Self-association of the Lentivirus protein, Nef

Background

The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization.

Results

By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123). Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIV_MAC239Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface.

Conclusions

We have demonstrated significant levels of intracellular Nef oligomers by immunoprecipitation from cellular extracts. However, our results are contrary to the identification of salt bridges between R105 and D123 as necessary for self-association. Importantly, binding between HIV-1Nef and SIVNef demonstrates evolutionary conservation and therefore significant function(s) for oligomerization. Based on modeling studies of Nef self-association, we propose a new dimerization interface. Finally, our findings support a stochastic model of Nef function with a dispersed intracellular distribution of Nef oligomers.     

Specific interactions by the N-terminal arm inhibit self-association of the AraC dimerization domain

Deletion of the regulatory N-terminal arms of the AraC protein from its dimerization domain fragments increases the susceptibility of the dimerization domain to form a series of higher order polymers by indefinite self-association. We investigated how the normal presence of the arm inhibits this self-association. One possibility is that arms can act as an entropic bristles to interfere with the approach of other macromolecules, thereby decreasing collision frequencies. We examined the repulsive effect of flexible arms by measuring the rate of trypsin cleavage of a specially constructed ubiquitin-arm protein. Adding an arm to ubiquitin or increasing its length produced only a modest repulsive effect. This suggests that arms such as the N-terminal arm of AraC do not reduce self-association by entropic exclusion. We consequently tested the hypothesis that the arm on AraC reduces self-association by binding to the core of the dimerization domain even in the absence of arabinose. The behaviors of dimerization domain mutants containing deletions or alterations in the N-terminal arms substantiate this hypothesis. Apparently, interactions between the N-terminal arm and the dimerization domain core position the arm to interfere with the protein-protein contacts necessary for self-association.     

Actin activates a cryptic dimerization potential of the vinculin tail domain

The tail domain of vinculin (V(t)) is an actin binding module containing two regions that interact with F-actin. Although intact V(t) purified from a bacterial expression system is a globular monomer, each actin binding region dimerizes when expressed individually, suggesting the presence of cryptic self-association sites whose exposure is regulated. We show that actin modulates V(t) self-association by inducing or stabilizing a conformational change in V(t) that allows dimerization. Chemical cross-linking studies implicate one of the actin binding regions in mediating dimerization in the presence of actin. Actin-induced V(t) dimers may play a role in the filament cross-linking activity of this protein. The V(t) dimers induced by actin are biochemically distinct from the V(t) dimers and higher oligomers induced by acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate, suggesting structural differences in V(t) bound to these two ligands that may provide a mechanistic basis for inhibition of F-actin binding by phosphatidylinositol 4,5-bisphosphate. The ability of actin to regulate the dimerization state of an actin binding protein suggests that, rather than serving a passive structural role, actin filaments may directly participate in signal transduction and other cellular events that are known to depend on cytoskeletal integrity.     

Sequence-specific dimerization of the transmembrane domain of the "BH3-only" protein BNIP3 in membranes and detergent

Mitochondria-mediated apoptosis is regulated by proteins of the Bcl-2 superfamily, most of which contain a C-terminal hydrophobic domain that plays a role in membrane targeting. Experiments with BNIP3 have implicated the transmembrane (TM) domain in its proapoptotic function, homodimerization, and interactions with Bcl-2 and Bcl-xL. We show that the BNIP3 TM domain self-associates strongly in Escherichia coli cell membranes and causes reversible dimerization of a soluble protein in the detergent SDS when expressed as an in-frame fusion. Limited mutational analysis identifies specific residues that are critical for BNIP3 TM self-association in membranes, and these residues are also important for dimerization in SDS micelles, suggesting that the self-association observed in membranes is preserved in detergent. The effects of sequence changes at positions Ala176 and Gly180 suggest that the BNIP3 TM domain associates using a variant of the GXXXG motif previously shown to be important in the dimerization of glycophorin A. The importance of residue His173 in BNIP3 TM domain dimerization indicates that polar residues, which have been implicated in self-association of model TM peptides, can act in concert with the AXXXG motif to stabilize TM domain interactions. Our results demonstrate that the hydrophobic C-terminal TM domain of the pro-apoptotic BNIP3 protein dimerizes tightly in lipidic environments, and that this association has a strong sequence dependence but is independent of the identity of flanking regions. Thus, the transmembrane domain represents another region of the Bcl-2 superfamily of proteins that is capable of mediating strong and specific protein-protein interactions.     

