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19. The protein-ligand X-ray structure of 24—bound memap-
sin 2 has been deposited in PDB (PDB ID: 2vkm).
Recombinant human BACE was expressed and purified as
previously described. Crystals of free BACE (APO form)
were grown using a hanging drop vapor diffusion method.
Monoclinic crystals suitable for structure determination
were obtained at 13% PEG 8000, pH 6.5, and 17 ꢁC with a
protein concentration of 18 mg/ml. The crystals grow to
full size (about 0.25 mm · 0.35 mm · 0.05 mm) in 4–5
weeks. Inhibitor 24-memapsin 2 complex crystal were
prepared by soaking the free memapsin 2 crystal in
concentrated inhibitor solution for 2 days.
Memapsin 2/inhibitor crystal was soaked in the mother
liquor plus 20% (v/v) glycerol and quickly frozen under a
cryogenic nitrogen gas stream. Cryogenic diffraction data
from a single crystal was recorded on a Mar 345 image
plate mounted on a MSC-Rigaku RU-300 X-ray genera-
tor. The structure was determined by molecular replace-
ment implemented with the program AmoRe using the
previously determined memapsin 2 structure (PDB ID:
1M4H) as a search model.20 Rotation and translation
functions followed by the rigid-body refinement with data
˚
˚
11. (a) Beaulieu, P.; Wernic, D.; Duceppe, J.-S.; Guindon, Y.
Tetrahedron Lett. 1995, 36, 3317; (b) Rotella, D. P.
Tetrahedron Lett. 1995, 36, 5453.
from 15 A to 3.5 A resolution in space group P21 gave
unambiguous solutions for the four memapsin 2 molecules
in the asymmetric unit. A random selection of 8% of
reflections was set aside as the test set for cross-validation
during the refinement. The refined model has well-defined
electron density for the inhibitor and its corresponding
structure was built into the active site. The four molecules
in the crystallographic asymmetric unit have essentially
identical structures. The crystal form was determined to be
12. Memapsin 2 inhibition was measured using recombinant
enzyme produced from E. coli expression as described in
Ref. 2a. A fluorogenic substrate Arg-Glu(EDANS)-Glu-
Val-Asn-Leu-Asp-Ala-Glu-Phe-Lys (Dabcyl)-Arg was
used with 0.47 lM of the enzyme in 0.1 M Na-ace-
tate + 5% dimethylsulfoxide, pH 4.5, at 37 ꢁC. The exci-
tation wavelength was 350 nm and the emission
wavelength was 490 nm.
˚
monoclinic with a resolution of 2.05 A. The unit cell
˚
˚
˚
parameters are a = 86.7 A, b = 130.3 A, c = 87.7 A,
b = 97.4ꢁ.
13. Chang, W. P.; Koelsch, G.; Wong, S.; Downs, D.; Da, H.;
Weerasena, V.; Gordon, B.; Devasamudram, T.; Bilcer,
G.; Ghosh, A. K.; Tang, J. J. Neurochem. 2004, 89, 1409.
20. Hong, L.; Turner, R. T.; Koelsch, G.; Shin, D.; Ghosh, A.
K.; Tang, J. Biochemistry 2002, 41, 10963.