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Three-dimensional structure of complement receptor type 2 and uses thereof

a technology of complement receptor and three-dimensional structure, which is applied in the field of three-dimensional structure of complement receptor type 2, can solve the problems of entanglement of the molecular recognition mechanism and achieve the effect of improving the molecular recognition efficiency and sensitivity of this class of proteins

Inactive Publication Date: 2006-01-19
UNIV OF COLORADO THE REGENTS OF
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0007] 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; ...

Problems solved by technology

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.

Method used

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  • Three-dimensional structure of complement receptor type 2 and uses thereof
  • Three-dimensional structure of complement receptor type 2 and uses thereof
  • Three-dimensional structure of complement receptor type 2 and uses thereof

Examples

Experimental program
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example 1

[0161] The following example describes the crystallization and structure determination of the complex of complement receptor type 2 (CR2) and C3d.

Crystallization and Structure Determination of Structure

[0162] The crystals of the complex of CR2-C3d were obtained by co-crystallization of CR2 and C3d, at a protein ratio where no free CR2 or C3d could be detected by native gel electrophoresis. The protein concentration of 20 mg / ml was used for crystallization by the method of hanging drop vapor diffusion. The crystallization buffer contained 17% PEG 2K, 0.2 M ZnAcetate, and 0.1 M NaCacodylate (pH 7.36). Crystals reached full size after 4-6 weeks at 4° C. Crystal was frozen under liquid nitrogen in the crystallization buffer containing 20% glycerol. Synchrotron data were collected at Brookhaven National Laboratory and was indexed, integrated and reduced using D*trek (licensed through MSC Inc., Table 1). The space group is R32, with unit cell a=b=170.5 Å, c=173.8 Å. AmoRe (CCP4, Acta C...

example 2

[0183] The following example describes the construction of C3d mutants that affect CR2 binding.

[0184] Based on the complex structure, mutagenesis of C3d around the interface to disrupt CR2 binding was predicted to be difficult. This is because the interaction between CR2 and C3d involves mostly main-chain H-bonding, and the side chain residues play relatively small roles in the binding. However, to confirm the accuracy of the CR2-C3d interaction seen in this co-crystal structure, two informative C3d mutants were constructed. In mutant 170 (mt170; SEQ ID NO:8), residue Asn170 was changed to Arg. Asn170 is located on H7 of C3d and is the only residue on C3d that more or less points directly toward CR2 in the interface (FIG. 3E). Asn170 also packs directly with Tyr81 of CR2 as well as forms an H-bond with CR2 (Lys 100) through a water molecule (FIG. 3E). In solution, this mutant protein behaved very similarly like the wild type C3d and showed the same apparent molecular weight as the ...

example 3

[0190] The following example describes the inhibition of CR2-C3d interaction by CR2-derived peptides.

[0191] Based on the structure of the CR2 L-C3d complex, the results from previously reported CR2 peptide inhibition and monoclonal antibody assays can now be explained. In the peptide inhibition tests using short synthetic peptides covering all of CR2 SCR1 and SCM, peptides from two regions were shown to inhibit CR2-C3 binding (H. Molina, et al., J Immunol 154,5426-35 (1995)). One of them contains sequences that are located right on the interaction interface of CR2 seen in the complex structure, namely the sequences from the B strand and B-C loop of SCR2 (FIGS. 3D & 3E). This independent result strongly supports the complex structure described by the present invention. However, the other peptide located on the B strand of SCR1 also showed a similar inhibition effect as the first one. Close examination of this fragment on CR2 structure within the SCR1 domain revealed a very similar 3...

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Abstract

Disclosed is a crystalline human CR2 protein in complex with C3d, and the three dimensional structure of the crystalline complex. Also disclosed are methods of use of the structure, particularly for structure-based identification of compounds that bind to CR2 and inhibit or enhance the binding of CR2 to a natural ligand, that bind to CR2 and agonize or antagonize the receptor, that bind to CR2 and inhibit or enhance CR2 dimerization, or that use the C3-binding ability of CR2 as a drug delivery vehicle. Also disclosed are therapeutic compounds obtained by such methods and uses for such compounds.

Description

GOVERNMENT SUPPORT [0001] This invention was made in part with government support under Grant No. NIH R0-1 CA53615 awarded by the National Institutes of Health. The government has certain rights to this invention.FIELD OF THE INVENTION [0002] This invention relates to the crystallization and resolution of the three-dimensional structure of the human complement receptor type 2 (CR9) protein, and to methods of using such structure, particularly for structure-based drug design of regulatory compounds. BACKGROUND OF THE INVENTION [0003] Complement receptor type 2 (CR2 or CD21) is a key interface between innate and adaptive immunity by serving as the receptor for complement component C3 d, 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 i 3, 127-49 (1995); D. T. Fearon, Semin Immunol 10, 355-61 (1998)). C3d and other CR2-binding C3 fragments that contai...

Claims

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Application Information

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IPC IPC(8): A61K38/17A61K9/14A61K31/00C07K14/705C07K16/28G06F19/00G16B15/30
CPCA61K31/00C07K14/70596C07K16/28C07K2299/00G06F19/16C07K16/2896G16B15/00G16B15/30
Inventor CHEN, XIAOJIANGHOLERS, VERNON
Owner UNIV OF COLORADO THE REGENTS OF
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