Dimerization Capacities of FGF2 Purified with or without Heparin-Affinity Chromatography

Fibroblast growth factor-2 (FGF2) is a pleiotropic growth factor exhibiting a variety of biological activities. In this article, we studied the capacity of FGF2 purified with or without heparin affinity chromatography to self-associate. Analyzing the NMR HSQC spectra for different FGF2 concentrations, heparin-affinity purified FGF2 showed perturbations that indicate dimerization and are a higher-order oligomerization state. HSQC perturbation observed with different FGF2 concentrations revealed a heparin-binding site and two dimer interfaces. Thus, with increasing protein concentrations, FGF2 monomers make contacts with each other and form dimers or higher order oligomers. On the contrary, FGF2 purified with ion-exchange chromatography did not show similar perturbation indicating that self-association of FGF2 is eliminated if purification is done without heparin-affinity chromatography. The HSQC spectra of heparin-affinity purified FGF2 can be reproduced to some extent by adding heparin tetra-saccharide to ion exchange chromatography purified FGF2. Heparin-affinity purified FGF2 bound to acceptor and donor beads in a tagged form using His-tagged or GST-tagged proteins, also dimerized in the AlphaScreen^™ assay. This assay was further validated using different experimental conditions and competitors. The assay constitutes an interesting tool to study dimerization of other FGF forms as well.     

Folding and homodimerization of wheat germ agglutinin

Wheat germ agglutinin (WGA) is emblematic of proteins that specialize in the recognition of carbohydrates. It was the first lectin reported to have a capacity for discriminating between normal and malignant cells. Since then, it has become a preferred model for basic research and is frequently considered in the development of biomedical and biotechnological applications. However, the molecular basis for the structural stability of this homodimeric lectin remains largely unknown, a situation that limits the rational manipulation and modification of its function. In this work we performed a thermodynamic characterization of WGA folding and self-association processes as a function of pH and temperature by using differential scanning and isothermal dilution calorimetry. WGA is monomeric at pH 2, and one of its four hevein-like domains is unfolded at room temperature. Under such conditions, the agglutinin exhibits a fully reversible thermal unfolding that consists of three two-state transitions. At higher pH values, the protein forms weak, nonobligate dimers. This behavior contrasts with that observed for the other plant lectins studied thus far, which form strong, obligate oligomers, indicating a distinctly different molecular basis for WGA function. For dimer formation, the four domains must be properly folded. Nevertheless, depending on the solution conditions, self-association may be coupled with folding of the labile domain. Therefore, dimerization may proceed as a rigid-body-like association or a folding-by-binding event. This hybrid behavior is not seen in other plant lectins. The emerging molecular picture for the WGA assembly highlights the need for a reexamination of existing ligand-binding data in the literature.     

Self-association of IQGAP1: characterization and functional sequelae

The scaffolding protein IQGAP1 participates in numerous cellular functions by binding to target proteins such as actin, calmodulin, E-cadherin, beta-catenin, Cdc42, Rac1, and CLIP-170. IQGAP1 regulates the cytoskeleton, promotes cell motility, and modulates E-cadherin-mediated cell-cell adhesion. However, how IQGAP1 exerts its functions in vivo is still unclear. In this study we investigate the self-association of IQGAP1 and its role in IQGAP1 function. Endogenous IQGAP1 co-immunoprecipitated from MCF-7 cells with IQGAP1 tagged with enhanced green fluorescent protein, indicating that IQGAP1 self-associates in cells. In vitro assays confirmed that IQGAP1 can self-associate and that this effect is mediated by the N-terminal half of the protein. Gel filtration analysis suggested that full-length IQGAP1 exists as a combination of monomers, dimers, and larger oligomers. Analysis performed with multiple fragments of IQGAP1 narrowed the self-association region to amino acids 763-863. In support of this observation, a peptide comprising residues 763-863 disrupted self-association of full-length IQGAP1 in a dose-dependent manner. Similarly, deleting this sequence from IQGAP1 abolished binding to full-length IQGAP1. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. Consistent with these findings, transfection into cells of a peptide containing the self-association domain significantly reduced the amount of active Cdc42 in cell lysates. These observations define a sequence of IQGAP1 that is necessary for its oligomerization and demonstrate that self-association is required for the normal cellular function of IQGAP1.     

