PDBsum entry 2ivt

Transferase

PDB id: 2ivt

Contents

Protein chain

289 a.a. *

Ligands

FMT ×2

AMP

Waters ×62

* Residue conservation analysis

PDB id:

2ivt

Name:

Transferase

Title:

Crystal structure of phosphorylated ret tyrosine kinase domain

Structure:

Proto-oncogene tyrosine-protein kinase receptor ret precursor. Chain: a. Fragment: tyrosine kinase domain, residues 705-1013. Synonym: ret receptor, c-ret. Engineered: yes. Other_details: ptr at 905

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: spodoptera frugiperda. Expression_system_taxid: 7108. Expression_system_cell_line: sf9.

Biol. unit:

Dimer (from PDB file)

Resolution:

2.60Å R-factor: 0.187 R-free: 0.249

Authors:

P.P.Knowles,J.Murray-Rust,N.Q.Mcdonald

Key ref:

P.P.Knowles et al. (2006). Structure and chemical inhibition of the RET tyrosine kinase domain. J Biol Chem, 281, 33577-33587. PubMed id: 16928683 DOI: 10.1074/jbc.M605604200

Date:

16-Jun-06 Release date: 14-Aug-06

Protein chain

P07949  (RET_HUMAN) -  Proto-oncogene tyrosine-protein kinase receptor Ret from Homo sapiens

Seq: 1114 a.a.

Struc: 289 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.7.10.1 - receptor protein-tyrosine kinase.
• Reaction: L-tyrosyl-[protein] + ATP = O-phospho-L-tyrosyl-[protein] + ADP + H⁺

L-tyrosyl-[protein] + ATP = O-phospho-L-tyrosyl-[protein] (Bound ligand (Het Group name = AMP) matches with 85.19% similarity) + ADP + H(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

DOI no: 10.1074/jbc.M605604200

J Biol Chem 281:33577-33587 (2006)

PubMed id: 16928683

Structure and chemical inhibition of the RET tyrosine kinase domain.

P.P.Knowles, J.Murray-Rust, S.Kjaer, R.P.Scott, S.Hanrahan, M.Santoro, C.F.Ibáñez, N.Q.McDonald.

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.

Selected figure(s)

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.

Figures were selected by an automated process.