Crystal structure of a plasma membrane proton pump

A computer-assisted method for identifying potential inhibitors of a type III P-type ATPase using a programmed computer processor, a data storage system, a data input devise and a data output devise, the method comprising:

• obtaining a crystal of a type III P-type ATPase of an amino acid sequence of residues 1-875 of SEQ ID NO:
• 1 fused at its C-terminus to the amino acids of SEQ ID NO:
• 34, the crystal being of space group P212121;
• with unit cell dimensions in which:
• a is 85±4Å,b is 144±4Å, c is 312±4Å,and α is 90° , β is 90° ,γ is 90°;
• and determining the three-dimensional structure to obtain the atomic coordinates of this crystal as presented in FIG. 13 or atomic coordinates selected from a three-dimensional structure that deviates from the three-dimensional structure as presented in FIG. 13 by a root mean square deviation over protein backbone atoms of not more than 3 Å;
• inputting into the programmed computer through said input device data comprising:
• a subset of the atoms of a type III P-type ATPase, thereby generating a criteria data set;
• wherein the atomic coordinates are selected from the three-dimensional structure as presented in FIG. 13 or atomic coordinates selected from a three-dimensional structure that deviates from the three-dimensional structures as presented in FIG. 13 by a root mean square deviation over protein backbone atoms of not more than 3 Å,
• comparing, using said processor, the criteria data set to a computer data base of low molecular weight organic chemical structures stored in the data storage system;
• and
• selecting from said data base, using computer methods, a chemical structure having a portion that is structurally complementary to the criteria data set and being free of steric interference with the ATPase.

A method for identifying a potential inhibitor capable of inhibiting the H+ translocating activity of a type III P-type ATPase, said method comprising:

• obtaining a crystal of a type III P-type ATPase of an amino acid sequence of residues 1-875 of SEQ ID NO:
• 1 fused at its C-terminus to the amino acids of SEQ ID NO:
• 34, the crystal being of space group P212121;
• with unit cell dimensions in which:
• a is 85±4Å, b is 144±4Å, c is 312±4Å, and α is 90° , β is 90° , γ is 90°;
• and determining the three-dimensional structure to obtain the atomic coordinates of this crystal as presented in FIG. 13 or atomic coordinates selected from a three-dimensional structure that deviates from the three-dimensional structure as presented in FIG. 13 by a root mean square deviation over protein backbone atoms of not more than 3 Å;
• selecting a potential inhibitor using atomic coordinates in conjunction with computer modelling, wherein said atomic coordinates are the atomic coordinates presented in FIG. 13 or wherein the atomic coordinates are selected from a three-dimensional structure that deviates from the three-dimensional structures presented in FIG. 13 by a root mean square deviation over protein backbone atoms of not more than 3 Å, by docking potential inhibitors into a set of binding interaction sites in a proton transfer pathway generated by computer modeling and selecting a potential inhibitor capable of binding to said proton transport pathway,
• providing said potential inhibitor and said ATPase,
• contacting the potential inhibitor with said ATPase and
• detecting inhibition of H+ translocating activity of said ATPase by the potential inhibitor.

A method for identifying a potential inhibitor capable of inhibiting the H+ translocating activity of a type III P-type ATPase, said method comprising:

• obtaining a crystal of a type III P-type ATPase of an amino acid sequence of residues 1-875 of SEQ ID NO:
• 1 fused at its C-terminus to the amino acids of SEQ ID NO:
• 34, the crystal being of space group P212121;
• with unit cell dimensions in which:
• a is 85±4Å,b is 144±4Å, c is 312±4Å,and α is 90° , β is 90° ,γ is 90°;
• and determining the three-dimensional structure to obtain the atomic coordinates of this crystal as presented in FIG. 13 or atomic coordinates selected from a three-dimensional structure that deviates from the three-dimensional structure as presented in FIG. 13 by a root mean square deviation over protein backbone atoms of not more than 3 Å;
• introducing into a computer information derived from atomic coordinates defining a conformation of a proton transport pathway, the proton transfer pathway comprising three regions, an inlet channel, an active proton binding site, and a proton release pathway, based on three-dimensional structure determination, whereby said program utilizes or displays on the computer screen the structure of said conformation, wherein the atomic coordinates are selected from the three-dimensional structure as presented in FIG. 13 or atomic coordinates selected from a three-dimensional structure that deviates from the three-dimensional structures as presented in FIG. 13 by a root mean square deviation over protein backbone atoms of not more than 3 Å,
• generating a three-dimensional representation of at least one of the three regions of the proton transport pathway of said ATPase by said computer program on a computer screen,
• superimposing a model of a potential inhibitor on the representation on at least one of the three regions of the proton transport pathway;
• assessing the possibility of bonding and the absence of steric interference of the potential inhibitor with the proton transport pathway;
• incorporation said potential compound in an activity assay of said ATPase and
• determining whether said potential compound inhibits H+ translocating activity of said ATPase.