Structural Characterization of the E2 Domain of APL-1, a Caenorhabditis elegans Homolog of Human Amyloid Precursor Protein, and Its Heparin Binding Site

Abstract

The amyloid ␤-peptide deposit found in the brain tissue of patients with Alzheimer disease is derived from a large heparinbinding protein precursor APP. The biological function of APP and its homologs is not precisely known. Here we report the x-ray structure of the E2 domain of APL-1, an APP homolog in Caenorhabditis elegans, and compare it to the human APP structure. We also describe the structure of APL-1 E2 in complex with sucrose octasulfate, a highly negatively charged disaccharide, which reveals an unexpected binding pocket between the two halves of E2. Based on the crystal structure, we are able to map, using site-directed mutagenesis, a surface groove on E2 to which heparin may bind. Our biochemical data also indicate that the affinity of E2 for heparin is influenced by pH: at pH 5, the binding appears to be much stronger than that at neutral pH. This property is likely caused by histidine residues in the vicinity of the mapped heparin binding site and could be important for the proposed adhesive function of APL-1. Amyloid precursor protein (APP)3 belongs to a gene family that in mammals includes two additional members, APLP1 and APLP2 (1-3). This gene family is important for viability because mice lacking all three members die shortly after birth (4). The function of APP and APP-like proteins and why their loss causes death are, however, not clear. Rare mutations in the human APP gene are also known to cause familial Alzheimer disease (for a review, see Ref. 5). The nematode Caenorhabditis elegans has a single APP-related gene, apl-1 (6). Inactivation of apl-1 prevents proper molting, a process that allows the worm to shed old cuticles between larval stages, and causes several other morphological defects (7).APP and APP-related proteins are type I membrane proteins. The majority of the full-length protein resides on the extracellular side of the membrane (Fig. 1A), and contains two conserved domains (E1, E2). E1 and E2 are retained in soluble forms of the protein, which are proteolytically released from the membrane by secretases. So far neither the primary sequence nor the three-dimensional structure of these two conserved domains has revealed any homology that would allow a convincing assignment of biological function to APP (8 -11). There are, however, a number of experimental observations suggesting that the APP family of proteins might play a role in regulating cell adhesion. For example, most triple knock-out mice developed cortical dysplasias, a condition that could result from defects in the adhesion of migrating neurons to extracellular matrix (4). Overexpression of apl-1 in the worm caused organ detachment (7), and transgenic flies expressing human APP developed a blistered-wing phenotype (12). Furthermore, in cell cultures, APP appears to concentrate to sites of adhesion (13-15).The structure of human E2/CAPPD has been solved by x-ray crystallography and NMR (10,11,22). In the x-ray structure, which contains the entire E2 region, the molecule forms a headto-tail dimer (10). This observation ...

Structure and biochemical analysis of the heparin-induced E1 dimer of the amyloid precursor protein

“…S1) (7). Experimental 3D structures are known for the N-terminal growth factor like (GFLD) (8) and the copper binding domains (CuBD) (9, 10) as well as for the C-terminal helical central APP domain (CAPPD) (11)(12)(13) and its Caenorhabditis elegans homologue (14). These two regions of defined secondary structure, also called E1 and E2 domain, are connected by a potentially flexible acidic domain (AcD).…”

Crystal structure of amyloid precursor-like protein 1 and heparin complex suggests a dual role of heparin in E2 dimerization

“…It is interesting to note that the footprint of the tetrasaccharide covers four histidines (His-307, His-376, His-426, and His-433) that are absolutely conserved from the worm homolog to the human proteins. At low pH, the protonation of these histidines is expected to increase the binding affinity between the E2 domain and the negatively charged sugar (23).…”

“…S1). The atomic structures of the E2 domains of APP and APP homologs obtained from different crystal forms suggest a conserved mode of protein dimerization (23)(24)(25). The E2 domain also contains the high affinity heparin binding site of the full-length molecule (26), and heparin binding induces the formation of E2 dimers in solution (25).…”

“…The E2 domain also contains the high affinity heparin binding site of the full-length molecule (26), and heparin binding induces the formation of E2 dimers in solution (25). Despite efforts to model heparin binding based on crystal structures of the apo proteins and complexes with sucrose octasulfate (23,27), the details of E2:heparin interaction and the mechanism of heparin-induced protein dimerization are not well understood. Here we report the crystal structure of a complex between the E2 domain of human APLP1 (NP_005157) and a heparin hexasaccharide.…”

Crystal Structure of the E2 Domain of Amyloid Precursor Protein-like Protein 1 in Complex with Sucrose Octasulfate

“…The E2 domain contains the higher affinity heparin binding site (19). Unlike the situation in a previous study (34), the ligand, as well as the protein side chains that interacted with it, has now been clearly defined in the electron density map. The crystal structure, combined with site-directed mutagenesis, permitted the identification of all key heparin binding residues in the E2 domain.…”