, UnicampCampinas, SP 13084-971Brazil

    3Laboratorio Nacional de LuzSncrotron, Campinas, SP13084-9701, Brazil

    4St. Vincents Institute ofMedical Research, 9 Princes St.Fitzroy, Vic. 3065, Australia

    5Department of PathologyCentre for NeuroscienceUniversity of Melbourne0022-2836/$ - see front matter q 2005 E

    M.G. and C.L.P.O. contributed eAbbreviations used: AD, Alzheim

    amyloid precursor protein; sAPPa,domain of the amyloid precursor psecretase cleavage; SAXS, small-ang

    E-mail address of the correspondferreira@bioqmed.ufrj.brtiano L. P. Oliveira2,3, Luiz H. Guerreiro1

    , Denise Galatis5,6, Colin L. Masters5,6,chael W. Parker4, Carlos H. I. Ramos3,rgio T. Ferreira1,3*

    Proteolytic cleavage of the amyloid precursor protein (APP) by b andg-secretases gives rise to the b-amyloid peptide, considered to be a causalfactor in Alzheimers disease. Conversely, the soluble extracellular domainof APP (sAPPa), released upon its cleavage by a-secretase, plays a numberof important physiological functions. Several APP fragments have beenstructurally characterized at atomic resolution, but the structures of intactAPP and of full-length sAPPa have not been determined. Here, ab initioreconstruction of molecular models from high-resolution solution X-rayscattering (SAXS) data for the two main isoforms of sAPPa (sAPPa695 andsAPPa770) provided models of sufficiently high resolution to identifydistinct structural domains of APP. The fragments for which structuresare known at atomic resolution were fitted within the solution models offull-length sAPPa, allowing localization of important functional sites (i.e.glycosylation, protease inhibitory and heparin-binding sites). Furthermore,combined results from SAXS, analytical ultracentrifugation (AUC) andsize-exclusion chromatography (SEC) analysis indicate that both sAPPaisoforms are monomeric in solution. On the other hand, SEC, bis-ANSfluorescence, AUC and SAXS measurements showed that sAPPa forms a2:1 complex with heparin. A conformational model for the sAPPa:heparincomplex was also derived from the SAXS data. Possible implications ofsuch complex formation for the physiological dimerization of APP andDomain of the Human Amyloid Precursor Protein

    1Solution Conformation andDimerization of the Full-lenlsevier Ltd. All rights reserve

    qually to this work.ers disease; APP,

    soluble extracellularrotein released by a-le X-ray scattering.ing author:eparin-inducedth Extracellular

    J. Mol. Biol. (2006) 357, 493508finding that mutations in APP cause hereditary,presenile AD indicated a causal connection of APPwith AD.3

    APP is present in the plasma membranes of allhuman cell types investigated so far, includingneurons, and has a single membrane-spanningdomain. APP can be cleaved in vivo by two different

    d.

  • proteolytic pathways. a-Secretase cleaves the extra-cellular domain of APP at a distance of 12 aminoacid residues from the membrane, in the middle ofthe amino acid sequence that corresponds to Ab.4

    This results in the release of a large extracellulardomain, known as sAPPa, encompassing up to 89%of the whole APP molecule. ADAM-10 has beenidentified as a protease necessary for a-secretaseactivity in vivo.5 Alternatively, and in much smalleramounts, APP can be cleaved by b-secretase (alsoknown as BACE1) at different residues upstream ofthe a-secretase cleavage site (for a review, seeVassar6). The membrane-inserted stub of APP leftafter b-secretase cleavage may be further processedby regulated intramembrane proteolysis by theg-secretase complex to release Ab and an intra-cellular fragment.7 While both Ab and the intra-

    spectroscopic and solution scattering tech-niques.26,27 Here, using a combination of high-resolution synchrotron small angle X-ray scattering(SAXS) and molecular modeling tools we proposestructural models for full-length sAPPa695 andsAPPa770, the main neuronal and non-neuronalisoforms of APP, respectively. These two isoformsdiffer by the presence or absence of a Kunitz-typeprotease inhibitor (KPI) domain, which is unique tothe longer isoform and necessary for some of thebiological functions of APP.28 Moreover, the state ofassociation of sAPPa in solution and its dimeri-zation induced by heparin were investigated.Possible implications of the interaction withheparin (or heparan sulfate) and dimerization ofAPP are discussed in terms of the regulation of itsbiological functions.

