Crystals and structures of members of the E. coli comA and yddB protein families (ComA)
OwnerGordon V. Louie, Ketan S. Gajiwala +1inventors
InventorsGordon V. Louie, Ketan S. Gajiwala, Sean Grant Buchanan
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Abstract
The present invention provides machine readable media embedded with the three-dimensional molecular structure coordinates of ComA proteins, and subsets thereof, including binding pockets, methods of using the structure to identify and design affecters, including inhibitors and activator, mutants of ComA, and compounds and compositions that affect ComA activity.
Claims111
A method of producing a computer readable database comprising the three-dimensional molecular structural coordinates of binding pocket of a ComA protein, said method comprising
- a) obtaining three-dimensional structural coordinates defining said protein or a binding pocket of said protein, from a crystal of said protein;
- and
- b) introducing said structural coordinates into a computer to produce a database containing the molecular structural coordinates of said protein or said binding pocket.
A method of producing a computer readable database comprising structural information about a molecule or a molecular complex of unknown structure comprising:
- a) generating an x-ray diffraction pattern from a crystallized form of said molecule or molecular complex;
- b) using a molecular replacement method to interpret the structure of said molecule;
- wherein said molecular replacement method uses the structural coordinates of FIG. 4, 5, or 6, or a subset thereof comprising a binding pocket, the structural coordinates of a binding pocket of FIG. 4, 5, or 6, or structural coordinates having a root mean square deviation for the alpha-carbon atoms of said structural coordinates of less than 2.0 Å;
- and
- c) storing the coordinates of the resulting structure in a computer readable database.
A method for homology modeling the structure of a ComA protein homolog comprising:
- a) aligning the amino acid sequence of a ComA protein homolog with an amino acid sequence of ComA protein;
- b) incorporating the sequence of the ComA protein homolog into a model of the structure of ComA protein, wherein said model has the same structural coordinates as the structural coordinates of FIG. 4, 5, or 6, or wherein the structural coordinates of said model's alpha-carbon atoms have a root mean square deviation from the structural coordinates of FIG. 4, 5, or 6, of less than 2.0 Å to yield a preliminary model of said homolog;
- c) subjecting the preliminary model to energy minimization to yield an energy minimized model;
- and
- d) remodeling regions of the energy minimized model where stereochemistry restraints are violated to yield a final model of said homolog.
A method for identifying a compound that binds ComA protein comprising:
- a) providing a computer modeling program with a set of structural coordinates or a three dimensional conformation for a molecule that comprises a binding pocket of ComA protein, or a homolog thereof;
- b) providing a said computer modeling program with a set of structural coordinates of a chemical entity;
- c) using said computer modeling program to evaluate the potential binding or interfering interactions between the chemical entity and said binding pocket;
- and
- d) determining whether said chemical entity potentially binds to or interferes with said protein or homolog.
A method for designing a compound that binds ComA protein comprising:
- a) providing a computer modeling program with a set of structural coordinates, or a three dimensional conformation derived therefrom, for a molecule that comprises a binding pocket comprising the structural coordinates of a binding pocket of ComA protein, or a homolog thereof;
- b) computationally building a chemical entity represented by set of structural coordinates;
- and
- c) determining whether the chemical entity is expected to bind to said molecule.
A ComA protein, or a functional ComA protein subunit, in crystalline form.
A machine-readable medium embedded with the molecular structural coordinates of FIG. 4, 5, or 6, or at least 50% of the coordinates thereof.
A machine-readable medium embedded with the molecular structural coordinates of FIG. 4, 5, or 6, or at least 80% of the coordinates thereof.
A machine-readable medium embedded with the molecular structural coordinates of a protein molecule comprising a ComA protein binding pocket, wherein said binding pocket comprises at least three amino acids selected from the group consisting of His56, Glu65, Gln50, Gly57, Gly58, Gly84, Ser69, Val83, His91, and Leu133 having the structural coordinates of FIG. 4, 5, or 6, or by the structural coordinates of a binding pocket homolog, wherein said the root mean square deviation of the backbone atoms of the amino acid residues of said binding pocket and said binding pocket homolog is less than 2.0 Å.
A method of producing a mutant ComA protein, having an altered property relative to ComA protein, comprising,
- a) constructing a three-dimensional structure of a molecule comprising a binding pocket, wherein said binding pocket comprises at least three amino acids selected from the group consisting of His56, Glu65, Gln50, Gly57, Gly58, Gly84, Ser69, Val83, His91, and Leu133 having the structural coordinates of FIG. 4, 5, or 6, or the structural coordinates of a binding pocket homolog, wherein said the root mean square deviation of the backbone atoms of the amino acid residues of said binding pocket and said binding pocket homolog is less than 2.0 Å;
- b) using modeling methods to identify in the three-dimensional structure at least one portion of said binding pocket wherein an alteration in said portion is predicted to result in said altered property;
- c) providing a nucleic acid molecule coding for a mutant ComA protein having a modified sequence that encodes a deletion, insertion, or substitution of one or more amino acids at a position corresponding to said portion;
- and
- d) expressing said nucleic acid molecule to produce said mutant;
- wherein said mutant has at least one altered property relative to the parent.
A method for determining whether a compound binds ComA protein, comprising,
- a) providing a computer modeling program with a set of structural coordinates or a three dimensional conformation for a molecule that comprises a binding pocket of ComA protein, or a homolog thereof;
- b) providing a said computer modeling program with a set of structural coordinates of a chemical entity;
- c) using said computer modeling program to evaluate the potential binding or interfering interactions between the chemical entity and said binding pocket;
- and
- d) determining whether said chemical entity potentially binds to or interferes with said protein or homolog.
A method of modulating thioesterase activity, comprising modulating the expression of a ComA protein.
A method of modulating thioesterase activity, comprising modulating the activity of a ComA protein.
Dependencies are inferred from the claim text.