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Analysis of the co-operative interaction between the allosterically regulated proteins GK and GKRP using tryptophan fluorescence.

Zelent B, Raimondo A, Barrett A, Buettger CW, Chen P, Gloyn AL, Matschinsky FM - Biochem. J. (2014)

Bottom Line: Titration of GKRP-WT by GK resulted in a sigmoidal increase in TF, suggesting co-operative PPIs (protein-protein interactions) perhaps due to the hysteretic nature of GK.The affinity of GK for GKRP was decreased and binding co-operativity increased by glucose, fructose 1-phosphate and GKA, reflecting disruption of the GK-GKRP complex.The results of the present TF-based biophysical analysis of PPIs between GK and GKRP suggest that hepatic glucose metabolism is regulated by a metabolite-sensitive drug-responsive co-operative molecular switch, involving complex formation between these two allosterically regulated proteins.

View Article: PubMed Central - PubMed

Affiliation: *Department of Biochemistry and Biophysics and Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, U.S.A.

ABSTRACT
Hepatic glucose phosphorylation by GK (glucokinase) is regulated by GKRP (GK regulatory protein). GKRP forms a cytosolic complex with GK followed by nuclear import and storage, leading to inhibition of GK activity. This process is initiated by low glucose, but reversed nutritionally by high glucose and fructose or pharmacologically by GKAs (GK activators) and GKRPIs (GKRP inhibitors). To study the regulation of this process by glucose, fructose-phosphate esters and a GKA, we measured the TF (tryptophan fluorescence) of human WT (wild-type) and GKRP-P446L (a mutation associated with high serum triacylglycerol) in the presence of non-fluorescent GK with its tryptophan residues mutated. Titration of GKRP-WT by GK resulted in a sigmoidal increase in TF, suggesting co-operative PPIs (protein-protein interactions) perhaps due to the hysteretic nature of GK. The affinity of GK for GKRP was decreased and binding co-operativity increased by glucose, fructose 1-phosphate and GKA, reflecting disruption of the GK-GKRP complex. Similar studies with GKRP-P446L showed significantly different results compared with GKRP-WT, suggesting impairment of complex formation and nuclear storage. The results of the present TF-based biophysical analysis of PPIs between GK and GKRP suggest that hepatic glucose metabolism is regulated by a metabolite-sensitive drug-responsive co-operative molecular switch, involving complex formation between these two allosterically regulated proteins.

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Co-operativity of GK–GKRP complex assembly(A and B) Relative change in TF for 0.3 μM GKRP in the presence of increasing amounts of GK-W99R/W167F/W257F (GK-Wfree) on the addition of 500 μM F6P (○) or 500 μM F6P and 100 mM D-glucose (●) for GKRP-WT (A) and GKRP-P446L (B). (C and D) The effect of increasing amounts of GK-W99R/W167F/W257F on the change in TF for 0.3 μM GKRP in the presence of 500 μM F1P (○) and 500 μM F1P and 100 mM D-glucose (●) can be seen for GKRP-WT (C) and GKRP-P446L (D) (λexc=295 nm; λem=340 nm).
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Figure 8: Co-operativity of GK–GKRP complex assembly(A and B) Relative change in TF for 0.3 μM GKRP in the presence of increasing amounts of GK-W99R/W167F/W257F (GK-Wfree) on the addition of 500 μM F6P (○) or 500 μM F6P and 100 mM D-glucose (●) for GKRP-WT (A) and GKRP-P446L (B). (C and D) The effect of increasing amounts of GK-W99R/W167F/W257F on the change in TF for 0.3 μM GKRP in the presence of 500 μM F1P (○) and 500 μM F1P and 100 mM D-glucose (●) can be seen for GKRP-WT (C) and GKRP-P446L (D) (λexc=295 nm; λem=340 nm).

