Structure of the Protease Domain of Memapsin 2 (β-Secretase) Complexed with Inhibitor
10.1126/science.290.5489.150
Crossref journal-article
American Association for the Advancement of Science (AAAS)
Science (221)
Abstract

Memapsin 2 (β-secretase) is a membrane-associated aspartic protease involved in the production of β-amyloid peptide in Alzheimer's disease and is a major target for drug design. We determined the crystal structure of the protease domain of human memapsin 2 complexed to an eight-residue inhibitor at 1.9 angstrom resolution. The active site of memapsin 2 is more open and less hydrophobic than that of other human aspartic proteases. The subsite locations from S 4 to S 2 ′ are well defined. A kink of the inhibitor chain at P 2 ′ and the change of chain direction of P 3 ′ and P 4 ′ may be mimicked to provide inhibitor selectivity.

Bibliography

Hong, L., Koelsch, G., Lin, X., Wu, S., Terzyan, S., Ghosh, A. K., Zhang, X. C., & Tang, J. (2000). Structure of the Protease Domain of Memapsin 2 (β-Secretase) Complexed with Inhibitor. Science, 290(5489), 150–153.

Authors 8
  1. Lin Hong (first)
  2. Gerald Koelsch (additional)
  3. Xinli Lin (additional)
  4. Shili Wu (additional)
  5. Simon Terzyan (additional)
  6. Arun K. Ghosh (additional)
  7. Xuenjun C. Zhang (additional)
  8. Jordan Tang (additional)
References 45 Referenced 604
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  15. OM99-2 is based on an octapeptide sequence of Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe in which the Leu-Ala bond is substituted by a hydroxyethylene transition-state isostere (25). The K i value reported for recombinant pro-memapsin 2 (6) was 9.8 nM (25). The K i value for recombinant memapsin 2 used in this structural study has been determined to be 1.6 nM (21).
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  17. The putative pro residues 28p–48p present in memapsin 2 used for structural determination are LRLPRETDEEPEEPGRRGSFV. The alignment of amino acid sequences and secondary structures of memapin 2 and human pepsin is shown in an additional Web figure (29). The numbering of residues in memapsin 2 used in this paper starts at Glu 1 in the sequence of EMVDN- and continues based on the published memapsin 2 sequence (6). The positions and marker sequences of the regions discussed in the text (insertions A through G) are: NH 2 -terminal lobe residues 1–180 starting at Glu 1 to the end of sequence IGGID 180 ; COOH-terminal lobe residues 181–385 starting at His 181 in the sequence of HSLYT to the end of COOH-terminal extension G (see below); flap residues VPYTQGK (residues 69–75); insertion A GFPLNQSEVL (residues 158–167); insertion B KEYN (residues 218–221); insertion C ASSTEKFP (residues 251–258); insertion D WQAG (residues 270–273); insertion E EVTNQS (residues 290–295); insertion F DVATSQD (residues 311–317); and COOH-terminal extension G CHVHDEFRTAAVEGPFVTLDMEDCGYNIPQTDEST (residues 359–393).
  18. Residues 1p to 393 of human pro-memapsin 2 [residues 28p to 393 are shown in additional Web figure (29); the complete pro sequence is described in (6)] was expressed from vector pET11a using an Escherichia coli host BL21(DE3) as previously described (6). The inclusion bodies were harvested washed and dissolved in 0.1 M tris 1 mM EDTA 1 mM glycine 8 M urea and 0.1 M β-mercaptoethanol (pH 10) and then refolded by rapid dilution as previously described (30). The recombinant pro-memapsin 2 was purified by gel filtration on Sepharose S-300 and FPLC on Resource-Q (Pharmacia) column (30). The NH 2 -terminal sequence of purified memapsin 2 established that it started at Leu 28p with a minor component starting at Leu 30p . Since the expressed pro-memapsin 2 had been determined to contain 48 putative pro residues [starting at the sequence of Ala-Gly-Val-Leu- (6)] activation with the removal of part of the pro peptide (residues 1p to 28p or 30p) had taken place during the preparation procedure. This purified enzyme form has the highest specific activity [using the assay described in (25); see also (6)] among all the activated memapsin 2 (21).
