Out of the loop

28 Sep 2010

Molecular dynamics simulations show why scientists have failed to generate an active form of the protein kinase PAK1

Fig. 1: The structure of active PAK1 kinase (gray), showing the Thr432 'clamp' that brings together the amino- and carbon-terminal lobes to activate the molecule.

Fig. 1: The structure of active PAK1 kinase (gray), showing the Thr432 ‘clamp’ that brings together the amino- and carbon-terminal lobes to activate the molecule.

PAKs — short for p21-activated kinases — are a family of enzymes that catalyze the transfer of phosphate groups from adenosine triphosphate (ATP) to proteins. They are involved in signal transduction pathways that regulate various cellular functions, including cytoskeletal dynamics and cell motility. There are six members in the PAK family and PAK1, the most studied member, is widely expressed in humans in the brain, muscle and spleen. PAK1-knockout mice have been shown to have defects in the central nervous and immune systems.

Scientists have recently synthesized PAK1(T423E) — a mutated form of PAK1 that has been widely reported to be active. However, Yuen Wai Ng at the A*STAR Institute of Medical Biology, Devanathan Raghunathan at the A*STAR BioInformatics Institute and co-workers have found that PAK1(T423E) is not active in cells and nor does it exhibit significant catalytic activity in vitro.

PAK1 has an activation loop that is important for maintaining it in its active form. PAK1 naturally occurs in pairs, with the autoinhibitory domain of one PAK1 molecule binding and inhibiting the catalytic domain of the other PAK1 molecule. When the small signaling proteins Cdc42 or Rac1 bind with one of the molecules, the structure of PAK1 changes, the pair dissociates and the catalytic domain becomes active again.

To make synthetic PAK1, scientists substituted glutamic acid in place of a threonine residue known as Thr423 to artificially activate the catalytic domain.

To fully understand the reason for the lack of activity in synthetic PAK1(T423E), the A*STAR researchers performed molecular dynamics simulations of PAK1(T423E) in complex with ATP and a substrate peptide. Their simulations revealed a key interaction between Lys308, a lysine residue at the end of the αC helix of PAK1, and Thr423 that has not been seen in protein crystallography studies. The interaction allows PAK1 to pack its amino- and carbon-terminal lobes into the active conformation (Fig. 1), but is missing when Thr423 is replaced by glutamic acid.

The simulations also predicted that Arg359, an arginine residue at the end of the αC helix of PAK4, may play a similar role to that of Lys308 in PAK1. The researchers validated this prediction by confirming the inactivity of experimentally mutated PAK4. The findings demonstrate not only the importance of the αC helix in maintaining PAKs in their active conformation, but also the possibility of using molecular dynamics simulations to accurately predict interactions between amino acids in protein kinases. “The work also cautions against the assumption that phosphomimetics necessarily simulate the effects of phosphorylation,” says Raghunathan.

The A*STAR-affiliated researchers contributing to this research are from the Bioinformatics Institute , the Institute of Medical Biology and the Institute of Molecular and Cell Biology.

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Ng, Y.W., Raghunathan, D., Chan, P.M., Baskaran, Y., Smith, D.J., Lee, C.H., Verma, C. & Manser, E. Why an A-loop phospho-mimetic fails to activate PAK1: understanding an inaccessible kinase state by molecular dynamics simulations. Structure 18, 879–890 (2010). | article

This article was made for A*STAR Research by Nature Research Custom Media, part of Springer Nature