 |
PDBsum entry 2ivt
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
Structure and chemical inhibition of the ret tyrosine kinase domain.
|
|
|
Authors
|
|
P.P.Knowles,
J.Murray-Rust,
S.Kjaer,
R.P.Scott,
S.Hanrahan,
M.Santoro,
C.F.Ibáñez,
N.Q.Mcdonald.
|
|
|
Ref.
|
|
J Biol Chem, 2006,
281,
33577-33587.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
Abstract
|
|
|
The RET proto-oncogene encodes a receptor tyrosine kinase for the glial cell
line-derived neurotrophic factor family of ligands. Loss-of-function mutations
in RET are implicated in Hirschsprung disease, whereas activating mutations in
RET are found in human cancers, including familial medullar thyroid carcinoma
and multiple endocrine neoplasias 2A and 2B. We report here the biochemical
characterization of the human RET tyrosine kinase domain and the structure
determination of the non-phosphorylated and phosphorylated forms. Both
structures adopt the same active kinase conformation competent to bind ATP and
substrate and have a pre-organized activation loop conformation that is
independent of phosphorylation status. In agreement with the structural data,
enzyme kinetic data show that autophosphorylation produces only a modest
increase in activity. Longer forms of RET containing the juxtamembrane domain
and C-terminal tail exhibited similar kinetic behavior, implying that there is
no cis-inhibitory mechanism within the RET intracellular domain. Our results
suggest the existence of alternative inhibitory mechanisms, possibly in trans,
for the autoregulation of RET kinase activity. We also present the structures of
the RET tyrosine kinase domain bound to two inhibitors, the pyrazolopyrimidine
PP1 and the clinically relevant 4-anilinoquinazoline ZD6474. These structures
explain why certain multiple endocrine neoplasia 2-associated RET mutants found
in patients are resistant to inhibition and form the basis for design of more
effective inhibitors.
|
|
|
|
|
|
|
|
Figure 2.
FIGURE 2. RET kinase structures. A, RET-KD-P (green) and
RET-KD-0P (cyan for molecule A and red for molecule B)
structures superimposed using the C-lobe C-  atoms. The bound
nucleotides are shown as sticks. B, RET-KD-P (green), activated
IRK (Protein Data Bank code 1IR3; orange), and Kit (Protein Data
Bank code 1PKG; blue) structures superimposed using equivalent
C-lobe C- atoms. C and D,
orthogonal views of the trans-inhibited RET-KD dimer with
molecule A in green and molecule B in light green. Their
N-terminal helices are red and cyan, respectively. Side chain
sticks are shown for Tyr^900 and Tyr^905 (orange), Met^918
(magenta), and Pro^766 (orange). E, main chain hydrogen bond
contacts in the complex structure (Protein Data Bank code 1IR3)
between IRK (orange) and substrate peptide (cyan). F, main chain
hydrogen bond contacts in RET-KD between the Met^918 pocket of
molecule A (green) and Pro^766 of molecule B (light green).
|
|
Figure 3.
FIGURE 3. Ligand-binding sites. Electron density maps
around ZD6474 (A) and PP1 (B) show REFMAC-calculated electron
density maps with 2mF[o] - DF[c] contoured at 1  in green
and mF[o] - DF[c] contoured at 3 in blue and (in A)
contoured at 8 in red. C and D are
Ligplot (79) schematic diagrams of ZD6474 and PP1 contacts with
RET. Water molecules (W) are shown in cyan. E-G show molecular
surfaces of the ligand-binding pockets in RET-KD-P, with the
solvent side of the surface white and the inside of the surface
blue-green. Ligands are shown in stick form, with carbon atoms
magenta for ZD6474 (E), green for AMP (F), and brown for PP1
(G). The Val^804 side chain is highlighted in yellow. The pocket
with access that is controlled by Val^804 is in the center of
each diagram; a second apparent pocket (^*) in E and G is the
result of the Phe^735 side chain being disordered in these two
complexes.
|
|
|
|
|
|
The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
33577-33587)
copyright 2006.
|
|
|
|
|
|
|
