Decifrando a base estrutural da regulação de proteína quinase eucariótica: mero acaso, fortuita necessidade ou design inteligente?

segunda-feira, agosto 20, 2018

Deciphering the Structural Basis of Eukaryotic Protein Kinase Regulation

Hiruy S. Meharena, Philip Chang, Malik M. Keshwani, Krishnadev Oruganty, Aishwarya K. Nene, Natarajan Kannan, Susan S. Taylor , Alexandr P. Kornev 


Eukaryotic protein kinases (EPKs) regulate numerous signaling processes by phosphorylating targeted substrates through the highly conserved catalytic domain. Our previous computational studies proposed a model stating that a properly assembled nonlinear motif termed the Regulatory (R) spine is essential for catalytic activity of EPKs. Here we define the required intramolecular interactions and biochemical properties of the R-spine and the newly identified “Shell” that surrounds the R-spine using site-directed mutagenesis and various in vitro phosphoryl transfer assays using cyclic AMP-dependent protein kinase as a representative of the entire kinome. Analysis of the 172 available Apo EPK structures in the protein data bank (PDB) revealed four unique structural conformations of the R-spine that correspond with catalytic inactivation of various EPKs. Elucidating the molecular entities required for the catalytic activation of EPKs and the identification of these inactive conformations opens new avenues for the design of efficient therapeutic EPK inhibitors.

Author Summary

Eukaryotic protein kinases (EPKs) have a highly conserved enzymatic kinase core that is involved in the regulation of numerous cell signaling processes through the transfer of a phosphate group from adenosine triphosphate (ATP) to more than 30% of human proteins. EPKs have been implicated in numerous human diseases, including cancer, cardiovascular diseases, and diabetes, making them one of the most sought-after therapeutic drug targets. The lack of structural diversity of the active kinase core has created a bottle-neck for designing successful therapeutic inhibitors. Here we describe the intramolecular interactions required for differentiating between the active and inactive states of EPKs. Kinases contain a hydrophobic regulatory spine (“R-spine”) that is disassembled in inactive kinases, and here we define an additional hydrophobic “Shell” that surrounds one end of the R-spine. Biochemical analysis of the five nonconsecutive R-spine residues and three nonconsecutive Shell residues shows that proper assembly of the R-spine and Shell is essential for maintaining kinase activity. Structural analysis of the 172 known structures of EPKs without bound ligands led to the identification of four inactive conformations that correlate with the disassembly of the R-spine. Understanding the molecular elements involved in the regulation of kinase activity and the identification of these diverse groups of inactive conformations should aid the design of more specific therapeutic EPK inhibitors.

Citation: Meharena HS, Chang P, Keshwani MM, Oruganty K, Nene AK, Kannan N, et al. (2013) Deciphering the Structural Basis of Eukaryotic Protein Kinase Regulation. PLoS Biol 11(10): e1001680.

Academic Editor: Gregory A. Petsko, Brandeis University, United States of America

Received: May 21, 2013; Accepted: August 29, 2013; Published: October 15, 2013

Copyright: © 2013 Meharena et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: National Institutes of Health Grant GM19301 and GM34921 (to SST) and National Science Foundation's Graduate Research Fellowship Program (GRFP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Abbreviations: EPK, eukaryotic protein kinase; R-spine, regulatory spine; RS0-4, R-spine residue 0–5; SH1–3, shell residue 1–3