Structural insights into the recognition of substrates and activators by the OSR1 kinase.
The oxidative-stress-responsive kinase 1 (OSR1) and the STE20/SPS1-related proline/alanine-rich kinase (SPAK) are key enzymes in a signalling cascade regulating the activity of Na(+)/K(+)/2Cl(-) co-transporters (NKCCs) in response to osmotic stress. Both kinases have a conserved carboxy-terminal (CCT) domain, which recognizes a unique peptide (Arg-Phe-Xaa-Val) motif present in OSR1- and SPAK-activating kinases (with-no-lysine kinase 1 (WNK1) and WNK4) as well as its substrates (NKCC1 and NKCC2). Here, we describe the structural basis of this recognition event as shown by the crystal structure of the CCT domain of OSR1 in complex with a peptide containing this motif, derived from WNK4. The CCT domain forms a novel protein fold that interacts with the Arg-Phe-Xaa-Val motif through a surface-exposed groove. An intricate web of interactions is observed between the CCT domain and an Arg-Phe-Xaa-Val motif-containing peptide derived from WNK4. Mutational analysis shows that these interactions are required for the CCT domain to bind to WNK1 and NKCC1. The CCT domain structure also shows how phosphorylation of a Ser/Thr residue preceding the Arg-Phe-Xaa-Val motif results in a steric clash, promoting its dissociation from the CCT domain. These results provide the first molecular insight into the mechanism by which the SPAK and OSR1 kinases specifically recognize their upstream activators and downstream substrates.
Villa
Fabrizio
F
Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
Goebel
Jürgen
J
Rafiqi
Fatema H
FH
Deak
Maria
M
Thastrup
Jacob
J
Alessi
Dario R
DR
van Aalten
Daan M F
DM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D020536:Enzyme Activators / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017510:Protein Folding; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D020536:Enzyme Activators; D050505:Mutant Proteins; D010455:Peptides; C118135:OXSR1 protein, human; D017346:Protein Serine-Threonine Kinases