Biochemical and structural characterization of the interaction of memapsin 2 (beta-secretase) cytosolic domain with the VHS domain of GGA proteins.
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.
Protein Studies Program, Oklahoma Medical Research Foundation, 825 Northeast 13th Street, Oklahoma City, Oklahoma 73104, USA.
Xiangyuan
X
He
Guangyu
G
Zhu
Gerald
G
Koelsch
Karla K
KK
Rodgers
Xuejun C
XC
Zhang
Jordan
J
Tang
D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D018076:DNA, Complementary; D010450:Endopeptidases; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010768:Phosphoserine / Q000378:metabolism; D011485:Protein Binding
D018076:DNA, Complementary; D010768:Phosphoserine; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human