    42.6(G0.3) A and 46.5(G2) A for sAPPa695and sAPPa770, respectively, have been previously

    494 Conformation and Dimerization of Human sAPPacellular fragment released by g-secretase may exertsignaling functions in the healthy brain,8,9 they mayalso contribute to the pathogenesis of AD.10 As aresult, increasing a-secretase cleavage of APP to thedetriment of b-secretase cleavage has been pro-posed as a possible treatment for AD.5,11

    Alongside the interest in APP generated by itsinvolvement in AD, several important physiolo-gical cellular functions of this protein have beendiscovered. Transmembrane APP may play roles inneurite outgrowth,12 neuronal adhesion,13 axonaltransport14 and copper metabolism.15 sAPPa, themain fragment of APP, may be neuroprotective,16

    modulate synaptic function,17 and regulate proli-feration of neuronal progenitors18 and other celltypes.19 The primary structure of APP has beenknown for 17 years. However, in spite of the greatinterest in APP kindled by its involvement in ADand in many important cellular processes, itscomplete 3D structure and molecular mechanismsof action are still largely unknown. Very recently,there has been substantial progress in the structuralbiology of isolated APP domains,2025 but atomic-resolution structures of full-length sAPP andAPP have not been obtained. This prompted usto pursue the characterization of sAPPa bydomain. Cyt: cytoplasmic domain. Cleavage by a-secretase (C-terminal fragment (CTFa), as indicated. Amino acid numbFigure 1. Domain structure ofAPP. Grey ellipses: APP domainsconserved in evolution35 for whichcomplete or partial structures areknown (see the text). 1mwp, 1owt,1aap, 1rw6, 1tkn correspond to thePDB access codes for the fragmentsof known structure. (DE)n, RC:intervening non-conservedstretches (Asp and Glu-rich; ran-dom coil) suggested to be largelyunstructured or to have non-stan-dard secondary structures.26

    HBD1, HBD2: heparin-bindingdomains. CuBD: copper-bindingdomain. Ab: amyloidogenicResults

    Overall analysis of SAXS data for sAPPa

    We have investigated the soluble extracellulardomains of the two main isoforms of APP, sAPPa695and sAPPa770. APP695 is predominant in neurons,while APP770 is expressed in most non-neuronal celltypes and contains an insert of 75 amino acidresidues, corresponding to a KPI domain, in themiddle of its sequence (Figure 1). High-resolutionsmall-angle X-ray scattering data were collected forsAPPa695 and sAPPa770 (Figure 2(a) and (b),respectively) and were used to calculate thepairwise distance distribution functions by inverseFourier transform (Figure 2(c) and (d), respec-tively). These functions allow the determination ofthe maximum dimensions of the proteins, Dmax,and their radii of gyration, Rg. The maximumlengths obtained from the P(r) functions werew135 A and w150 A, respectively, for sAPPa695and sAPPa770, while radii of gyration (Rg) of

    sequence. TM: transmembranevertical arrow) releases sAPPa and the membrane-boundering is for APP770.

  • calculated from the same data set.27 Analysis of thedata using Guinier plots (insets to Figure 2(a) and(b)) yields Rg estimates of 46(G3) A and 47(G3) A,for sAPPa695 and sAPPa770, respectively, in good

    Conformation and Dimerization of Human sAPPaagreement with the values obtained from P(r)distribution analysis. The similarity of molecularweights obtained from SAXS data with the valuesexpected for both isoforms27 rules out the presenceof aggregates or other oligomers in the samples. Theshapes of the P(r) functions and the ratios betweenDmax and Rg for both APP isoforms clearly indicateelongated (prolate) shapes for the proteins. Con-sidering the high extent of sequence homologybetween them (the only difference being thepresence of the KPI domain in the longer APPisoform), one would indeed expect to encountersignificant similarity in their overall three-dimen-sional structures. Interestingly, however, theshoulder observed at rw100 A in the P(r) functionfor sAPPa695 indicates that the overall shape of thatisoform is somewhat different from that ofsAPPa770.

    Ab initio modeling

    The choice of the right strategy for ab initiomodeling using SAXS data depends on the qualityand information content of the scattering curve.29

    For momentum transfer values larger than 0.5 AK1,in addition to the determination of the overall shapeof the protein it is generally possible to obtaininformation on its fold and structural domains. Thepresent data set has an excellent signal-to-noiseratio and reaches qw0.8 AK1.27 This allowed usto analyze the experimental results employinga procedure capable of retrieving relatively detailedstructural information on the protein. To this end,we have represented the protein as a chain ofdummy residues and carried out simulated annea-

