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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