A fine balance of hydrophobic-electrostatic communication pathways in a pH-switching protein

Proc Natl Acad Sci U S A. 2022 Jun 28;119(26):e2119686119. doi: 10.1073/pnas.2119686119. Epub 2022 Jun 22.


Allostery is the phenomenon of coupling between distal binding sites in a protein. Such coupling is at the crux of protein function and regulation in a myriad of scenarios, yet determining the molecular mechanisms of coupling networks in proteins remains a major challenge. Here, we report mechanisms governing pH-dependent myristoyl switching in monomeric hisactophilin, whereby the myristoyl moves between a sequestered state, i.e., buried within the core of the protein, to an accessible state, in which the myristoyl has increased accessibility for membrane binding. Measurements of the pH and temperature dependence of amide chemical shifts reveal protein local structural stability and conformational heterogeneity that accompany switching. An analysis of these measurements using a thermodynamic cycle framework shows that myristoyl-proton coupling at the single-residue level exists in a fine balance and extends throughout the protein. Strikingly, small changes in the stereochemistry or size of core and surface hydrophobic residues by point mutations readily break, restore, or tune myristoyl switch energetics. Synthesizing the experimental results with those of molecular dynamics simulations illuminates atomistic details of coupling throughout the protein, featuring a large network of hydrophobic interactions that work in concert with key electrostatic interactions. The simulations were critical for discerning which of the many ionizable residues in hisactophilin are important for switching and identifying the contributions of nonnative interactions in switching. The strategy of using temperature-dependent NMR presented here offers a powerful, widely applicable way to elucidate the molecular mechanisms of allostery in proteins at high resolution.

Keywords: NMR temperature dependence; allostery; myristoylation; pH dependence; switch.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Genes, Switch
  • Hydrogen-Ion Concentration
  • Hydrophobic and Hydrophilic Interactions
  • Microfilament Proteins* / chemistry
  • Microfilament Proteins* / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Protozoan Proteins* / chemistry
  • Protozoan Proteins* / metabolism
  • Signal Transduction
  • Static Electricity


  • Microfilament Proteins
  • Protozoan Proteins
  • hisactophilin protein, Protozoan