Photon Factory Activity Report 2004 #22 Part B (2005)
Biological Science

6A, NW12/2002G149

Localization of β-secretase through interaction of its
cytosolic C-terminal tail with GGA-VHS
1Tomoo SHIBA, 2Satoshi KAMETAKA, 1Masato KAWASAKI, 2Masahiro SHIBATA,
2
Satoshi WAGURI, 2Yasuo UCHIYAMA and 1Soichi WAKATSUKI
1
KEK-PF, Tsukuba, Ibaragi 305-0801, Japan
2
Osaka Univ. Graduate School of Medicine, Suita, Osaka 565-0871, Japan

Introduction
BACE, β-site amyloid precursor protein (APP)
cleaving enzyme is a novel membrane-bound aspartic
protease with the β-secretase function in the production ofβ-amyloid (Aβ) peptide leading to the Alzheimer’s
disease. Its cytosolic tail has a characteristic acidiccluster dileucine (ACLL) motif recognized by the VHS
domain of adaptor proteins GGA (Golgi-localizing, γadaptin ear homology domain, ARF-interacting) for exitfrom the trans-Golgi network (TGN). Here we show that
BACE is colocalized with GGAs in the TGN andperipheral structures, and phosphorylation of a serine
residue in the cytoplasmic tail enhances interaction withthe VHS domain of GGA1 by about three folds. Toreveal the difference of the affinity between
phosphorylated and unphosphorylated BACE against
GGA1-VHS, we report the crystal structure of the VHS
domain of GGA1 in complex with a phosphorylated orunphosphorylated peptide of the BACE-tail [1].
Materials and Methods
Crystals of the GGA1 VHS domain complexed with theunphosphorylated BACE peptide were grown against areservoir containing 20 % (w/v) PEG 5000 MME, 0.2 M(NH4)2HPO4 and 0.1 M Tris-HCl (pH 8.4 – 9.0). Crystalsof the GGA1 VHS domain complexed with thephosphorylated BACE peptide were grown against areservoir containing 15 % (w/v) PEG 5000 MME, 0.2 M
NH4I, 0.3 M 1,6-hexanediol and 0.1 M MES-NaOH (pH6.0 – 6.2).
A data set of the complex with unphosphorylated
peptide was collected at PF-NW12. Data processing gave
an Rmerge of 0.059 for intensities and these data were99.5 % complete (50 – 2.6 Å). A data set of the complex
with phosphorylated peptide was collected at PF-BL6A.
Data processing gave an Rmerge of 0.055 for intensities andthese data were 99.2 % complete (50 – 1.9 Å).
The crystal structures of the GGA1 VHS domain
complexed with unphosphorylated and phosphorylated
BACE peptide were solved by the molecular-replacementwith the human GGA1 VHS domain structure as a searchmodel. The final model of unphosphorylated complex
has an R-factor of 24.4 % and an Rfree of 29.5 %. The final
model of phosphorylated complex has an R-factor of
21.3 % and an Rfree of 24.5 % for the resolution range
between 40 and 1.9 Å. Data processing and refinement
statistics are summarized in Table 1.

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Users' Report

Results and discussion
The X-ray crystal structures of the complex between
the GGA1-VHS domain and the BACE C- terminal
peptides (unphosphorylated; BACE and phosphorylated;BACE-P) illustrate a similar recognition mechanism as
mannose 6-phophate receptors (MPRs) except that a
glutamine residue closes in to fill the gap created by theshorter BACE peptide. The serine and lysine of theBACE peptide point their side chains towards the solvent(Fig. 1). However, phosphorylation of the serine affects
the lysine side chain and the peptide backbone resulting
in one additional hydrogen bond and a stronger
electrostatic interaction with the VHS domain, hence thereversible increase in affinity.
Table 1 Data processing and refinement statistics
BACE
BACE-PSpace group
I4
P212121
Cell constants (Å)
a114.848.5
b114.8
72.3
c53.6
103.1
Wavelength (Å) 1.00
0.97
Observed
77,496
189,494
Unique10,872
28,983
Resolution (Å)
50 – 2.6
50 – 1.9
Rmerge (%)
5.9
5.5
I/σ(I)
20.7
17.6
Completeness (%) 99.5
99.2
BACE
BACE-P
Resolution (Å)
40 – 2.6
40 – 1.9
Reflections
10,85728,939Rfactor (%)
24.4
21.3
Rfree (%)
29.524.5pS
KLDL
α6α8Fig. 1 Electron density map of BACE-P
References[1] T. Shiba, et al., Traffic, 5, 437, (2004)*soichi.wakatsuki@kek.jp