    Figure 2. Upper panels. SAXSintensity data for sAPPa695 (a) andsAPPa770 (b). Data measured atconcentrations up to 12.5 mg/ml(sAPPa695) or 9.6 mg/ml (sAPPa770)(open circles) were used to constructthe curves over the complete q rangeup to 0.8 AK1.27 These data areshown in order to allow evaluationof the quality of the fits derived fromab initio SAXS models (continuouslines) and from fitting the domains ofknown structure of APP into the abinitio SAXS models of sAPPa (brokenlines, shown up to qZ0.4 AK1). Fordescription of these models, seelegends to Figures 3 and 5. Insets:Guinier plots of the data showing themonodispersity of the samples.Lower panels, open circles: pairwisedistance distribution functions, p(r),calculated from the full range ofexperimental data for sAPPa695 (c)and sAPPa770 (d) using GNOM.

    57

    Continuous lines: fits from ab initioSAXS models (see Figure 3).

    495ling optimization to search for the configurationof the backbone that minimizes the discrepancybetween the scattering intensity or the P(r) functioncalculated from the ab initio model and theexperimental SAXS data.30 We have analyzed ourmodels in both real space and reciprocal space, andthe best results were obtained using real space P(r)fitting. The models generated in this way seemed torender more reliable values of radii of gyration ofthe proteins with a better fit of the low q region. Aspreviously reported, real space fitting relies less onthe high-angle scattering data than reciprocal spacefitting.31 The P(r) functions for both sAPPa isoformswere very well described by each of 20 indepen-dently calculated models that yielded the best fits tothe data. For evaluation of the quality of the fits,Figure 2 also shows the mean P(r) functions andstandard deviations (calculated using the 20 inde-pendent P(r) curves that gave best fits to the data)along with the experimental data for both sAPPaisoforms. It is apparent that the fits yielded verygood descriptions of the P(r) functions for both APPforms.

    Due to the use of a high number of dummyresidues in the model calculation, as well as to thepresence of flexible domains in the structure ofAPP,27 the results of the simulation lead to therecovery of slightly different independent structuralmodels even using our high-resolution SAXS data.Consequently, in order to retrieve the most probablemodel for each isoform, an averaging procedure

  • (a)d Mo y

    496 Conformation and Dimerization of Human sAPPawas carried out.32 In this procedure, the differentdummy residue models encountered as indepen-dent solutions to the fits are compared with eachother, and those having higher similarity areaveraged. The final 3D configuration is representedas a space-filling model with a close packing ofspheres (Figure 3, filled spheres), along with thesuperposition of all 20 independent solutions(semitransparent spheres) for each isoform.

    A useful check for the validity of ab initio modelscalculated from SAXS data is to compare thevolume and molecular weight ratios obtained fortwo proteins. Hydrodynamic volumes (V) werecalculated from the models shown in Figure 3 usingHYDROPRO,33 while molecular weights (MW)were determined directly from the zero-angleintensities in the SAXS measurements. If the modelsare compatible with the experimental data, the

    Figure 3. Solution conformational models for sAPPa695using a chain model of dummy residues (see Materials aneach isoform (shown as a light grey envelope) and filtered tThe models are rotated as indicated in the Figures.ratios between V(sAPPa695)/V(sAPPa770) andMW(sAPPa695)/MW(sAPPa770) should yield simi-lar values. For the present analysis, the valuesobtained were V(sAPPa695)/V(sAPPa770)Z0.89G0.05 and MW(sAPPa695)/MW(sAPPa770)Z0.90.The excellent agreement between those valuesindicates that the models correctly retrieve theexpected volume ratios for the two sAPPa isoforms.

    Table 1. Hydrodynamic data on HC, sAPPa and sAPPahep

    SampleBuoyant weight

    (kDa)Molecular weight

    (kDa)

    HC n.d. n.d.sAPPa695 19 69sAPPa770 n.d. n.d.sAPPa695heparin 44 155sAPPa770heparin n.d. n.d.

    The values of buoyant weight correspond to the best fits to equilibriumconcentrations ranging from 2.3 mM to 18.5 mM and heparin:sAPPa695from buoyant weights using the correct partial specific volume, as dcoefficients, s (in Svedberg units), correspond to the best fits to sedimpresence of 3.5 mM heparin or for HC at 140 mM, and for serial dicoefficients calculated from the ab initio SAXS models. Maximum smolecular weights. n.d., not determined.Calculation of sedimentation coefficients from theSAXS-derived ab initio models using HYDROPRO33

    gave values of 3.77 S for sAPPa695 and 3.93 S forsAPPa770. For both sAPPa isoforms, these valuesare considerably lower than the maximum sedi-mentation coefficient...

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