E6 Proteins from Diverse Papillomaviruses Self-Associate Both InVitro and In Vivo

Papillomavirus E6 oncoproteins bind and often provoke the degradation of many cellular proteins important for the control of cell proliferation and/or cell death. Structural studies on E6 proteins have long been hindered by the difficulties of obtaining highly concentrated samples of recombinant E6. Here, we show that recombinant E6 proteins from eight human papillomavirus strains and one bovine papillomavirus strain exist as oligomeric and multimeric species. These species were characterized using a variety of biochemical and biophysical techniques, including analytical gel filtration, activity assays, surface plasmon resonance, electron microscopy and Fourier transform infrared spectroscopy. The characterization of E6 oligomers is facilitated by the fusion to the maltose binding protein, which slows the formation of higher-order multimeric species. The proportion of each oligomeric form varies depending on the viral strain considered. Oligomers appear to consist of folded units, which, in the case of high-risk mucosal human papillomavirus E6, retain binding to the ubiquitin ligase E6-associated protein and the capacity to degrade the proapoptotic protein p53. In addition to the small-size oligomers, E6 proteins spontaneously assemble into large organized multimeric structures, a process that is accompanied by a significant increase in the β-sheet secondary structure content. Finally, co-localisation experiments using E6 equipped with different tags further demonstrate the occurrence of E6 self-association in eukaryotic cells. The ensemble of these data suggests that self-association is a general property of E6 proteins that occurs both in vitro and in vivo and might therefore be functionally relevant.     

The central region of human T-cell leukemia virus type 1 Tax protein contains distinct domains involved in subunit dimerization

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) can form homodimers. Tax dimerization contributes to optimal Tax activity involved in transactivation of the HTLV-1 promoter. The mechanisms used to form specific Tax dimers are poorly understood because the domains that mediate such interactions have not been clearly characterized. Here we have used different approaches (the two-hybrid assay in yeast, the glutathione S-transferase pull-down assay, and the Spot method) to study Tax-Tax interactions. Our results indicate that the integrity of the sequence of Tax, except for the last 16 amino acids (residues 338 to 353), is critical, suggesting that Tax dimerization is dictated more by secondary structure than by primary structure. We were, however, able to delimit a central region involved in Tax self-association that encompasses the residues 127 to 228. This region can be divided into three subdomains of dimerization: DD1 (residues 127 to 146), DD2 (residues 181 to 194), and DD3 (residues 213 to 228). Moreover, the Tax mutants M22 (T130A and L131S) and M29 (K189A and R190S), with amino acid substitutions located in DD1 and DD2, respectively, were found to be impaired in Tax self-association.     

Determinants of the higher order association of the restriction factor TRIM5alpha and other tripartite motif (TRIM) proteins

Many tripartite motif (TRIM) proteins self-associate, forming dimers and higher order complexes. For example, dimers of TRIM5α, a host factor that restricts retrovirus infection, assemble into higher order arrays on the surface of the viral capsid, resulting in an increase in avidity. Here we show that the higher order association of different TRIM proteins exhibits a wide range of efficiencies. Homologous association (self-association) was more efficient than the heterologous association of different TRIM proteins, indicating that specificity determinants of higher order self-association exist. To investigate the structural determinants of higher order self-association, we studied TRIM mutants and chimeras. These studies revealed the following: 1) the RING domain contributes to the efficiency of higher order self-association, which enhances the binding of TRIM5α to the human immunodeficiency virus (HIV-1) capsid; 2) the RING and B-box 2 domains work together as a homologous unit to promote higher order association of dimers; 3) dimerization is probably required for efficient higher order self-association; 4) the Linker 2 region contributes to higher order self-association, independently of effects of Linker 2 changes on TRIM dimerization; and 5) for efficiently self-associating TRIM proteins, the B30.2(SPRY) domain is not required for higher order self-association. These results support a model in which both ends of the core TRIM dimer (RING-B-box 2 at one end and Linker 2 at the other) contribute to the formation of higher order arrays.     

