Molecular Dynamics Simulations Predict A pH-Dependent Conformational Change in the C-Helix of Cell Cycle Checkpoint Kinase Wee1

Abstract

Wee1 is the cell cycle checkpoint kinase that phosphorylates Tyr15 of Cdk1, maintaining it in an inactive state. Cdk1 regulates the transition from S to G2/M phases in eukaryotic cells and is activated upon dephosphorylation of residues Thr14 and Tyr15 and cyclin B1 binding. We previously reported that increased intracellular pH (pHi) (> 7.2) promotes G2/M transition. Moreover, we found that in cells having low pHi (< 7.2) the abundance of phosphorylated Cdk1 (pTyr15) is aberrantly sustained. We are testing the hypothesis that Wee1 kinase activity might be pH sensitive, with higher activity at lower pH. The C-helix in Wee1 contains four contiguous residues unique to the Wee1 family centered around His350, which faces the ATP binding site. Based on this observation, we hypothesized that changes in pHi could affect the C-helix conformation and hence catalytic activity (C-helix conformation modulates activity in many other kinases). Molecular dynamics simulations of Wee1 were performed while allowing His350 to be either neutral or charged, capturing the hypothetical protonation state of His350 at high and low pHi, respectively. Most portions of the kinase domain showed only small changes at the end of the two 10 ns simulations. However, when His350 was neutral, the C-helix adopted a conformation that closely mimics the inactive state of other kinases. In contrast, when His350 was charged, the C-helix adopted a conformation similar to the active state of other kinases wherein the C-helix was rotated towards the active site. These data suggest that pH-sensitivity in Wee1 may be mediated by the influence of the protonation state of His350 on the C-helix conformation. We are currently testing these predictions using NMR and biochemical approaches.