Mentions: In order to mimic the concentrations of GK and GKRP likely to occur in the cytosol, assembly of these two proteins was studied at a constant GKRP concentration of 0.3 μM with increasing amounts of GK ranging from 0.5 to 5 μM. Sigmoidal TF dose–response curves were observed with increasing amounts of GK-W99R/W167F/W257F, as indicated by indistinguishable Hill coefficients of 1.74 and 1.86 in the presence of GKRP-WT and GKRP-P446L respectively (Figures 5A and 5B, and Table 1). Saturating levels of F6P had no significant effect on the binding of GKRP-WT, but increased the affinity of GKRP-P446L for GK and lowered the degree of co-operativity of the PPI (H=1.31 compared with 1.86). This conclusion was supported by curve fits using simple saturation or sigmoidal functions (Figure 5 and Supplementary Table S6 at http://www.biochemj.org/bj/459/bj4590551add.htm). However, on average, the S0.5 value of GK for complex formation of approximately 1.2 μM in the presence of GKRP-WT was 35% lower than that obtained with GKRP-P446L (Figure 5). Saturating concentrations of glucose, F1P or GKA, either alone or in combination, markedly increased the sigmoidicity of these dose–response curves (Figures 6–8 and Table 1), indicating enhanced co-operativity. In the case of GKRP-WT the effect was most pronounced in the presence of high glucose and GKA, resulting in a 4-fold increase in the S0.5 value for GK and a 1.84-fold increase in the Hill coefficient (Figure 6C and Table 1). GKRP-P446L was less responsive to the combined action of saturating glucose and GKA, since the GK S0.5 value increased by only 1.73-fold and the Hill coefficient by 1.44-fold (Figure 6D and Table 1). Furthermore, the maximal ΔTF value of GKRP-P446L was generally constant at approximately 0.41–0.44 under all conditions tested, compared with a wider range of 0.29–0.42 for GKRP-WT, depending on the assay conditions. When titration of GKRP-WT with GK was performed in the presence of glucose, F1P or GKA, the TF dose–response curves were shifted to the right and corresponded to changes in the biophysical constants of these ligands largely consistent with known effects (Figures 6–8 and Table 1). The strikingly high efficacy of GKA alone (known not to activate unmodified GK in the absence of glucose [3]) is readily explained by the W99R substitution in GK, the effect of which would be to facilitate access of the GKA to its binding site ([50,51] and Supplementary Online Data). These qualitative and quantitative differences between GKRP-WT and GKRP-P446L are strikingly displayed in Figure 6. The co-operative nature of the GK–GKRP interaction and the extent to which this interaction is modified by physiological ligands and GKAs is further illustrated in Hill plots (Supplementary Figure S9 at http://www.biochemj.org/bj/459/bj4590551add.htm). Since the TF spectra of proteins are influenced by their basic structure and conformational rearrangements upon ligand binding, these results strongly suggest that the P446L substitution on GKRP results in an overall change in structure. We propose that the association of GKRP-P446L with multiple metabolic traits, including reduced T2D risk, can be attributed to its altered overall structure, which acts to hinder GK binding and nuclear import. This hypothesis is supported by the close approximation of Pro446 to Asp413 (as proposed by Choi et al. [47]) or Gln443 (as proposed by Beck et al. [49]), which appear to form a critical salt bridge with Arg186 of GK (Figures 1C and 1D).


Analysis of the co-operative interaction between the allosterically regulated proteins GK and GKRP using tryptophan fluorescence.

Zelent B, Raimondo A, Barrett A, Buettger CW, Chen P, Gloyn AL, Matschinsky FM - Biochem. J. (2014)

