Crystal structure of the large ribosomal subunit from s. aureus

Abstract

A composition-of-matter comprising a crystallized form of a large ribosomal (50S) subunit of a pathogenic bacterium, and the atomic coordinates of the three-dimensional structure thereof are provided herein, as well as methods for crystallizing the same, and using the atomic coordinates of the same to design de novo ligands with high specificity thereto.

What is claimed is: 
     
         1 . A method of screening for a ligand having an affinity to a large ribosomal subunit of a pathogenic bacterium, the method comprising:
 (a) obtaining positioning data indicative of atomic coordinates determined from an electron density map having a resolution of at least 4 □ calculated from X-rays diffraction data obtained using at least one of a composition-of-matter that comprises a crystallized  Staphylococcus aureus  50S large ribosomal subunit, wherein the crystallized 50S large ribosomal subunit effectively diffracts X-rays for calculating an electron density map and determination of atomic coordinates to a resolution of at least 4 □ and forms in a hexagonal space group with unit cell dimensions of a=279.6±10 Å, b=279.6±10 Å, c=872.7±10 Å, α=90, β=90, γ=120;   (b) calculating at least a portion of a molecular surface of said large ribosomal subunit;   (c) computationally screening a library of molecules by determining a matching/binding score for each of said molecules with respect to said portion of a molecular surface of said large ribosomal subunit; and   (d) based on said matching/binding score, selecting the ligand having an affinity to the large ribosomal subunit.   
     
     
         2 . The method of  claim 1 , wherein said portion of said molecular surface of said large ribosomal subunit is a binding site of a pre-existing ligand of said large ribosomal subunit, said binding site is determined from atomic coordinates of a complex of said pre-existing ligand bound to said large ribosomal subunit. 
     
     
         3 . The method of  claim 2 , wherein said binding site is selected from the group consisting of a inter-subunit interface, a peptidyl transferase site, a GTPase center, an mRNA binding site, an A-site, a P-site, an E-site, a polypeptide exit tunnel, a translation initiation factor (IF1) binding site, a translation initiation factor (IF2) binding site, a translation initiation factor (IF3) binding site, an elongation factor G (EF-G) binding site, elongation factor Tu (EF-Tu) binding site, hibernation factor HPF binding site, hibernation factor RMF binding site, hibernation factor YfiA binding site, a GTP binding site and a ricin binding site. 
     
     
         4 . The method of  claim 1 , wherein each of said molecules is having a molecular weight of less than 1,500 g/mol. 
     
     
         5 . A ligand comprising:
 a first binding moiety having a molecular surface that mimics or duplicates a molecular surface of a first pre-existing ligand that binds to a first binding site in a large ribosomal subunit; and   a second binding moiety having a molecular surface that mimics or duplicates a surface of a second pre-existing ligand that binds with a second binding site in said ribosomal subunit,   wherein:   said first pre-existing ligand is not said second pre-existing ligand;   said first binding site is not said second binding site;   said first binding moiety is attached to said second binding moiety via a linking moiety so as to permit both the first moiety and the second moiety to bind simultaneously each with its respective binding site, wherein a positioning data of said first binding moiety relative to a positioning data of said second binding moiety, is determined according to atomic coordinates indicative of atomic coordinates determined from an electron density map having a resolution of at least 4 □ calculated from X-rays diffraction data obtained using at least one of a composition-of-matter that comprises a crystallized  Staphylococcus aureus  50S large ribosomal subunit, wherein the crystallized 50S large ribosomal subunit effectively diffracts X-rays for calculating an electron density map and determination of atomic coordinates to a resolution of at least 4 □ and forms in a hexagonal space group with unit cell dimensions of a=279.6±10 Å, b=279.6±10 Å, c=872.7±10 Å, α=90, β=90, γ=120.   
     
     
         6 . The ligand of  claim 5 , having a molecular weight of less than about 1,500 g/mol. 
     
     
         7 . The ligand of  claim 5 , wherein an affinity constant of the ligand is greater than each of an affinity constant of said first pre-existing ligand and said second pre-existing ligand, with respect to said ribosomal subunit. 
     
     
         8 . The ligand of  claim 5 , wherein said pre-existing ligand is an antibacterial agent. 
     
     
         9 . The ligand of  claim 8 , wherein each of said first pre-existing ligand and said second pre-existing ligand is selected from the group consisting of linezolid, BC-3205, telithromycin, lefamulin and lincomycin. 
     
     
         10 . A pharmaceutical composition comprising, as an active ingredient, the ligand of  claim 5 , and a pharmaceutically acceptable carrier. 
     
     
         11 . A method of treating an infection associated with a pathogenic bacterium, the method comprising administering to a subject in need thereof a therapeutically effective amount of the ligand of  claim 5 , wherein the pathogenic bacterium is Gram positive and/or exhibiting a degree of 23S rRNA sequence identity of at least 80% compared to rRNA of  Staphylococcus aureus  and a degree of 23S rRNA sequence identity of less than 99.9% compared to rRNA of  Escherichia coli.    
     
     
         12 . A method of treating an infection associated with a pathogenic bacterium, the method comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of  claim 10 , wherein the pathogenic bacterium is Gram positive and/or exhibiting a degree of 23S rRNA sequence identity of at least 80% compared to rRNA of  Staphylococcus aureus  and a degree of 23S rRNA sequence identity of less than 99.9% compared to rRNA of  Escherichia coli.