Biochemical and Structural Characterization of the Interaction of Memapsin 2 (β-Secretase) Cytosolic Domain with the VHS Domain of GGA Proteins

Abstract

Memapsin 2 (beta-secretase) is a membrane-associated aspartic protease that initiates the hydrolysis of beta-amyloid precursor protein (APP) leading to the production of amyloid-beta and the onset of Alzheimer's disease (AD). Both memapsin 2 and APP are transported from the cell surface to endosomes where APP hydrolysis takes place. Thus, the intracellular transport mechanism of memapsin 2 is important for understanding the pathogenesis of AD. We have previously shown that the cytosolic domain of memapsin 2 contains an acid-cluster-dileucine (ACDL) motif that binds the VHS domain of GGA proteins (He et al. (2002) FEBS Lett. 524, 183-187). This mechanism is the presumed recognition step for the vesicular packaging of memapsin 2 for its transport to endosomes. The phosphorylation of a serine residue within the ACDL motif has been reported to regulate the recycling of memapsin 2 from early endosomes back to the cell surface. Here, we report a study on the memapsin 2/VHS domain interaction. Using isothermal titration calorimetry, the dissociation constant, K(d), values are 4.0 x 10(-4), 4.1 x 10(-4), and 3.1 x 10(-4) M for VHS domains from GGA1, GGA2, and GGA3, respectively. With the serine residue replaced by phosphoserine, the K(d) decreased about 10-, 4-, and 14-fold for the same three VHS domains. A crystal structure of the complex between memapsin 2 phosphoserine peptide and GGA1 VHS was solved at 2.6 A resolution. The side chain of the phosphoserine group does not interact with the VHS domain but forms an ionic interaction with the side chain of the C-terminal lysine of the ligand peptide. Energy calculation of the binding of native and phosphorylated peptides to VHS domains suggests that this intrapeptide ionic bond in solution may reduce the change in binding entropy and thus increase binding affinity.

Internalization of Exogenously Added Memapsin 2 (β-Secretase) Ectodomain by Cells Is Mediated by Amyloid Precursor Protein

“…The endocytosis of memapsin 2 is thought to require its cytosolic domain and a dileucine motif contained therein (11,26). It was demonstrated recently that the dileucine motif is part of the acid cluster-dileucine (ACDL) sequence DDISLL that binds to the VHS domain of GGA (Golgi-localized ␥-ear-containing ADP-ribosylation factor binding) proteins (17,18). Because GGA proteins also bind clathrin, this interaction appears to mediate the packaging of memapsin 2 to the coated pit for vesicular transport.…”

“…Both proteins are endocytosed into the endosomes, the major site for the hydrolysis of APP by memapsin 2 (9, 11), leading to the production of A␤. The intracellular domain of memapsin 2 contains a signal motif that binds three GGA proteins (17,18). This interaction apparently serves to package memapsin 2 into clathrin-coated vesicles for transportation in the endocytic or/and recycling pathways (17,18).…”

“…The intracellular domain of memapsin 2 contains a signal motif that binds three GGA proteins (17,18). This interaction apparently serves to package memapsin 2 into clathrin-coated vesicles for transportation in the endocytic or/and recycling pathways (17,18). Being located in the lipid raft at the plasma membrane (19,20), memapsin 2 endocytosis is influenced by other components having direct or indirect contact with the protease.…”

Direct binding of the Kex2p cytosolic tail to the VHS domain of yeast Gga2p facilitates TGN to prevacuolar compartment transport and is regulated by phosphorylation

“…An alternative manual alignment of the Kex2p and Vps10p sequences with the mammalian GBS motifs aligns Asp residues in the yeast proteins with the conserved Asp of the mammalian GBS motifs and aligns at least one Leu in each yeast sequence with the conserved aliphatic residues of the mammalian GBS motifs. The crystal structures of these motifs bound to the VHS domain of GGA1 (sortilin, BACE, and SorLA) or GGA3 (CI-M6PR) identify key conserved residues in the mammalian VHS domains that are required for interaction with the Asp and Leu residues (or Asp, Val, and Met residues in the case of SorLA) of the GBS motifs Shiba et al, 2002;He et al, 2003;Cramer et al, 2010). As observed by Misra et al (2002), several of these VHS residues are not conserved in the yeast Gga1&2p VHS domains.…”

“…The key residues in the C-tails of the cation-dependent MPR (CD-MPR), cation-independent MPR (CI-MPR), sortilin, memapsin 2 (β-secretase [BACE]), and low-density-lipoprotein receptor-related protein 3 for binding to the VHS domain of human GGAs were shown to constitute acidic dileucine motifs with the consensus sequence DXXLL (Nielsen et al, 2001;Puertollano et al, 2001;Takatsu et al, 2001;Zhu et al, 2001;He et al, 2002;Misra et al, 2002). Phosphorylation of a Ser residue within or adjacent to the acidic dileucine motif enhanced the binding of CI-MPR, BACE, sorLA, and sortilin C-tail sequences to human GGAs, suggesting a phosphoregulated mechanism for binding and cargo sorting He et al, 2003;Cramer et al, 2010). Comparison of the sequences of the yeast Gga proteins to the structures of the mammalian GGAs, however, suggested that the yeast proteins might not be able to bind the classic acidic dileucine motif .…”