Crystallization and structure determination of beta secretase and/or beta secretase-like proteins

Abstract

The x-ray crystal structure of human BACE or BACE-like proteins is useful for solving the structure of other molecules or molecular complexes, and identifying and/or designing potential modifiers of human BACE activity.

1 . A crystal of beta secretase having tetragonal space group symmetry P4 3 2 1 2. 
     
     
         2 . The crystal of  claim 1  wherein the beta secretase comprises glycosylated beta secretase. 
     
     
         3 . A crystal of beta secretase according to  claim 1  comprising a unit cell defined by the dimensions a, b, c, α, β, and γ, wherein a is about 94 Å to about 134 Å, b is about 94 Å to about 134 Å, c is about 170 Å to about 210 Å, and α=β=γ=90°. 
     
     
         4 . A crystal of beta secretase according to  claim 1  and having a unit cell defined by the dimensions a, b, c, α, β, and γ, wherein a is about 94 Å to about 134 Å, b is about 94 Å to about 134 Å, c is about 170 Å to about 210 Å, and α=β=γ=90°. 
     
     
         5 . The crystal of  claim 4  wherein the beta secretase has an amino acid sequence comprising SEQ ID NO:2. 
     
     
         6 . The crystal of  claim 4  wherein the beta secretase has an amino acid sequence comprising SEQ ID NO:2, with the proviso that a methionine is replaced with selenomethionine. 
     
     
         7 . A crystal of beta secretase preferable by:
 modifying a human beta secretase by contacting the human beta secretase with an HIV protease under conditions sufficient to produce a proteolytically cleaved human beta secretase, wherein the N-terminus of the proteolytically cleaved human beta secretase differs from the N-terminus of the non-cleaved human beta secretase; and   crystallizing the proteolytically cleaved human beta secretase.   
     
     
         8 . The crystal of  claim 7  wherein the beta secretase is expressed in CHO cells. 
     
     
         9 . The crystal of  claim 7  wherein the crystal has tetragonal space group symmetry P4 3 2 1 2. 
     
     
         10 . A molecule or molecular complex that forms a crystal having tetragonal space group symmetry P4 3 2 1 2 and comprises at least a portion of a human beta secretase or beta secretase-like binding pocket, wherein the binding pocket comprises the amino acids listed in Table 3 and the binding pocket is defined by a set of points having a root mean square deviation of less than about 0.45 Å from points representing the backbone atoms of said amino acids as represented by the structure coordinates listed in Table 1. 
     
     
         11 . The molecule or molecular complex of  claim 10  wherein the molecule or molecular complex comprises glycosylated beta secretase. 
     
     
         12 . The molecule or molecular complex of  claim 10  wherein the binding pocket comprises the amino acids listed in Table 4. 
     
     
         13 . The molecule or molecular complex of  claim 10  wherein the binding pocket comprises the amino acids listed in Table 5. 
     
     
         14 . A computer-assisted method for identifying a potential modifier of human beta secretase activity comprising:
 supplying a computer modeling application with a set of structure coordinates of a molecule or molecular complex that forms a crystal having tetragonal space group symmetry P4 3 2 1 2, the molecule or molecular complex comprising at least a portion of a human beta secretase or beta secretase-like binding pocket, the binding pocket comprising the amino acids listed in Table 3;   supplying the computer modeling application with a set of structure coordinates of a chemical entity; and   determining whether the chemical entity is expected to bind to or interfere with the molecule or molecular complex, wherein binding to or interfering with the molecule or molecular complex is indicative of potential modification of human beta secretase activity.   
     
     
         15 . The method of  claim 14  wherein the binding pocket comprises the amino acids listed in Table 3, the binding pocket being defined by a set of points having a root mean square deviation of less than about 0.45 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in Table 1. 
     
     
         16 . The method of  claim 14  wherein the binding pocket comprises the amino acids listed in Table 4, the binding pocket being defined by a set of points having a root mean square deviation of less than about 0.45 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in Table 1. 
     
     
         17 . The method of  claim 14  wherein determining whether the chemical entity is expected to bind to or interfere with the molecule or molecular complex comprises performing a fitting operation between the chemical entity and a binding pocket of the molecule or molecular complex, followed by computationally analyzing the results of the fitting operation to quantify the association between the chemical entity and the binding pocket. 
     
     
         18 . The method of  claim 14  further comprising screening a library of chemical entities. 
     
     
         19 . A method of using the crystal of  claim 1  in a drug screening assay comprising:
 selecting a potential modifier by performing rational drug design with a three-dimensional structure determined for the crystal, wherein selecting is performed in conjunction with computer modeling;   adding the potential modifier to a binding assay; and   detecting a measure of binding, wherein a potential modifier that binds is selected as a potential drug.