Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Biological or biochemical
Reexamination Certificate
2001-04-11
2004-11-16
Brusca, John S. (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Biological or biochemical
C702S019000, C702S020000, C435S006120, C530S300000, C530S350000
Reexamination Certificate
active
06820011
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the crystallization and resolution of the three-dimensional structure of the human complement receptor type 2 (CR2) protein, and to methods of using such structure, particularly for structure-based drug design of regulatory compounds.
BACKGROUND OF THE INVENTION
Complement receptor type 2 (CR2 or CD21) is a key interface between innate and adaptive immunity by serving as the receptor for complement component C3d, as well as for C3 and fragments of C3 that contain the C3d domain or a portion thereof, including but not limited to C3dg, iC3b and C3b (D. T. Fearon and R. H. Carter,
Annu Rev Immunol
13, 127-49 (1995); D. T. Fearon,
Semin Immunol
10, 355-61 (1998)). C3d and other CR2-binding C3 fragments that contain C3d or a portion thereof are covalently attached to foreign antigens (such as invading microorganisms) through the action of the classical, alternative or lectin complement pathways (S. K. A. Law and K. B. M. Reid,
Complement
,. D. Male, Ed., In Focus (Oxford, UK: IRL Press., ed. second edition, 1995)). C3d- or other CR2-binding C3 fragment-bound antigens then greatly amplify B cell responses by binding to CR2 through these C3 fragments at the same time as engaging the B cell receptor (BCR) via the bound antigen (R. H. Carter and D. T. Fearon,
Science
256, 105-7 (1992); J. C. Cambier,
Biochem Soc Trans
25, 441-5 (1997)). The cross-linking of CR2 to the BCR by C3d, C3, or other CR2-binding fragments of C3 that contain C3d or a portion thereof greatly amplifies a signal transduction cascade through the CR2/CD19/CD81 co-activation complex (D. T. Fearon, 1995 ibid.; D. T. Fearon, 1998, ibid.; J. C. Cambier, 1997, ibid.; A. K. Matsumoto, et al.,
J Exp Med
173, 55-64 (1991)).
Human CR2 is also the obligate receptor for the Epstein-Barr virus (EBV) through its interactions with the gp350/220 viral membrane protein (J. D. Fingeroth, et al.,
Proc Natl Acad Sci USA
81, 4510-4 (1984)). EBV causes infectious mononucleosis, and is associated with Burkitt's Lymphoma and several other lymphomas and non-lymphoid tumors (M. Okano,
Acta Paediatr
87, 11-8 (1998)). In addition, human CR2 serves as a receptor for CD23 (J. P. Aubry et al.,
Nature
358, 505-7 (1992)) and is thus a receptor for at least three biologically important ligands. Using genetically manipulated mice and animal models, CR2 has been shown to be essential for the development of normal humoral immunity to T-dependent antigens (T. Hebell et al.,
Science
254, 102-5 (1991); J. M. Ahearn, et al.,
Immunity
4, 251-62 (1996); H. Molina, et al.,
Proc Natl Acad Sci USA
93, 3357-61 (1996)) as well as possibly play an important role in the maintenance of B cell self-tolerance and the development of autoimmunity (A. P. Prodeus, et al.,
Immunity
9, 721-31 (1998)). CR2 has also been shown to mediate the interaction of C3-bound HIV-1 as an immune complex with B cells in a fashion that promotes transfer of virus and infection of CD4 T cells (S. Moir, et al.,
J Exp Med
192, 637-46 (2000)). CR2 also mediates direct infection of CR2-expressing T cells or other CR2-expressing cell lineages that are bound by HIV-1 immune complexes containing C3, C3d or other CR2-binding C3 fragments (including, but not limited to, HIV-1 complexed with C3d).