Membrane Protein Dimerization in Cell-Derived Lipid Membranes Measured by FRET with MC Simulations

Many membrane proteins are thought to function as dimers or higher oligomers, but measuring membrane protein oligomerization in lipid membranes is particularly challenging. Förster resonance energy transfer (FRET) and fluorescence cross-correlation spectroscopy are noninvasive, optical methods of choice that have been applied to the analysis of dimerization of single-spanning membrane proteins. However, the effects inherent to such two-dimensional systems, such as the excluded volume of polytopic transmembrane proteins, proximity FRET, and rotational diffusion of fluorophore dipoles, complicate interpretation of FRET data and have not been typically accounted for. Here, using FRET and fluorescence cross-correlation spectroscopy, we introduce a method to measure surface protein density and to estimate the apparent Förster radius, and we use Monte Carlo simulations of the FRET data to account for the proximity FRET effect occurring in confined two-dimensional environments. We then use FRET to analyze the dimerization of human rhomboid protease RHBDL2 in giant plasma membrane vesicles. We find no evidence for stable oligomers of RHBDL2 in giant plasma membrane vesicles of human cells even at concentrations that highly exceed endogenous expression levels. This indicates that the rhomboid transmembrane core is intrinsically monomeric. Our findings will find use in the application of FRET and fluorescence correlation spectroscopy for the analysis of oligomerization of transmembrane proteins in cell-derived lipid membranes.     

Analysis of self-association of West Nile virus capsid protein and the crucial role played by Trp 69 in homodimerization

The understanding of capsid (C) protein interactions with itself would provide important data on how the core is organized in flaviviruses during assembly. In this study, West Nile (WN) virus C protein was shown to form homodimers using yeast two-hybrid analysis in conjunction with mammalian two-hybrid and in vivo co-immunoprecipitation assays. To delineate the region on the C protein which mediates C-C dimerization, truncation studies were carried out. The results obtained clearly showed that the internal hydrophobic segment flanked by helix I and helix III of WN virus C protein is essential for the self-association of C protein. The crucial role played by Trp 69 in stabilizing the self-association of C protein was also demonstrated by mutating Trp to Gly/Arg/Phe. Substitution of the Trp residue with Gly/Arg abolished the dimerization, whereas substitution with Phe decreased the self-association significantly. The results of this study pinpoint a critical residue in the C protein that potentially plays a role in stabilizing the homotypic interaction.     

Nucleotide-induced switch in oligomerization of the AAA+ ATPase ClpB

ClpB is a member of the bacterial protein-disaggregating chaperone machinery and belongs to the AAA(+) superfamily of ATPases associated with various cellular activities. The mechanism of ClpB-assisted reactivation of strongly aggregated proteins is unknown and the oligomeric state of ClpB has been under discussion. Sedimentation equilibrium and sedimentation velocity show that, under physiological ionic strength in the absence of nucleotides, ClpB from Escherichia coli undergoes reversible self-association that involves protein concentration-dependent populations of monomers, heptamers, and intermediate-size oligomers. Under low ionic strength conditions, a heptamer becomes the predominant form of ClpB. In contrast, ATP gamma S, a nonhydrolyzable ATP analog, as well as ADP stabilize hexameric ClpB. Consistently, electron microscopy reveals that ring-type oligomers of ClpB in the absence of nucleotides are larger than those in the presence of ATP gamma S. Thus, the binding of nucleotides without hydrolysis of ATP produces a significant change in the self-association equilibria of ClpB: from reactions supporting formation of a heptamer to those supporting a hexamer. Our results show how ClpB and possibly other related AAA(+) proteins can translate nucleotide binding into a major structural transformation and help explain why previously published electron micrographs of some AAA(+) ATPases detected both six- and sevenfold particle symmetry.     

