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

Yi Xue,Sangwon Lee,Yongcheng Wang,Ya Ha

doi:10.1074/jbc.m111.219659

Abstract

Missense mutations in the amyloid precursor protein (APP) gene can cause familial Alzheimer disease. It is thought that APP and APP-like proteins (APLPs) may play a role in adhesion and signal transduction because their ectodomains interact with components of the extracellular matrix. Heparin binding induces dimerization of APP and APLPs. To help explain how these proteins interact with heparin, we have determined the crystal structure of the E2 domain of APLP1 in complex with sucrose octasulfate (SOS). A total of three SOS molecules are bound to the E2 dimer. Two SOSs are bound inside a narrow intersubdomain groove, and the third SOS is bound near the two-fold axis of the protein. Mutational analyses show that most residues interacting with SOS also contribute to heparin binding, although in varying degrees; a deep pocket, defined by His-376, Lys-422, and Arg-429, and an interfacial site between Lys-314 and its symmetry mate are most important in the binding of the negatively charged polysaccharide. Comparison with a lower resolution APP structure shows that all key heparin binding residues are conserved and identically positioned, suggesting that APLP1 and APP may bind heparin similarly. In transfected HEK-293 cells, mutating residues responsible for heparin binding causes little change in the proteolysis of APP by the secretases. However, mutating a pair of conserved basic residues (equivalent to Arg-414 and Arg-415 of APLP1) immediately adjacent to the heparin binding site affects both the maturation and the processing of APP.

Highlights

The affinity for heparin was expressed empirically as the molar salt concentration required for eluting the protein from a heparin column
The percentages of the salt concentration relative to the wild-type (WT) value are shown in parentheses
The mutation of Gln-421, whose side chain helped to anchor the C terminus on ␣D, increased the affinity of the protein for heparin slightly. These results suggested that the folded back peptide interfered with heparin binding

Summary

EXPERIMENTAL PROCEDURES

Reagents—The sodium salt of SOS was obtained from Toronto Research Chemicals Inc. The heparin disaccharides were obtained from Sigma-Aldrich (H9267 and H9392). The N-terminal hexahistidine tag was removed by thrombin The protein without His tag was concentrated and further purified on a Superdex S-200 column (GE Healthcare) in a buffer containing 20 mM HEPES, pH 7.5, 0.5 M NaCl, and 5% glycerol. Heparin Binding Assay—After exchanging into a buffer of 10 mM sodium phosphate/acetate, pH 7.5, the protein solutions were loaded onto the 1-ml HiTrap heparin column (GE Healthcare) and eluted with a linear salt gradient (0 –2 M NaCl). After 36 h, the transfected cells were harvested and lysed in a lysis buffer containing 10 mM Tris, pH 7.4, 150 mM NaCl, 0.1% sodium deoxycholate, 1% Nonidet P-40, and the Complete protease inhibitor mixture (Roche Applied Science). The mutants were generated using the QuikChange௡ site-directed mutagenesis kit and expressed in HEK-293 cells

RESULTS

Summary of mutant data

DISCUSSION