PDBsum entry 4bn1

Transferase

PDB id: 4bn1

Contents

Protein chain

263 a.a.

Ligands

ADP

Metals

_CA ×2

Waters ×44

PDB id:

4bn1

Name:

Transferase

Title:

Crystal structure of v174m mutant of aurora-a kinase

Structure:

Aurora kinase a. Chain: a. Fragment: residues 122-403. Synonym: aurora 2, aurora/ipl1-related kinase 1, ark-1, aurora-relat ed kinase 1, hark1, breast tumor-amplified kinase, serine/threonin e- protein kinase 15, serine/threonine-protein kinase 6, serine/thr eonine-protein kinase aurora-a, aurora-a. Engineered: yes. Mutation: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

2.50Å R-factor: 0.195 R-free: 0.244

Authors:

R.A.Bibby,R.Bayliss

Key ref:

F.C.Rowan et al. (2013). Insights into Aurora-A kinase activation using unnatural amino acids incorporated by chemical modification. Acs Chem Biol, 8, 2184-2191. PubMed id: 23924325 DOI: 10.1021/cb400425t

Date:

13-May-13 Release date: 26-Mar-14

Protein chain

O14965  (AURKA_HUMAN) -  Aurora kinase A from Homo sapiens

Seq: 403 a.a.

Struc: 263 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.7.11.1 - non-specific serine/threonine protein kinase.
• Reaction:
  1. L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] + ADP + H⁺
  2. L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] + ADP + H⁺

L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] (Bound ligand (Het Group name = ADP) corresponds exactly) + ADP + H(+)

L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] (Bound ligand (Het Group name = ADP) corresponds exactly) + ADP + H(+)

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

reference

DOI no: 10.1021/cb400425t

Acs Chem Biol 8:2184-2191 (2013)

PubMed id: 23924325

Insights into Aurora-A kinase activation using unnatural amino acids incorporated by chemical modification.

F.C.Rowan, M.Richards, R.A.Bibby, A.Thompson, R.Bayliss, J.Blagg.

ABSTRACT

Most protein kinases are regulated through activation loop phosphorylation, but the contributions of individual sites are largely unresolved due to insufficient control over sample phosphorylation. Aurora-A is a mitotic Ser/Thr protein kinase that has two regulatory phosphorylation sites on its activation loop, T287 and T288. While phosphorylation of T288 is known to activate the kinase, the function of T287 phosphorylation is unclear. We applied site-directed mutagenesis and selective chemical modification to specifically introduce bioisosteres for phospho-threonine and other unnatural amino acids at these positions. Modified Aurora-A proteins were characterized using a biochemical assay measuring substrate phosphorylation. Replacement of T288 with glutamate and aspartate weakly stimulated activity. Phospho-cysteine, installed by chemical synthesis from a corresponding cysteine residue introduced at position 288, showed catalytic activity approaching that of the comparable phospho-serine protein. Unnatural amino acid residues, with longer side chains, inserted at position 288 were autophosphorylated and supported substrate phosphorylation. Aurora-A activity is enhanced by phosphorylation at position 287 alone but is suppressed when position 288 is also phosphorylated. This is rationalized by competition between phosphorylated T287 and T288 for a binding site composed of arginines, based on a structure of Aurora-A in which phospho-T287 occupies this site. This is, to our knowledge, the first example of a Ser/Thr kinase whose activity is controlled by the phosphorylation state of adjacent residues in its activation loop. Overall we demonstrate an approach that combines mutagenesis and selective chemical modification of selected cysteine residues to investigate otherwise impenetrable aspects of kinase regulation.