Refolded recombinant β-secretase crystals and methods for preparing and using the same
View Patent ↗The present application relates to methods for growing crystals of both the uncomplexed and complexed forms of β-secretase (BACE) polypeptide. The present application also relates to crystalline forms of uncomplexed BACE and the three-dimensional structure of BACE, as determined from the crystals. In addition, the present application relates to the use of crystalline forms of BACE to identify ligands, preferably inhibitors (antagonists), which bind to, and preferably inhibit the enzymatic activity of, BACE. Furthermore, the present application relates to nucleic acid sequences encoding BACE polypeptide, and methods for making BACE in greater quantity than prior methods, resulting in more effective crystallization.
1. A method for growing an uncomplexed β-secretase (apo BACE) crystal comprising: (a) mixing an equal volume of apo BACE solution comprising 20 mM Hepes pH 7.5 and 150 mM NaC1, wherein the apo BACE polypeptides consist of SEQ 1D NO:20 and SEQ ID NO:22, with a crystallization solution comprising 20% PEG3350, 0.6M ammonium iodide, and (b) crystallizing said apo BACE polypeptides at about 4° C. using a hanging drop method.
2. A crystal made by the method of claim 1 , and wherein the apo BACE crystal comprises space group C2 with unit cell dimensions wherein a ranges from 231.3 Å to 240.7 Å, b ranges from 101.5 Å to 105.7 Å, and c ranges from 63.7 Å to 66.3 Å.
3. The crystal of claim 2 , wherein the polypeptide is characterized by the structural coordinates of Table 1.
4. The crystal of claim 2 , wherein the crystal comprises space group C2 with unit cell dimensions of a=236.0 Å, b=103.6 Å and c=65.0 Å.
5. The crystal of claim 2 , wherein the crystal diffracts X-rays for determination of atomic coordinates of the crystal to a resolution value of about 5 Å or less.
6. A crystal of apo BACE polypeptide consists of the amino acid sequence set forth in SEQ ID NO:20 and SEQ ID NO: 22, and wherein said crystal comprises space group C2 with unit cell dimensions wherein a ranges from 231.3 Å to 240.7 Å, b ranges from 101.5 Å to 105.7 Å, and c ranges from 63.7 Å to 66.3 Å.
7. The crystal of claim 6 , wherein the polypeptide is characterized by the structural coordinates of Table 1.
8. The crystal of claim 6 , wherein the crystal comprises space group C2 with unit cell dimensions of a=236.0 Å, b=103.6 Å and c=65.0 Å.