PDBsum entry 1ksw

Transferase

PDB id: 1ksw

Contents

Protein chain

450 a.a. *

Ligands

NBS

Waters ×46

* Residue conservation analysis

PDB id:

1ksw

Name:

Transferase

Title:

Structure of human c-src tyrosine kinase (thr338gly mutant) in complex with n6-benzyl adp

Structure:

Proto-oncogene tyrosine-protein kinase src. Chain: a. Fragment: sh3, sh2 and kinase domains. Synonym: c-src. Engineered: yes. Mutation: yes

Source:

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

Resolution:

2.80Å R-factor: 0.231 R-free: 0.294

Authors:

L.A.Witucki,X.Huang,K.Shah,Y.Liu,S.Kyin,M.J.Eck,K.M.Shokat

Key ref:

L.A.Witucki et al. (2002). Mutant tyrosine kinases with unnatural nucleotide specificity retain the structure and phospho-acceptor specificity of the wild-type enzyme. Chem Biol, 9, 25-33. PubMed id: 11841936 DOI: 10.1016/S1074-5521(02)00091-1

Date:

14-Jan-02 Release date: 27-Feb-02

Protein chain

P12931  (SRC_HUMAN) -  Proto-oncogene tyrosine-protein kinase Src from Homo sapiens

Seq: 536 a.a.

Struc: 450 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.2.7.10.2 - non-specific 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 = NBS) matches with 79.41% similarity) + ADP + H(+)

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

Added reference

DOI no: 10.1016/S1074-5521(02)00091-1

Chem Biol 9:25-33 (2002)

PubMed id: 11841936

Mutant tyrosine kinases with unnatural nucleotide specificity retain the structure and phospho-acceptor specificity of the wild-type enzyme.

L.A.Witucki, X.Huang, K.Shah, Y.Liu, S.Kyin, M.J.Eck, K.M.Shokat.

ABSTRACT

The direct substrates of one protein kinase in a cell can be identified by mutation of the ATP binding pocket to allow an unnatural ATP analog to be accepted exclusively by the engineered kinase. Here, we present structural and functional assessment of peptide specificity of mutant protein kinases with unnatural ATP analogs. The crystal structure (2.8 A resolution) of c-Src (T338G) with N(6)-(benzyl) ADP bound shows that the creation of a unique nucleotide binding pocket does not alter the phospho-acceptor binding site of the kinase. A panel of optimal peptide substrates of defined sequence, as well as a degenerate peptide library, was utilized to assess the phospho-acceptor specificity of the engineered "traceable" kinases. The specificity profiles for the mutant kinases were found to be identical to those of their wild-type counterparts.

Selected figure(s)

Figure 1.
Figure 1. Chemical Structures of A*TP Analogs Used in This Study1: N^6-(benzyl) ATP; 2: N^6-(cyclopentyl) ATP. Definitions of analog-sensitive (as) kinase mutants used in this study.

Figure 2.
Figure 2. Comparison of Wild-Type and Analog-Specific c-Src Crystal Structures(A) The crystal structure of c-Src-as1 superimposed on wild-type c-Src. c-Src-as1 is shown in gray, and c-Src is in red. The rmsd for the overlay is 0.35 Å.(B) The binding of the A*TP analog, N^6-(benzyl) ADP to the mutant c-Src (T338G) kinase. The surface corresponding to the glycine residue at the 338 position is colored red. The benzyl ring of the A*TP analog projects into a pocket in the nucleotide binding cleft. This pocket is made accessible by the c-Src (T338G) point mutation. For clarity, the 11 residues that bind over the nucleotide at the front of the nucleotide cleft are omitted from the figure in order to more clearly show the surface at the back of the nucleotide binding pocket where the 338 residue lies. The omitted residues are c-Src 272–282.(C) The steric clash of the wild-type c-Src threonine residue at the 338 position, shown in red, with the N^6-(benzyl) ATP analog (blue). The gray surface was built over the crystal structure of the mutant kinase overlayed with the wild-type c-Src crystal structure, and the surface was rendered over threonine 338 (red). The N^6-(benzyl) ADP (blue) is superimposed on the AMP-PNP ligand (yellow).

The above figures are reprinted by permission from Cell Press: Chem Biol (2002, 9, 25-33) copyright 2002.

Figures were selected by an automated process.