Copyright © 2019 American Chemical Society. Kelch-like 3 (KLHL3) is a substrate adaptor of an E3 ubiquitin ligase complex that regulates the degradation of its substrates, including with-no-lysine [K] kinase 4 (WNK4). Mutations in KLHL3 are associated with pseudohypoaldosteronism type II (PHAII), a hereditary form of hypertension. Many PHAII-causing mutations are located in the Kelch domain of KLHL3 that binds with WNK4; however, detailed mechanisms by which these mutations disrupt the binding are not well-understood. In the present study we use molecular dynamics simulations and Western blot analyses to examine the effects of these mutations on the interaction between the Kelch domain of KLHL3 and the acidic motif (AM) of WNK4. The simulation results correlated well with those from Western blot analyses with the exception of the L387P mutation, which led to deregulation of AM degradation by KLHL3 but not recapitulated by simulations. On the basis of the simulation results, a mutation on the binding surface of the Kelch domain affected the Kelch-AM interaction through two major mechanisms: altering the electrostatic potential of the AM binding site and disrupting the Kelch-AM hydrogen bonds. The mutations buried inside the Kelch domain were predicted by our simulations to have no or modest effects on the Kelch-AM interaction. Buried mutations R384Q and S410L disrupted intramolecular hydrogen bonds within the Kelch domain and affected the Kelch-AM interaction indirectly. No significant effect of buried mutation A340V or A494T on the AM degradation or Kelch-AM interaction was observed, implying these mutations may disrupt mechanisms other than Kelch-AM interaction.