Kinase crystal structures
AssigneeThe Institute of Cancer Research
InventorsDavid Barford, Jing Yang, Brian Arthur Hemmings, Peter David Cron
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Abstract
Disclosed are mutants of protein kinase B/Akt which can be crystallised in an enzymatically active conformation, crystals of these mutants and X-ray coordinate data for the crystals. Also disclosed are methods of using the coordinate data provided for identification of modulators of protein kinase activity and for structural analysis of other protein kinases, in particular AGC kinases.
Claims32
A crystal of PKBβ having a tetragonal space group P212121, and unit cell dimensions of a=44.94±0.5 Å, b=61.00±0.5 Å, c=131.32±0.5 Å.
A crystal according to any one of the preceding claims, wherein the PKBβ is co-complexed with one or more of a substrate peptide and a nucleotide or nucleotide analogue.
A crystal of PKBβ having the three dimensional atomic coordinates of either of Tables 6 or 7.
A method of determining the structure of a PKB derivative comprising the step of X-ray diffraction analysis of a crystal according to any one of the preceding claims.
A method of analysing a PKBβ-ligand complex comprising the step of employing (i) X-ray crystallographic diffraction data from the PKBβ-ligand complex and (ii) a three-dimensional structure of PKBβ to generate a difference Fourier electron density map of the complex, the three-dimensional structure being defined by atomic coordinate data according to either of Tables 6 or 7.
A method of determining a three dimensional structure for a target kinase comprising the steps of:
- (a) aligning a representation of an amino acid sequence of a target kinase of unknown structure with the amino acid sequence of PKBβ to match homologous regions of the amino acid sequences;
- (b) modelling the structure of the matched homologous regions of the target kinase on the structure of the corresponding regions of PKBβ as defined by either of Tables 6 or 7;
- and
- (c) determining a conformation for the target kinase which substantially preserves the structure of said matched homologous regions.
A method for determining a three-dimensional structure for a target kinase, comprising the steps of;
- providing the co-ordinates of either of Tables 6 or 7, and positioning the co-ordinates in the crystal unit cell of said target kinase so as to provide a structure for said target kinase.
A computer system or computer-readable media containing either (a) atomic coordinate data of either of Tables 6 or 7, said data defining the three-dimensional structure of PKB, or at least selected coordinates thereof;
- (b) structure factor data for PKB, said structure factor data being derivable from the atomic coordinate data of either of Tables 6 or 7;
- (c) a Fourier transform of atomic coordinate data according to either of Tables 6 or 7, or at least selected coordinates thereof;
- (d) atomic coordinate data of a target kinase generated by homology modelling of the target based on the data of either of Tables 6 or 7;
- (e) atomic coordinate data of a target kinase generated by interpreting X-ray crystallographic data or NMR data by reference to the data of either of Tables 6 or 7;
- or (f) structure factor data derivable from the atomic coordinate data of (c), (d) or (e).
A method for modelling the interaction between PKB and an agent compound which modulates PKB activity, comprising the steps of:
- (a) employing three-dimensional atomic coordinate data of either of Tables 6 or 7 to characterise at least one PKBβ binding site;
- (b) providing the structure of said agent compound;
- and
- (c) fitting said agent compound to the binding site.
A method for identifying an agent compound which modulates PKB activity, comprising the steps of:
- (a) employing three-dimensional atomic coordinate data according to either of Tables 6 or 7 to characterise at least one PKBβ binding site;
- (b) providing the structure of a candidate agent compound;
- (c) fitting the candidate agent compound to the binding site;
- and
- (d) selecting the candidate agent compound.
Dependencies are inferred from the claim text.