Distinct Stabilities of the Structurally Homologous Heptameric Co-Chaperonins GroES and gp31

Abstract

The GroES heptamer is the molecular co-chaperonin that partners with the tetradecamer chaperonin GroEL, which assists in the folding of various nonnative polypeptide chains in Escherichia coli. Gp31 is a structural and functional analogue of GroES encoded by the bacteriophage T4, becoming highly expressed in T4-infected E. coli, taking over the role of GroES, favoring the folding of bacteriophage proteins. Despite being slightly larger, gp31 is quite homologous to GroES in terms of its tertiary and quaternary structure, as well as in its function and mode of interaction with the chaperonin GroEL. Here, we performed a side-by-side comparison of GroES and gp31 heptamer complexes by (ion mobility) tandem mass spectrometry. Surprisingly, we observed quite distinct fragmentation mechanisms for the GroES and gp31 heptamers, whereby GroES displays a unique and unusual bimodal charge distribution in its released monomers. Not only the gas-phase dissociation but also the gas-phase unfolding of GroES and gp31 were found to be very distinct. We rationalize these observations with the similar discrepancies we observed in the thermal unfolding characteristics and surface contacts within GroES and gp31 in the solution. From our data, we propose a model that explains the observed simultaneous dissociation pathways of GroES and the differences between GroES and gp31 gas-phase dissociation and unfolding. We conclude that, although GroES and gp31 exhibit high homology in tertiary and quaternary structure, they are quite distinct in their solution and gas-phase (un)folding characteristics and stability. Graphical Abstract.

Temperature Regulates Stability, Ligand Binding (Mg2+ and ATP) and Stoichiometry of GroEL/GroES Complexes

“…48 In fact, R47 and K55 residues form salt bridges with neighboring residues in only two subunits and these stabilizing interactions are missing in the other five subunits. This structural heterogeneity 35 might also explain the observation of monomer and dimer ions in native mass spectrum of GroES7 (Figure 3) and previous studies. 32,35 The energy requirement for disassembly of the GroES complex can be compensated by the binding energy to GroEL14.…”

“…This structural heterogeneity 35 might also explain the observation of monomer and dimer ions in native mass spectrum of GroES7 (Figure 3) and previous studies. 32,35 The energy requirement for disassembly of the GroES complex can be compensated by the binding energy to GroEL14. Previous studies have shown that both ATP and GroES binding are necessary for apical domain movement and for transition from substrate-binding mode (hydrophobic surface exposed) to the folding-active state (hydrophilic chamber).…”

“…Duckworth and coworkers assigned the fragment ions to gas phase collisional activation reactions; 32 however, based on a series of studies we are confident that under these experimental conditions these product ions results from reactions that occur in the solution. 25,33 The midpoint in the melting curve (Figure 2E) is used to estimate the melting point (Tm) of ~ 48.5 °C, which is much lower than that reported by Boudker et al 34 of ~74 o C as well as the Tm reported by Dyachenko et al 35 It is important to note, however, that their DCp vs. T curve revealed evidence for a low temperature endotherm at ~40 o C, which agrees well with our Tm for GroES7. Moreover, the DSC and vT-ESI results reveal strong evidence for T-dependent instability, and the vT-ESI results suggest that this transition may serve as the early steps leading to disassembly of GroES7 complex.…”

Temperature Regulates Stability, Ligand Binding (Mg2+ and ATP) and Stoichiometry of GroEL/GroES Complexes

“…26,34 The midpoint in the melting curve (Figure 2E) is used to estimate the melting point (Tm) of ~ 48.5 °C, (see experimental section) which is significantly lower than the Tm of ~74 °C reported by Boudker et al 35 It is important to note, however, that their DCp vs. T curve revealed evidence for a low temperature endotherm at ~40 o C, which agrees well with our Tm for GroES7. Geels et al 36 and Dyachenko et al 37 have reported Tm data for GroES using mass spectrometry, and they report similar Tm values (~70 o C), but both of these studies were performed using ammonium acetate buffer. Moreover, the DSC and vT-ESI results reveal strong evidence for T-dependent instability, and the vT-ESI results suggest that this transition may serve as the early steps leading to disassembly of GroES7 complex.…”

“…This structural heterogeneity 37 might also explain the observation of monomer and dimer ions in native mass spectrum of GroES7 (Figure 3) and previous studies. 33,37 The energy requirement for disassembly of the GroES complex can be compensated by the binding energy to GroEL14. Previous studies have shown that both ATP and GroES binding are necessary for apical domain movement and for transition from substrate-binding mode (hydrophobic surface exposed) to the folding-active state (hydrophilic chamber).…”