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Functional Adaptations of the Bacterial Chaperone Trigger Factor to Extreme

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Functional Adaptations of the Bacterial Chaperone Trigger Factor to Extreme Amandine Godin, Philipp Schmidpeter*, Franz X. Schmid*, Georges Feller Laboratory of Biochemistry, Centre for Protein Engineering, Institute of Chemistry B6, University of Liège, B-4000 Liège, Belgium *Laboratorium fu r̈ Biochemie, Universita ẗ Bayreuth, D-95440 Bayreuth, Germany Comparative study of three trigger factors E. coli trigger factor ribosome from different thermal origins “head” PPIase A key determinant of protein adaptation to temperature is the acquisition of the final, biologically active conformation aided by chaperones and folding catalysts, trigger factor (TF) which remains almost unexplored. The aim of this work was to identify the functional adaptations that enable trigger factor (TF) to be active in the wide range of biological temperatures. As TF is the GroES “arm1” “arm2” first molecular chaperone interacting with virtually all nascent polypeptides DnaJ synthesized by the bacterial ribosome and also possesses a PPIase activity, it native represents a suitable model for this study. To cover nearly all temperatures encountered by living organisms, we compared three structurally homologous TFs. DnaK GroEL “tail” N-term domain C-term domain (+GrpE, ATP) (+ ATP) Protein Source Estimated environ. T° Proteomic context Ph TF P. haloplanktis TAC125 < 0°C Cold Acclimation Protein “ribosome binding” Ec TF E. coli RR1 37°C Cold Shock Protein native native Ferbitz et al., Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins. Tm TF T. maritima DSM3109 85-90°C undetermined Nature, 2004. 431 (7008): p. 590-6. Hoffmann et al., Structure and function of the molecular chaperone Trigger Factor. BBA, 2010. 1803 (6): p. 650-61 Chaperone activities of TFs Interaction TFs -unfolded protein Foldase activity: effect of the experimental temperature Isothermal Titration Calorimetry Holdase activity Time (min) Time (min) 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Acid-denatured green fluorescent protein refolding assay 0.10 0.05 performed at different temperatures: 0.00 0.00 100 Ph TF 20°C -0.10 Physiological T° (5°C for PhTF, 37°C for EcTF, 50°C for -0.05 -0.20 -0.10 TmTF): consistent with GAPDH reactivation assay -0.30 -0.15 80 µcal/sec 15°C: similar results except for EcTF (no inhibition at high µcal/sec -0.40 -0.20 [TF]) consistent with its role of cold shock protein -0.50 -0.60 -0.25 60 0.00 160 0.00 -2.00 150 Ec TF -4.00 140 -2.00 130 -6.00 Aggregation (%) Aggregation 40 Ec TF+GroELS of injectant Ph TF+GroELS injectant of -8.00 -1 -4.00 Ph TF 15°C 120 Ph TF -1 110 GroELS -10.00 100 -12.00 -6.00 kcal mol 20 No chaperone mol kcal 15°C 15°C 90 -14.00 0.0 0.5 1.0 Ec TF 20°C 80 0.0 0.5 1.0 1.5 Molar Ratio Molar Ratio Tm TF 20°C 70 0 60 Interaction Ec TF-α-casein Interaction Tm TF-α-casein 0,0 0,5 1,0 1,5 2,0 2,5 3,0 50 40 [TF]/[GAPDH] EcTF – H-bonds and VDW interactions with the 30 Effects of TFs on GdmCl-denatured D-glyceraldehyde-3- 20 natively unstructured substrate 10 15°C Tm TF - Dominant hydrophobic effect in α-casein phosphate dehydrogenase aggregation: 0 Time (sec) 130 100 200 300 400 500 600 700 800 binding, confirmed by 8-Anilinonaphthalene-1- Ec TF and Tm TF - Gradual prevention of aggregation at 20°C Tm TF+GroELS 120 TF - No protection against aggregation at 20°C, holdase sulfonic acid titration, up to 4 Tm TF bound to 1 α- Ph 110 GroELS GFP (%) Fluorescence casein, corroborating its main role of holdase activity maintained for 10-20 min at 15°C after cold incubation 100 No chaperone 90 Ph TF - Very low affinity for non-native proteins + GroELS Foldase activity 80 70 PPIase activity 30 60 Ec TF 15°C - GroELS 50 Peptide and protein substrate assays 40 _________________________________________________ 25 30 Tm TF 20 5°C 20 10 0 Time (sec) 100 200 300 400 500 600 700 800 15 Cooperation of TFs with the chaperonin GroEL/ES+ATP: Ph TF GroELS - Improvement of GFP refolding yield 10 Ec TF +GroELS - Competition between the 2 chaperones, GAPDH activity recovery (%) recovery activity GAPDH Ec TF is a more effective foldase 5 Tm TF Ph TF +GroELS - Additive effects, independent catalysis of refolding 0 20 40 60 0 [TF] (nM) 0 1 2 3 4 5 6 7 8 910 Tm TF +GroELS - Folding recovery, cooperative effect [TF]/[GAPDH] highlighting a sequential action of chaperones, Tm TF as Catalytic efficiency: Effects on the reactivation of GdmCl-denatured GAPDH: holdase and GroELS as foldase Ec TF - The most effective Ph TF - Moderate foldase activity Ph TF - Similar to Ec TF at low temperature PPIase Ec TF - Good foldase activity at low [TF], inhibition at high [TF] function not adapted Tm TF - Inhibition Tm TF - Very weak PPIase activity Conclusions Adaptations of the chaperone function In P. haloplanktis , only Ph TF and GroELS are chaperones expressed significantly at low temperature and they catalyze protein folding independently. As cold prevents misfolding and aggregation, Ph TF is a weak foldase in vitro . It probably only retains its basic function of foldase associated with the ribosome. Ec TF possesses a complete chaperone activity, essential at E. coli biological temperature which promotes misfolding and aggregation. As two efficient foldases, Ec TF and GroELS compete for the binding of the substrate. At low temperature and high [ Ec TF], foldase activity persists, which is consistent with its role of cold shock protein. Tm TF acts mainly as holdase, which can be related to the hydrophobicity of its chaperone cavity. Its cooperation with GroELS reveals that Tm TF forms a complex with proteins to protect them from high environmental temperatures that promote aggregation, before the transfer of the substrate to downstream chaperones for folding. Adaptations of the PPIase function Ph TF PPIase function is not specially adapted to cold. However, P. haloplanktis genome possesses 14 PPIases, and Ph TF is largely overexpressed ( ~40 x) at low temperature, which constitutes a peculiar adaptation of the PPIase function. E. coli possesses 8 PPIases and needs a highly active TF because prolyl isomerisation is a rate-limiting step for protein folding at 37°C. Tm TF is a weak PPIase, probably because prolyl isomerisation is not limiting at high temperature, as exemplified by the unique PPIase found in T. maritima genome. .
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