Crystallization and structure determination of glycosylated human beta secretase, an enzyme implicated in alzheimer's disease

Abstract

An inhibitor bound form of human beta secretase, also known as memapsin 2 and BACE, particularly in a glycosylated form as expressed in Chinese hamster ovary (CHO), HEK293 cells, or in insect cells as part of a Baculovirus expression system has been crystallized, and the three-dimensional x-ray crystal structure has been solved to 3.2 Å resolution. The x-ray crystal structure is useful for solving the structure of other molecules or molecular complexes, and designing inhibitors of human beta secretase activity.

1 . A glycosylated molecule or molecular complex that comprises at least a portion of a human beta secretase binding pocket, wherein the binding pocket comprises the amino acids listed in Table 5, the binding pocket being defined by a set of points having a root mean square deviation of less than about 0.43 Å from points representing the backbone atoms of said amino acids as represented by the structure coordinates listed in Table 1. 
     
     
         2 . The molecule or molecular complex of  claim 1 , wherein the binding pocket comprises the amino acids listed in Table 6. 
     
     
         3 . The molecule or molecular complex of  claim 1 , wherein the binding pocket comprises the amino acids listed in Table 7. 
     
     
         4 . A molecule or molecular complex that is structurally homologous to a human beta secretase molecule or molecular complex that forms a crystal having unit cell dimensions a=112.0±35 Å, b=112±35 Å, c=110±35 Å, α=β=90°, and γ=120°, wherein the human beta secretase molecule or molecular complex is represented by at least a portion of the structure coordinates listed in Table 1. 
     
     
         5 . A scalable three-dimensional configuration of points, at least a portion of said points derived from structure coordinates of at least a portion of a human beta secretase molecule or molecular complex listed in Table 1 comprising at least one of a human beta secretase or beta secretase-like binding pocket, wherein the human beta secretase molecule or molecular complex forms a crystal having the trigonal space group symmetry P3 2 21. 
     
     
         6 . The scalable three-dimensional configuration of points of  claim 5 , wherein substantially all of said points are derived from structure coordinates of a human beta secretase molecule or molecular complex listed in Table 1. 
     
     
         7 . The scalable three-dimensional configuration of points of  claim 5  wherein at least a portion of the points derived from the human beta secretase structure coordinates are derived from structure coordinates representing the locations of at least the backbone atoms of amino acids defining a human beta secretase binding pocket comprising the amino acids listed in Table 5. 
     
     
         8 . The scalable three-dimensional configuration of points of  claim 7 , wherein the binding pocket comprises the amino acids listed in Table 6. 
     
     
         9 . The scalable three-dimensional configuration of points of  claim 7 , wherein the binding pocket comprises the amino acids listed in Table 7. 
     
     
         10 . The scalable three-dimensional configuration of points of  claim 5  displayed as a holographic image, a stereodiagram, a model or a computer-displayed image. 
     
     
         11 . A scalable three-dimensional configuration of points, at least a portion of said points derived from structure coordinates of at least a portion of a human beta secretase molecular complex listed in Table 1 comprising at least one of a human beta secretase or beta secretase-like binding pocket. 
     
     
         12 . A scalable three-dimensional configuration of points, at least a portion of the points derived from structure coordinates of at least a portion of a molecule or a molecular complex that is structurally homologous to a human beta secretase molecule or molecular complex and comprises at least one of a human beta secretase or beta secretase-like binding pocket, wherein the human beta secretase molecule or molecular complex forms a crystal having the trigonal space group symmetry P3 2 21. 
     
     
         13 . The scalable three-dimensional configuration of points of  claim 12  displayed as a holographic image, a stereodiagram, a model or a computer-displayed image 
     
     
         14 . A machine-readable data storage medium comprising a data storage material encoded with machine readable data which, when using a machine programmed with instructions for using said data, is capable of displaying a graphical three-dimensional representation of at least one glycosylated molecule or molecular complex selected from the group consisting of:
 (i) a glycosylated molecule or molecular complex comprising at least a portion of a human beta secretase or beta secretase-like binding pocket comprising the amino acids listed in Table 5, the binding pocket defined by a set of points having a root mean square deviation of less than about 0.43 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in Table 1;   (ii) a glycosylated molecule or molecular complex comprising at least a portion of a human beta secretase or beta secretase-like binding pocket comprising the amino acids listed in Table 6, the binding pocket defined by a set of points having a root mean square deviation of less than about 0.43 Å from points representing the backbone atoms of said amino acids as represented by structure coordinates listed in Table 1; and   (iii) a glycosylated molecule or molecular complex that is structurally homologous to an human beta secretase molecule or molecular complex, wherein the human beta secretase molecule or molecular complex is represented by at least a portion of the structure coordinates listed in Table 1.