4BN1

Crystal structure of V174M mutant of Aurora-A kinase

Summary for 4BN1

• Entry DOI: 10.2210/pdb4bn1/pdb
• Descriptor: AURORA KINASE A, ADENOSINE-5'-DIPHOSPHATE, CALCIUM ION, ... (4 entities in total)
• Functional Keywords: transferase, protein kinase, mitosis
• Biological source: HOMO SAPIENS (HUMAN)
• Cellular location: Cytoplasm, cytoskeleton, microtubule organizing center, centrosome: O14965
• Total number of polymer chains: 1
• Total formula weight: 33648.17

Authors

Bibby, R.A.,Bayliss, R. (deposition date: 2013-05-13, release date: 2014-03-26, Last modification date: 2024-11-06)

Primary citation

Rowan, F.C.,Richards, M.,Bibby, R.A.,Thompson, A.,Bayliss, R.,Blagg, J.
Insights Into Aurora-A Kinase Activation Using Unnatural Amino Acids Incorporated by Chemical Modification.
Acs Chem.Biol., 8:2184-, 2013

PubMed 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.

PubMed: 23924325
DOI: 10.1021/CB400425T

Experimental method

X-RAY DIFFRACTION (2.499 Å)