PDBsum entry 1lum

Transferase

PDB id: 1lum

Contents

Protein chain

110 a.a. *

* Residue conservation analysis

PDB id:

1lum

Name:

Transferase

Title:

Nmr structure of the itk sh2 domain, pro287trans, 20 low energy structures

Structure:

Tyrosine-protein kinase itk/tsk. Chain: a. Fragment: src homology 2 (sh2) domain (residues 238-344). Synonym: interleukin-2 tyrosine kinase, t-cell-specific kinase, il-2- inducible t-cell kinase, kinase emt, kinase tlk. Engineered: yes

Source:

Mus musculus. House mouse. Organism_taxid: 10090. Expressed in: escherichia coli bl21. Expression_system_taxid: 511693.

NMR struc:

20 models

Authors:

R.J.Mallis,K.N.Brazin,D.B.Fulton,A.H.Andreotti

Key ref:

R.J.Mallis et al. (2002). Structural characterization of a proline-driven conformational switch within the Itk SH2 domain. Nat Struct Biol, 9, 900-905. PubMed id: 12402030 DOI: 10.1038/nsb864

Date:

22-May-02 Release date: 27-Nov-02

Protein chain

Q03526  (ITK_MOUSE) -  Tyrosine-protein kinase ITK/TSK from Mus musculus

Seq: 625 a.a.

Struc: 109 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

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⁺

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

Added reference

DOI no: 10.1038/nsb864

Nat Struct Biol 9:900-905 (2002)

PubMed id: 12402030

Structural characterization of a proline-driven conformational switch within the Itk SH2 domain.

R.J.Mallis, K.N.Brazin, D.B.Fulton, A.H.Andreotti.

ABSTRACT

Interleukin-2 tyrosine kinase (Itk) is a T cell-specific kinase required for a proper immune response following T cell receptor engagement. In addition to the kinase domain, Itk is composed of several noncatalytic regulatory domains, including a Src homology 2 (SH2) domain that contains a conformationally heterogeneous Pro residue. Cis-trans isomerization of a single prolyl imide bond within the SH2 domain mediates conformer-specific ligand recognition that may have functional implications in T cell signaling. To better understand the mechanism by which a proline switch regulates ligand binding, we have used NMR spectroscopy to determine two structures of Itk SH2 corresponding to the cis and trans imide bond-containing conformers. The structures indicate that the heterogeneous Pro residue acts as a hinge that modulates ligand recognition by controlling the relative orientation of protein-binding surfaces.

Selected figure(s)

Figure 1.
Figure 1. NMR structures of the cis and trans Itk SH2 conformers. a, Stereo view of 20 low energy structures of the cis (coral) and trans (turquoise) conformations of the Itk SH2 domain. Backbone heavy atoms within the secondary structural elements over the entire sequence were used for superpositions. b, Ribbon diagrams of the energy minimized average structures of the cis (left) and trans (right) conformers. Secondary structural elements and ligand-binding pockets are labeled in (a,b) according to standard nomenclature for SH2 domains8. Pro 287 is labeled in each structure. c, Sequence of the Itk SH2 domain and sequence alignment of the CD loop regions in the SH2 domains of several tyrosine kinases. The residues that give rise to nondegenerate chemical shifts2 are bold and underlined, and Pro 287 is labeled. d, Solvent-accessible surface plot of the cis conformer. The residues that give rise to dual resonances because of Pro cis-trans isomerization are highlighted in white. The trans conformer shows a similar contiguous surface for the heterogeneous residues (data not shown). e, Overlay of the energy minimized average structures of the cis (coral) and trans (turquoise) conformers. Expanded views of the CD loop (left), the central -sheet (right) and the BG loop regions (middle) are shown. All structures are rendered using MolMol31.

Figure 3.
Figure 3. Hydrophobic packing involving residues in the CD loop of the cis SH2 structure provides stabilization energy. a, Overlay of 20 lowest energy structures including the CD loop, the central -sheet and A of the cis (left) and trans (right) conformers. Side chains of Leu 254 and Pro 287 are yellow. His 291 and Glu 250 are also labeled. b, Overlay of 20 lowest energy structures (rotated clockwise with respect to (a)) showing the CD loop of the cis (left) and trans (right) conformers. Side chains of Ile 282, Ala 281 and Cys 288 are labeled and shown in yellow. Additional side chains are included without labels for clarity. c, Three-dimensional 13C-edited NOESY experiment showing through-space proximity between the -methyl protons of Ile 282 and one of the -methylene protons of Cys 288. The NOE is observed only for the cis conformer (left panel). The total number of NOEs unique to the cis and trans structures is shown in Table 1. d, Three-dimensional 15N-edited TOCSY experiment illustrating the nondegenerate resonance frequencies for the Cys 288 -methylenes in the cis conformer (left). The same protons resonate at a single frequency in the trans conformer (right). e, Expansion of the 1H-15N HSQC spectra showing the amide signal of Gly 260 (6260) in the cis and trans forms. Left, unmodified, reduced Itk SH2 domain. Middle, spectrum acquired following reaction of the Itk SH2 domain with glutathione disulfide (GSSG) (20 mM GSSG, pH 7.4, 40 min, 25 °C). Right, spectrum acquired following reaction with methyl methane thiosulfonate (MMTS) (5 mM MMTS, pH 7.4, 20 min). The percentage of SH2 domain in the cis conformation in each of these experiments as measured by peak volumes of Gly 260 (cis) and Gly 260 (trans) was 48, 5 and 32% for the reduced, GSSG-treated and MMTS-treated proteins, respectively. The backbone amide resonance of Cys 288 was monitored over the course of both reactions and the reactions were allowed to proceed until no further chemical shift changes occurred. The completeness of the S-glutathiolation reaction was also assessed by separation of the domain with nondenaturing isoelectric focusing (IEF) gel electrophoresis over a pH range of 3.5 -10 as described^33.

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Biol (2002, 9, 900-905) copyright 2002.

Figures were selected by an automated process.