Co-operativity of GK–GKRP complex assembly(A and B) Relative change in TF for 0.3 μM GKRP in the presence of increasing amounts of GK-W99R/W167F/W257F (GK-Wfree) on the addition of 500 μM F6P (○) or 500 μM F6P and 100 mM D-glucose (●) for GKRP-WT (A) and GKRP-P446L (B). (C and D) The effect of increasing amounts of GK-W99R/W167F/W257F on the change in TF for 0.3 μM GKRP in the presence of 500 μM F1P (○) and 500 μM F1P and 100 mM D-glucose (●) can be seen for GKRP-WT (C) and GKRP-P446L (D) (λexc=295 nm; λem=340 nm).
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Figure 8: Co-operativity of GK–GKRP complex assembly(A and B) Relative change in TF for 0.3 μM GKRP in the presence of increasing amounts of GK-W99R/W167F/W257F (GK-Wfree) on the addition of 500 μM F6P (○) or 500 μM F6P and 100 mM D-glucose (●) for GKRP-WT (A) and GKRP-P446L (B). (C and D) The effect of increasing amounts of GK-W99R/W167F/W257F on the change in TF for 0.3 μM GKRP in the presence of 500 μM F1P (○) and 500 μM F1P and 100 mM D-glucose (●) can be seen for GKRP-WT (C) and GKRP-P446L (D) (λexc=295 nm; λem=340 nm).
Mentions: In order to mimic the concentrations of GK and GKRP likely to occur in the cytosol, assembly of these two proteins was studied at a constant GKRP concentration of 0.3 μM with increasing amounts of GK ranging from 0.5 to 5 μM. Sigmoidal TF dose–response curves were observed with increasing amounts of GK-W99R/W167F/W257F, as indicated by indistinguishable Hill coefficients of 1.74 and 1.86 in the presence of GKRP-WT and GKRP-P446L respectively (Figures 5A and 5B, and Table 1). Saturating levels of F6P had no significant effect on the binding of GKRP-WT, but increased the affinity of GKRP-P446L for GK and lowered the degree of co-operativity of the PPI (H=1.31 compared with 1.86). This conclusion was supported by curve fits using simple saturation or sigmoidal functions (Figure 5 and Supplementary Table S6 at http://www.biochemj.org/bj/459/bj4590551add.htm). However, on average, the S0.5 value of GK for complex formation of approximately 1.2 μM in the presence of GKRP-WT was 35% lower than that obtained with GKRP-P446L (Figure 5). Saturating concentrations of glucose, F1P or GKA, either alone or in combination, markedly increased the sigmoidicity of these dose–response curves (Figures 6–8 and Table 1), indicating enhanced co-operativity. In the case of GKRP-WT the effect was most pronounced in the presence of high glucose and GKA, resulting in a 4-fold increase in the S0.5 value for GK and a 1.84-fold increase in the Hill coefficient (Figure 6C and Table 1). GKRP-P446L was less responsive to the combined action of saturating glucose and GKA, since the GK S0.5 value increased by only 1.73-fold and the Hill coefficient by 1.44-fold (Figure 6D and Table 1). Furthermore, the maximal ΔTF value of GKRP-P446L was generally constant at approximately 0.41–0.44 under all conditions tested, compared with a wider range of 0.29–0.42 for GKRP-WT, depending on the assay conditions. When titration of GKRP-WT with GK was performed in the presence of glucose, F1P or GKA, the TF dose–response curves were shifted to the right and corresponded to changes in the biophysical constants of these ligands largely consistent with known effects (Figures 6–8 and Table 1). The strikingly high efficacy of GKA alone (known not to activate unmodified GK in the absence of glucose [3]) is readily explained by the W99R substitution in GK, the effect of which would be to facilitate access of the GKA to its binding site ([50,51] and Supplementary Online Data). These qualitative and quantitative differences between GKRP-WT and GKRP-P446L are strikingly displayed in Figure 6. The co-operative nature of the GK–GKRP interaction and the extent to which this interaction is modified by physiological ligands and GKAs is further illustrated in Hill plots (Supplementary Figure S9 at http://www.biochemj.org/bj/459/bj4590551add.htm). Since the TF spectra of proteins are influenced by their basic structure and conformational rearrangements upon ligand binding, these results strongly suggest that the P446L substitution on GKRP results in an overall change in structure. We propose that the association of GKRP-P446L with multiple metabolic traits, including reduced T2D risk, can be attributed to its altered overall structure, which acts to hinder GK binding and nuclear import. This hypothesis is supported by the close approximation of Pro446 to Asp413 (as proposed by Choi et al. [47]) or Gln443 (as proposed by Beck et al. [49]), which appear to form a critical salt bridge with Arg186 of GK (Figures 1C and 1D).

Bottom Line: Titration of GKRP-WT by GK resulted in a sigmoidal increase in TF, suggesting co-operative PPIs (protein-protein interactions) perhaps due to the hysteretic nature of GK.The affinity of GK for GKRP was decreased and binding co-operativity increased by glucose, fructose 1-phosphate and GKA, reflecting disruption of the GK-GKRP complex.The results of the present TF-based biophysical analysis of PPIs between GK and GKRP suggest that hepatic glucose metabolism is regulated by a metabolite-sensitive drug-responsive co-operative molecular switch, involving complex formation between these two allosterically regulated proteins.

View Article: PubMed Central - PubMed

Affiliation: *Department of Biochemistry and Biophysics and Institute for Diabetes, Obesity and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, U.S.A.

ABSTRACT
Hepatic glucose phosphorylation by GK (glucokinase) is regulated by GKRP (GK regulatory protein). GKRP forms a cytosolic complex with GK followed by nuclear import and storage, leading to inhibition of GK activity. This process is initiated by low glucose, but reversed nutritionally by high glucose and fructose or pharmacologically by GKAs (GK activators) and GKRPIs (GKRP inhibitors). To study the regulation of this process by glucose, fructose-phosphate esters and a GKA, we measured the TF (tryptophan fluorescence) of human WT (wild-type) and GKRP-P446L (a mutation associated with high serum triacylglycerol) in the presence of non-fluorescent GK with its tryptophan residues mutated. Titration of GKRP-WT by GK resulted in a sigmoidal increase in TF, suggesting co-operative PPIs (protein-protein interactions) perhaps due to the hysteretic nature of GK. The affinity of GK for GKRP was decreased and binding co-operativity increased by glucose, fructose 1-phosphate and GKA, reflecting disruption of the GK-GKRP complex. Similar studies with GKRP-P446L showed significantly different results compared with GKRP-WT, suggesting impairment of complex formation and nuclear storage. The results of the present TF-based biophysical analysis of PPIs between GK and GKRP suggest that hepatic glucose metabolism is regulated by a metabolite-sensitive drug-responsive co-operative molecular switch, involving complex formation between these two allosterically regulated proteins.

Show MeSH
Related in: MedlinePlus