Abstract 2669: Crystal structure of the active N-terminal kinase domain of p90 ribosomal S6 protein kinase 2
Authors: Malakhova Margarita , Kurinov Igor , Liu Kangdong , Zheng Duo , Shim Jung-Hyun , Bode Ann , Dong Zigang 
Published: February 2011

The p90 ribosomal S6 kinase 2 (RSK2) is a serine-threonine kinase, which is activated in response to a variety of stimuli, including insulin, growth factors, neurotransmitters, and chemokines. RSK2 possesses two non-identical kinase domains, and its N-terminal domain (NTD) is responsible for endogenous substrate phosphorylation. We have determined the crystal structure of the NTD RSK2 at 1.8 Å resolution in complex with AMP-PNP. The comparison of ATP-binding site residues showed similarity with active PKA and differences with inactive homologous NTD MSK1 and NTD RSK1. The N-lobe of the NTD RSK2, traditionally composed of twisted, five stranded β-sheets, has an inserted novel βB-sheet comprised of three antiparallel β-strands. The βB-sheet insertion resulted in displacement of the αC-helix and disruption of the important Lys-Glu interaction, classifying the kinase conformation as inactive. However, the presence of the βB-sheet in the NTD RSK2 does not impair the ability of the enzyme to exhibit phosphotransferase in vitro activity or to strongly bind ATP in the crystal form. The isolated protein, which was phosphorylated at Ser227 in the T-activation loop, showed substrate specificity. It phosphorylated the S6 (AKRRRLSSLRA) and Crosstide (GRPRTSSFAEG) peptides and did not phosphorylate the CREBtide (KRREILSRRPSYR) or Kemptide (LRRASLG) peptide.

We suggest that a new βB-sheet insert, instead of the αC-helix, stabilizes the N-terminal kinase lobe active conformation in the purified protein. Examination of the active site revealed a Lys216 residue located on the βB-sheet that interacts with the β-phosphate (distance 2.66 Å) and the conserved Asp211 (distance 2.86 Å) from the DFG-motif. The Lys216 occupies a central position in the deep cavity formed inside the N-lobe. The lack of a glutamic acid residue (Glu118 from the αC-helix) in RSK2 due to disruption of the Lys-Glu salt bridge creates a void of charges, and Lys216 compensates by introducing a positive charge to the hydrophobic cavity. Mutation of Lys216 to alanine decreased protein activity ∼30% as was shown by [γ32-ATP] and luminescent assay. The slightly reduced enzyme activity of the K216A mutant indicates that a single point mutation did not introduce global conformational changes into the N-lobe, and confirmed the structural finding that Lys216 participates in phosphate binding. The involvement of one more residue, in addition to the conserved Lys100 from the β3-strand, for the coordination of the β-phosphate comprises a novel binding contact never before seen in other kinases complexed with ATP. The NTD RSK2 adopted a unique active conformation showing its structural diversity compared to other kinases. The presence of a unique βB-sheet insert in the N-lobe suggests a different type of activation mechanism for RSK2. The possibility of the βB-sheet unfolding and restoring the αC-helix position and the Lys-Glu ionic interaction in vivo is not inconceivable.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2669.