Self-association studies on the EphB2 receptor SAM domain using analytical ultracentrifugation

The self-association behavior of the Eph-kinases SAM domain has been studied in phosphate buffer, pH 7.4, containing 0.14 M NaCl using concentration-dependent sedimentation equilibrium experiments. Only weak interactions typical for a monomer-dimer equilibrium up to at least 12 mg/mL were observed. Such concentrated solutions require a consideration of the non-ideality expressed by virial coefficients. A special centrifuge equation was used for the global analysis to estimate equilibrium constants based on the thermodynamic activities of the reactants. When neglecting this, the parameters deviate by about 20%. Association constants for dimerization of the EphB2-SAM domain vary between 163 M(-1) at 10 degrees C and 395 M(-1) at 32 degrees C, indicating hydrophobic forces are involved in the dimerization process. In solutions of about 12 mg/mL, less than 50% dimers are in solution and higher oligomers can be excluded.     

Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. II. Stoichiometry of holoenzyme association and energetics of subunit association

The self-association pattern of D-amino acid oxidase holoenzyme in 0.1 M sodium pyrophosphate, pH 8.3, at 25 degrees C was examined by the low-angle laser light-scattering method. As to the results of nonlinear least-squares analysis of the apparent weight-average molecular weight (Mwapp) versus protein concentration (c) data, the following three models fitted equally well the data over the concentration range of 0.03-11.4 mg/ml: 1) the model of isodesmic indefinite self-association of the monomer where the dimerization constant differs from the isodesmic association constant, 2) the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, and 3) the model which involves the trimerization of the monomer and isodesmic indefinite self-association of the trimer. In a more limited concentration range (0.3-11.4 mg/ml), a model of isodesmic indefinite self-association of the stable dimer where the dimer does not dissociate into the monomers cannot be excluded from the above three models. Measurements with the concentration range lowered to 0.03 mg/ml enabled us to exclude unequivocally the model involving such a stable dimer and to extrapolate the Mwapp data to the Mr of the monomer at infinite dilution as in the case of the apoenzyme. The observed sedimentation boundary profiles were qualitatively consistent with the idealized boundary profiles calculated with the model which involves the dimerization of the monomer and isodesmic indefinite self-association of the dimer, so this model is the most probable of the models examined. These results provide the first evidence that the association mode of the holoenzyme is different from that of the apoenzyme, i.e. isodesmic indefinite self-association of the monomer (Tojo, H., Horiike, K., Shiga, K., Nishina, Y., Watari, H., and Yamano, T. (1985) J. Biol. Chem. 260, 12607-12614). The overall linkage scheme, between binding of coenzyme FAD and subunit association, was considered, and the overall free energy change in each process in the scheme was calculated. The total stabilization energies of the intersubunit interaction in the holoenzyme relative to the apoenzyme were found to be -2.2 kcal/mol at the dimerization step and -0.5 kcal/mol at the step of the addition of the dimer to any 2i-mer (i = 1,2, ...).     

Roles of the N- and C-terminal sequences in Hsp27 self-association and chaperone activity

The small heat shock protein 27 (Hsp27 or HSPB1) is an oligomeric molecular chaperone in vitro that is associated with several neuromuscular, neurological, and neoplastic diseases. Although aspects of Hsp27 biology are increasingly well known, understanding of the structural basis for these involvements or of the functional properties of the protein remains limited. As all 11 human small heat shock proteins (sHsps) possess an α-crystallin domain, their varied functional and physiological characteristics must arise from contributions of their nonconserved sequences. To evaluate the role of two such sequences in Hsp27, we have studied three Hsp27 truncation variants to assess the functional contributions of the nonconserved N- and C-terminal sequences. The N-terminal variants Δ1-14 and Δ1-24 exhibit little chaperone activity, somewhat slower but temperature-dependent subunit exchange kinetics, and temperature-independent self-association with formation of smaller oligomers than wild-type Hsp27. The C-terminal truncation variants exhibit chaperone activity at 40 °C but none at 20 °C, limited subunit exchange, and temperature-independent self-association with an oligomer distribution at 40 °C that is very similar to that of wild-type Hsp27. We conclude that more of the N-terminal sequence than simply the WPDF domain is essential in the formation of larger, native-like oligomers after binding of substrate and/or in binding of Hsp27 to unfolding peptides. On the other hand, the intrinsically flexible C-terminal region drives subunit exchange and thermally-induced unfolding, both of which are essential to chaperone activity at low temperature and are linked to the temperature dependence of Hsp27 self-association.     

