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US 2006/0079669 A1Published application
Crystal structures of NH3-dependent NAD synthetases
AssigneeAffinium Pharmaceuticals, Inc.
InventorsAled Edwards, Akil Dharamsi, Masoud Vedadi, Matthew Kimber, Robert Lam
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Abstract
The present invention relates to novel drug targets for pathogenic bacteria. Accordingly, the invention provides purified protein comprising the amino acid sequence set forth in SEQ ID NO: 4. The invention also provides biochemical and biophysical characteristics of the polypeptides of the invention.
Claims35
A composition comprising an isolated, recombinant polypeptide, wherein the polypeptide comprises:
- (a) an amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- (b) an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- or (c) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide having SEQ ID NO:
- 1 or SEQ ID NO:
- 3 and has at least one biological activity of NH3-dependent NAD synthetase from P. aeruginosa;
- and wherein the polypeptide of (a), (b) or (c) is at least about 90% pure in a sample of the composition.
7 more elements
A sample comprising an isolated, recombinant polypeptide, wherein the polypeptide comprises:
- (a) an amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- (b) an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- or (c) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide having SEQ ID NO:
- 1 or SEQ ID NO:
- 3 and has at least one biological activity of NH3-dependent NAD synthetase from P. aeruginosa;
- and wherein the polypeptide of (a), (b) or (c) is labeled with a heavy atom.
7 more elements
A crystallized, recombinant polypeptide comprising:
- (a) an amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- (b) an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- or (c) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide having SEQ ID NO:
- 1 or SEQ ID NO:
- 3 and has at least one biological activity of NH3-dependent NAD synthetase from P. aeruginosa;
- wherein the polypeptide of (a), (b) or (c) is in crystal form.
7 more elements
A sample comprising an isolated, recombinant polypeptide, wherein the polypeptide comprises:
- (a) an amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- (b) an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- or (c) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide having SEQ ID NO:
- 1 or SEQ ID NO:
- 3 and has at least one biological activity of NH3-dependent NAD synthetase from P. aeruginosa;
- and wherein the polypeptide of (a), (b) or (c) is enriched in at least one NMR isotope.
7 more elements
A host cell comprising a nucleic acid encoding a polypeptide comprising:
- (a) an amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- (b) an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- or (c) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide having SEQ ID NO:
- 1 or SEQ ID NO:
- 3 and has at least one biological activity of NH3-dependent NAD synthetase from P. aeruginosa;
- wherein a culture of the host cell produces at least about 1 mg of the polypeptide per liter of culture and the polypeptide is at least about one-third soluble as measured by gel electrophoresis.
7 more elements
An isolated, recombinant polypeptide, comprising:
- (a) an amino acid sequence having at least about 90% identity with the amino acid sequence set forth in SEQ ID NO:
- 4;
- or (b) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide having SEQ ID NO:
- 1 or SEQ ID NO:
- 3 and has at least one biological activity of NH3-dependent NAD synthetase from P. aeruginosa;
- and wherein the polypeptide comprises one or more of the following amino acid residues at the specified position of the polypeptide:
- Leu at position 49, Gly at position 50, Ile at position 51, Ser at position 52, Ser at position 57, Val at position 85, Arg at position 86, Leu at position 87, Pro at position 88, Gln at position 92, Asp at position 94, Ala at position 146, Arg at position 149, Met at position 150, Gln at position 153, Tyr at position 154, Val at position 164, Ile at position 165, Gly at position 166, Thr at position 167, Asp at position 168, Glu at position 172, Gly at position 182, Asp at position 183, Gly at position 184, Ala at position 185 and Cys at position 186.
4 more elements
A method for obtaining structural information of a crystallized polypeptide, the method comprising:
- (a) crystallizing a recombinant polypeptide, wherein the polypeptide comprises:
- (1) an amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- (2) an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- or (3) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide having SEQ ID NO:
- 1 or SEQ ID NO:
- 3 and has at least one biological activity of NH3-dependent NAD synthetase from P. aeruginosa;
- and wherein the crystallized polypeptide is capable of diffracting X-rays to a resolution of 3.5 Å or better;
- and
- (b) analyzing the crystallized polypeptide by X-ray diffraction to determine the three-dimensional structure of at least a portion of the crystallized polypeptide.
10 more elements
A method for identifying a druggable region of a polypeptide, the method comprising:
- (a) obtaining crystals of a polypeptide comprising (1) an amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- (2) an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- or (3) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide having SEQ ID NO:
- 1 or SEQ ID NO:
- 3 and has at least one biological activity of NH3-dependent NAD synthetase from P. aeruginosa, such that the three dimensional structure of the crystallized polypeptide may be determined to a resolution of 3.5 Å or better;
- (b) determining the three dimensional structure of the crystallized polypeptide using X-ray diffraction;
- and
- (c) identifying a druggable region of the crystallized polypeptide based on the three-dimensional structure of the crystallized polypeptide.
9 more elements
Crystalline NH3-dependent NAD synthetase from P. aeruginosa comprising a crystal having unit cell dimensions of a=111.657 Å, b=40.751 Å, c=63.884 Å, β=113.020° and space group C2 with two molecules per asymmetric unit.
A crystallized polypeptide comprising (1) an amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- (2) an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO:
- 2 or SEQ ID NO:
- 4;
- or (3) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide having SEQ ID NO:
- 1 or SEQ ID NO:
- 3 and has at least one biological activity of NH3-dependent NAD synthetase from P. aeruginosa;
- wherein the crystal has a C2 space group.
6 more elements
A crystallized polypeptide comprising a structure of a polypeptide that is defined by a substantial portion of the atomic coordinates set forth in FIG. 9.
Dependencies are inferred from the claim text.