PDBsum entry 2yn8

Transferase

PDB id: 2yn8

Contents

Protein chains

253 a.a.

Ligands

STU ×2

Waters ×419

PDB id:

2yn8

Name:

Transferase

Title:

Ephb4 kinase domain inhibitor complex

Structure:

Ephrin type-b receptor 4. Chain: a, b. Fragment: kinase domain, residues 598-892. Synonym: hepatoma transmembrane kinase, tyrosine-protein kinase tyro11. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: spodoptera frugiperda. Expression_system_taxid: 7108. Expression_system_cell_line: sf21.

Resolution:

2.11Å R-factor: 0.197 R-free: 0.234

Authors:

J.Read,C.A.Brassington,R.Overmann

Key ref:

R.C.Overman et al. (2013). Stability and solubility engineering of the EphB4 tyrosine kinase catalytic domain using a rationally designed synthetic library. Protein Eng Des Sel, 26, 695-704. PubMed id: 23840071 DOI: 10.1093/protein/gzt032

Date:

13-Oct-12 Release date: 23-Oct-13

Protein chains

P54760  (EPHB4_HUMAN) -  Ephrin type-B receptor 4 from Homo sapiens

Seq: 987 a.a.

Struc: 253 a.a.*

* PDB and UniProt seqs differ at 2 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⁺

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

Added reference

DOI no: 10.1093/protein/gzt032

Protein Eng Des Sel 26:695-704 (2013)

PubMed id: 23840071

Stability and solubility engineering of the EphB4 tyrosine kinase catalytic domain using a rationally designed synthetic library.

R.C.Overman, I.Green, C.M.Truman, J.A.Read, K.J.Embrey, M.S.McAlister, T.K.Attwood.

ABSTRACT

The inability to generate soluble, correctly folded recombinant protein is often a barrier to successful structural and functional studies. Access to affordable synthetic genes has, however, made it possible to design, make and test many more variants of a target protein to identify suitable constructs. We have used rational design and gene synthesis to create a controlled randomised library of the EphB4 receptor tyrosine kinase, with the aim of obtaining soluble, purifiable and active catalytic domain material at multi-milligram levels in Escherichia coli. Three main parameters were tested in designing the library-construct length, functional mutations and stability grafting. These variables were combined to generate a total of 9720 possible variants. The screening of 480 clones generated a 3% hit rate, with a purifiable solubility of up to 15 mg/L for some EphB4 constructs that was largely independent of construct length. Sequencing of the positive clones revealed a pair of hydrophobic core mutations that were key to obtaining soluble material. A minimal kinase domain construct containing these two mutations exhibited a +4.5°C increase in thermal stability over the wild-type protein. These approaches will be broadly applicable for solubility engineering of many different protein target classes. Atomic coordinates and structural factors have been deposited in PDB under the accession 2yn8 (EphB4 HP + staurosporine).