  19. Purified recombinant memapsin 2 in the presence of fivefold molar excess of OM99-2 was crystallized in 0.2 M ammonium sulfate and 22.5% PEG 8000 buffered with 0.1 M Na-cacodylate (pH 7.4) at 20°C using the hanging drop vapor diffusion method. The typical crystal size was about 0.4 mm by 0.4 mm by 0.2 mm. Diffraction data were collected at 95 K with an Raxis-IV image plate integrated and reduced with the HKL program package [
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  21. ]. The crystals belong to the space group P2 1 with two memapsin 2/OM99-2 complexes per crystallographic asymmetric unit and 56% solvent content. The molecular replacement calculations were performed using pepsin (Protein Data Bank accession number 1psn) as the search model with the program AMoRe [
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  23. ]. The refinement was carried out using the program CNS (31)]. The molecular graphic program O [
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  25. ] was used for interactive map fitting. The initial solution had a correlation coefficient of 22% and an R factor of 0.51 for data in the range of 15 to 3.5 Å. The ‖ F o ‖ − ‖ F c ‖ omit map from early stages of refinement revealed surplus electron density within the active-site cleft which was fitted with the inhibitor structure. After multiple cycles of refinement and model building the R working and R free values of the final structure were 18.0 and 22.4% respectively. Crystallographic refinement used molecular dynamics and energy minimization functions of CNS (31). No solvent molecules were added to the model until the R factor was reduced below 0.25. Solvent molecules 529 were then added as identified in the ‖ F o ‖ − ‖ F c ‖ map contoured at the 3 σ level. Noncrystallographic symmetry restriction and averaging were used in early stages of refinement and model building. Bulk solvent and anisotropic overall B factor corrections were applied through the refinement. In the final model no backbone φ ψ torsion angles were located in the disallowed region of the Ramachandran plot whereas 90% of the residues were located in the most favored regions as defined by the program PROCHECK [R. A. Laskowski M. W. MacArthur D. S. Moss J. M. Thornton. J. Appl. Crystallogr. 26 283 (1993)]. The two memapsin 2/OM99-2 complexes in the crystallographic asymmetric unit are essentially identical.
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  42. Supplementary Web figure is available at www.sciencemag.org/feature/data/1052536.shl.
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  45. We thank D. R. Davies A. Wlodawer and J. D. Capra for critical reading of this manuscript and D. Downs and A. Irwin for assisting in enzyme preparation. Coordinates have been deposited in the Protein Data Bank (accession number 1FKN). J.T. is holder of the J. G. Puterbaugh Chair in Biomedical Research at the Oklahoma Medical Research Foundation. G.K. is a Scientist Development Awardee of the American Heart Association.
Dates
Type When
Created 23 years, 1 month ago (July 27, 2002, 5:53 a.m.)
Deposited 1 year, 7 months ago (Jan. 13, 2024, 5:20 a.m.)
Indexed 4 weeks, 1 day ago (July 30, 2025, 9:58 a.m.)
Issued 24 years, 10 months ago (Oct. 6, 2000)
Published 24 years, 10 months ago (Oct. 6, 2000)
Published Print 24 years, 10 months ago (Oct. 6, 2000)
Funders 0

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@article{Hong_2000, title={Structure of the Protease Domain of Memapsin 2 (β-Secretase) Complexed with Inhibitor}, volume={290}, ISSN={1095-9203}, url={http://dx.doi.org/10.1126/science.290.5489.150}, DOI={10.1126/science.290.5489.150}, number={5489}, journal={Science}, publisher={American Association for the Advancement of Science (AAAS)}, author={Hong, Lin and Koelsch, Gerald and Lin, Xinli and Wu, Shili and Terzyan, Simon and Ghosh, Arun K. and Zhang, Xuenjun C. and Tang, Jordan}, year={2000}, month=oct, pages={150–153} }