Activation of c-Abl kinase activity and transformation by a chemical inducer of dimerization

c-Abl is a non-receptor tyrosine kinase that is activated in human leukemias by the fusion of Bcr or Tel sequences to the Abl NH(2) terminus. Although Bcr and Tel have little in common, both contain oligomerization domains. To determine whether oligomerization alone is sufficient to activate c-Abl, we have generated and characterized an Abl protein that can be activated selectively with the chemical inducer of dimerization, AP1510. Mutant Abl proteins with one (c4F1) or two (c4F2) copies of the AP1510 binding motif (FKBP) transformed NIH 3T3 cells in a ligand-dependent manner with the c4F2 protein 60-fold more potent than c4F1. Both chimeric proteins exhibited ligand-dependent dimerization in vivo, suggesting that the increased transformation efficiency of the c4F2 mutant reflects more effective dimerization rather than formation of higher order oligomers. In the absence of ligand, c4F2-expresssing fibroblasts morphologically reverted and arrested in G(1). In Ba/F3 cells, the c4F2 chimera exhibited ligand-dependent kinase activation, transformation to interleukin 3-independent growth, and relocalization of the fusion protein from nucleus to cytoplasm. These results demonstrate that dimerization alone is sufficient to activate the Abl kinase and provide a method to regulate conditionally c-Abl activity that will be useful for studying the normal physiological role of c-Abl and the mechanism of transformation and leukemogenesis.     

Effect of self-association of alphas1-casein and its cleavage fractions alphas1-casein(136-196) and alphas1-casein(1-197),1 on aromatic circular dichroic spectra: comparison with predicted models.

The self-association of native alphas1-casein is driven by a sum of interactions which are both electrostatic and hydrophobic in nature. The dichroism of aromatic side chains was used to derive regio-specific evidence in relation to potential sites of alphas1-casein polymerization. Near-ultraviolet circular dichroism (CD) revealed that both tyrosine and tryptophan side chains play a role in alphas1-casein associations. Spectral evidence shows these side chains to be in an increasingly nonaqueous environment as both ionic strength and protein concentration lead to increases in the degree of self-association of the protein from dimer to higher oligomers. Near-UV CD investigation of the carboxypeptidase A treated peptide, alphas1-casein(1-197), indicated that the C-terminal residue (Trp199) may be superficial to these interactions, and that the region surrounding Trp164 is more directly involved in an aggregation site. Similar results for the cyanogen bromide cleavage peptide alphas1-casein(136-196) indicated the presence of strongly hydrophobic interactions. Association constants for the peptides of interest were determined by analytical ultracentrifugation, and also were approximated from changes in the near-UV CD curves with protein concentration. Sedimentation equilibrium experiments suggest the peptide to be dimeric at low ionic strength; like the parent protein, the peptide further polymerizes at elevated (0.224 M) ionic strength. The initial site of dimerization is suggested to be the tyrosine-rich area near Pro147, while the hydrophobic region around Pro168, containing Trp164, may be more significant in the formation of higher-order aggregates.     

The amyloid precursor protein and its homologues: structural and functional aspects of native and pathogenic oligomerization

Over the last 25 years, remarkable progress has been made not only in identifying key molecules of Alzheimer's disease but also in understanding their meaning in the pathogenic state. One hallmark of Alzheimer pathology is the amyloid plaque. A major component of the extracellular deposit is the amyloid-β (Aβ) peptide which is generated from its larger precursor molecule, i.e., the amyloid precursor protein (APP) by consecutive cleavages. Processing is exerted by two enzymes, i.e., the β-secretase and the γ-secretase. We and others have found that the self-association of the amyloid peptide and the dimerization and oligomerization of these proteins is a key factor under native and pathogenic conditions. In particular, the Aβ homodimer represents a nidus for plaque formation and a well defined therapeutic target. Further, dimerization of the APP was reported to increase generation of toxic Aβ whereas heterodimerization with its homologues amyloid precursor like proteins (APLP1 and APLP2) decreased Aβ formation. This review mainly focuses on structural features of the homophilic and heterophilic interactions among APP family proteins. The proposed contact sites are described and the consequences of protein dimerization on their functions and in the pathogenesis of Alzheimer's disease are discussed.