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

Abstract

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.

Engineering the kinetic stability of a β-trefoil protein by tuning its topological complexity

“…The hisactophilin point mutant H90G (HisH90G) was identified in previous equilibrium denaturation experiments to be thermodynamically stabilized compared to wtHis ( MacKenzie et al, 2022 ). Here, HisH90G was used as a pseudo wild type parent protein for the core-swap design, and the H90G point mutation was included in the core-swapped hisactophilin variant (see Results ).…”

“…The nickel affinity column was equilibrated with 50 mM Tris buffer pH 8.0 with 0.1 M NaCl, 1 mM MgCl 2 , and 0.1 mM PMSF. Following loading of filtered lysate, the column was washed with 50 mM sodium phosphate buffer pH 6.3 with 0.1 M NaCl, 20 mM imidazole, and 0.1 mM PMSF ( MacKenzie et al, 2022 ) (Profinity™ IMAC, BioRad Laboratories Inc, CA, United States). Then, wtHis, HisH90G, or csHisH90G was eluted using 50 mM sodium phosphate buffer pH 6.3 with 0.1 M NaCl, 0.25–0.5 M imidazole, and 0.1 mM PMSF.…”

“…3Foil and wtHis core residues were identified as promising targets for modulating LRO and ACO in a residue-swapped wtHis/3Foil hybrid since wtHis core residues make significantly fewer long-range contacts than 3Foil core residues (Figure 2). The hisactophilin point mutant H90G (HisH90G) was identified in previous equilibrium denaturation experiments to be thermodynamically stabilized compared to wtHis (MacKenzie et al, 2022). Here, HisH90G was used as a pseudo wild type parent protein for the core-swap design, and the H90G point mutation was included in the core-swapped hisactophilin variant (see Results).…”

“…In contrast, the natural β-trefoil protein wtHis has markedly fewer long-range contacts, a low unfolding barrier ( Gosavi, 2013 ; Broom et al, 2015b ), and moderate kinetic stability with a typical unfolding half-life of minutes to hours ( Smith et al, 2010 ). Notably, core residues in wtHis form a functional cavity that spans the protein core ( Smith et al, 2010 ; Shental-Bechor et al, 2012 ; MacKenzie et al, 2022 ). This cavity precludes the formation of long-range contacts between core residues and contributes to wtHis’ low LRO and unfolding free energy barrier.…”

Electrostatics in Computational Biophysics and Its Implications for Disease Effects

“…The pH-dependence is also pronounced in allosteric regulation. A recent work reported combined experimental with computation investigations and showed that allostery in pH-switching proteins is guided by coupling throughout the protein, featuring a large network of hydrophobic interactions that work in concert with key electrostatic interactions [ 94 ].…”