Interactions with all three human CR2 ligands require the first two of 15 or 16 short consensus repeat (SCR) domains (C. A. Lowell, et al.,
J Exp Med
170, 1931-46 (1989); J. C. Carel et al.,
J Biol Chem
265, 12293-9(1990)). SCR domains, like Ig domains, are found in many proteins from both complement and non-complement families, and mediate diverse biological functions (A. P. Wiles, et al.,
J Mol Biol
272, 253-65 (1997)). Several of the SCR proteins also serve as receptors for important human pathogens. For example, in addition to CR2, CD46 is a Measles Virus receptor (R. E. Dorig et al.,
Cell
75, 295-305 (1993)), and CD55 is an echovirus receptor (T. Ward, et al.,
EMBO J
13, 5070-4 (1994); J. M. Bergelson, et al.,
Proc Natl Acad Sci USA
91, 6245-9 (1994)). Previously determined structures of SCR proteins containing two or four SCR domains have revealed a conserved core structure but variable orientations between domains mediated in part by relatively short 3-8 amino acid inter-SCR linker peptides (A. P. Wiles, et al., 1997, ibid.; P. N. Barlow, et al.,
J Mol Biol
232, 268-84 (1993); J. M. Casasnovas et al.,
EMBO J
18, 2911-22 (1999); R. Schwarzenbacher, et al.,
EMBO J
18, 6228-39 (1999)). As one of the major functions of SCR domains is to mediate protein-protein (such as receptor-ligand) interactions, and at least two SCRs have been found to be required for these interactions, the relative angle and orientation unique to each SCR-containing protein is likely to contribute to both biologic diversity as well as specificity. However, the lack of a high-resolution structure of a receptor-ligand complex in this family has hindered the understanding of the molecular recognition mechanisms of this class of proteins. With regard to the structure of CR2 and the molecular interactions with its ligands, C3d and EBVgp350/220, variable results have been obtained using mutagenesis, monoclonal antibody, and peptide strategies (C. A. Lowell, et al.,
J Exp Med
170, 1931-46 (1989); D. R. Martin et al.,
J Exp Med
174, 1299-311 (1991); H. Molina, et al.,
J Biol Chem
266, 12173-9(1991); H. Molina et al.,
J Immunol
153, 789-95 (1994); D. R. Martin et al.,
J Virol
68, 4716-26 (1994); H. Molina, et al.,
J Immunol
154, 5426-35 (
1995)).
Therefore, there is a need in the art for a three dimensional structure of CR2 in order to better understand the molecular recognition mechanisms of the protein and to enable the identification and/or design of compounds that mimic, enhance, disrupt or compete with the interactions of CR2 with its ligands.
SUMMARY OF THE INVENTION
One embodiment of the present invention relates to a method of structure-based identification of compounds which potentially bind to complement receptor type 2 (CR2) proteins or to a complex of CR2 and its ligand. This method includes the steps of: (a) providing a three dimensional structure of a CR2 short consensus repeat (SCR) 1-2 region; and, (b) identifying a candidate compound for binding to the CR2 SCR 1-2 region by performing structure based drug design with the structure of (a). The three dimensional structure of a CR2 short consensus repeat (SCR) 1-2 region is selected from: (i) a structure defined by atomic coordinates of a three dimensional structure of a crystalline CR2 SCR1-2 region in complex with C3d; (ii) a structure defined by atomic coordinates selected from: (1) atomic coordinates represented in a table selected from the group consisting of Table 2 (CR2-C3d) and Table 3 (CR2 only); and, (2) atomic coordinates that define a three dimensional structure, wherein at least 50% of the structure has an average root-mean-square deviation (RMSD) from backbone atoms in secondary structure elements in at least one domain of a three dimensional structure represented by the atomic coordinates of (1) of equal to or less than about 1.0 Å; and (ii) a structure defined by atomic coordinates derived from CR2 protein molecules arranged in a crystalline manner in a space group R32 so as to form a unit cell of dimensions a=b=170.5 Å, c=173.8 Å.
In one aspect of this embodiment, the step of identifying comprises selecting candidate compounds that potentially bind to and activate CR2.
In another aspect of this embodiment, the method further includes the step of: (c) selecting candidate compounds of (b) that inhibit the binding of CR2 to its ligand. The step (c) of selecting can include: (i) contacting the candidate compound identified in step (b) with CR2 or a fragment thereof and a CR2 ligand or a fragment thereof under conditions in which a CR2-CR2 ligand complex can form in the absence of the candidate compound; and (ii) measuring the binding affinity of the CR2 or fragment thereof to the CR2 ligand or fragment thereof; where
Chen Xiaojiang
Holers Vernon Michael
Brusca John S.
Sheridan & Ross P.C.
The Regents of the University of Colorado
Zhou Shubo
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