Thermodynamic profiles for cotranslational trigger factor substrate recognition

Abstract

Molecular chaperones are central to the maintenance of proteostasis in living cells. A key member of this protein family is trigger factor (TF), which acts throughout the protein life cycle and has a ubiquitous role as the first chaperone encountered by proteins during synthesis. However, our understanding of how TF achieves favorable interactions with such a diverse substrate base remains limited. Here, we use microfluidics to reveal the thermodynamic determinants of this process. We find that TF binding to empty 70S ribosomes is enthalpy-driven, with micromolar affinity, while nanomolar affinity is achieved through a favorable entropic contribution for both intrinsically disordered and folding-competent nascent chains. These findings suggest a general mechanism for cotranslational TF function, which relies on occupation of the exposed TF-substrate binding groove rather than specific complementarity between chaperone and nascent chain. These insights add to our wider understanding of how proteins can achieve broad substrate specificity.

Fig. 1.. Microfluidic analysis of TF-substrate interactions.

(A) Network of TF interactions includes binding to isolated proteins, empty ribosomes, RNCs, and dimerization (3, 29). (B) Microfluidic diffusional sizing enables the hydrodynamic radius of biomolecules to be determined in free solution. The microfluidic chip is used in conjunction with a temperature-controlled stage to characterize the thermodynamics of protein interactions. (C) Fluorescence image of 200 nM Alexa Fluor 488–labeled TF in the measurement region of the diffusional sizing chip. Bottom: the corresponding fluorescence profiles in blue, with a fit to the data in orange to obtain D and R_H. A.U., arbitrary units. (D) Analysis of multiple components in a mixture 200 nM TF (left) and the intrinsic fluorescence from 4 μM luciferase RNC (right) (40). Binding to the RNC is measured through the increase in TF R_H.

Fig. 2.. Temperature-dependent TF interactions with ribosome constructs.

(A) Schematic of the NC constructs used in this study, comprising the SecM stall sequence, protein of interest, and a His₆ tag for purification. The estimated range that can be contacted by the chaperone is shaded in green (residues 1 to 118 outside the exit tunnel) (19, 36). The R_H of 200 nM TF as a function of RNC concentration and temperature (10° to 27°C) for (B) αsyn, (C) αsyn(luc87–100), and (D) firefly luciferase. The estimated complex sizes from the intrinsic RNC fluorescence (purple dashed line) and R_H for TF (green dashed line) are shown. (E) TF binding to the empty 70S ribosome at 22° to 37°C.

Fig. 3.. K_{d app} from fits to thermodynamic parameters.

(A) K_{d app} for global fits of ΔH and ΔS to the binding curves in Fig. 2 (B to E). (B) TF and ribosome distribution in slow and (C) fast growing cells based on the K_d values measured here at 22°C (K_d = 1.5 μM for TF dimerization; average K_{d app} = 482 nM for RNC binding; K_d = 2.7 μM for binding to empty ribosomes) (, –47). The total TF and ribosome concentrations used were 50 and 30 μM, respectively (46, 47). The equilibria would be shifted by TF binding to isolated proteins.

Fig. 4.. A favorable ΔS drives TF-NC interactions.

(A) The thermodynamic profiles for TF binding to three diverse RNC substrates show that binding is favored by both ΔH and ΔS contributions, whereas TF binding to empty ribosomes is enthalpy-driven. (B) Summary schematic showing the thermodynamic driving forces and equilibrium parameters measured in this study in the context of TF function. The dimer conformation acts as a reservoir, which can release TF to compensate for a reduction in the monomer concentration. TF occupies a fraction of empty ribosomes due to the high total concentrations of TF and 70S (50 and 30 μM) (46, 47). TF undergoes a conformational change upon binding to the ribosome (indicated by the yellow surface), exposing hydrophobic patches and priming the chaperone for NC interactions (3, 42, 43). The similar thermodynamic profiles for the TF-RNC interactions suggest that TF uses a general strategy when associating with NCs at the ribosome surface, leading to similar K_d values. In contrast, when TF remains associated with an elongating NC and leaves the ribosome surface during protein synthesis, a report has found the duration of the complex to be substrate-dependent (3).