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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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Fructose phosphate ester binding to GKRP and 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 F6P and F1P for GKRP-WT (A) and GKRP-P446L (B). (C and D) The effect of increasing amounts of GK-W99R/W167F/W257F (GK-Wfree) on the change in TF for 0.5 μM GKRP-WT in the absence (○) or presence (●) of 500 μM F6P or 500 μM F1P for GKRP-WT (C) and for GKRP-P446L (D) (λexc=295 nm; λem=340 nm).
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Figure 7: Fructose phosphate ester binding to GKRP and 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 F6P and F1P for GKRP-WT (A) and GKRP-P446L (B). (C and D) The effect of increasing amounts of GK-W99R/W167F/W257F (GK-Wfree) on the change in TF for 0.5 μM GKRP-WT in the absence (○) or presence (●) of 500 μM F6P or 500 μM F1P for GKRP-WT (C) and for GKRP-P446L (D) (λexc=295 nm; λem=340 nm).

Mentions: Disassembly of the GK–GKRP complex was studied by TF using F1P, glucose and GKA with a GK/GKRP ratio of 3:1, as this ratio most closely mimics the ratio of these two proteins in the cytosol and provided optimal conditions to observe TF changes (see Figure 6). The concentration-dependency curves for these agents were hyperbolic for both GKRP-WT and GKRP-P446L (Tables 2–4 and Supplementary Figures S10–S12 at http://www.biochemj.org/bj/459/bj4590551add.htm). The glucose-dependency curve was monophasic for GKRP-WT, but biphasic for P446L-GKRP (Supplementary Figure S10). An apparent Kd value of 12 mM is consistent with the lower glucose Kd value of 5 mM for unbound GK-W99R/W167F/W257F under these conditions, and reflects the fact that GK-mediated glucose uptake and disposal by the intact hepatocyte is less efficient than expected based on the observed S0.5 value for purified GK [9]. The observation of two distinct glucose Kd values in the presence of GKRP-P446L (13.6 and 99.5 mM) is striking. This result is perhaps due to the altered ability of GKRP-P446L to bind glucose besides fructose phosphates (Supplementary Figure S7), which would result in increased, but separate, binding constants for the complex (one for GK and the other for GKRP). GKA was also highly effective in dissociating the GK–GKRP complex, with practically equal Kd values for GKRP-WT and GKRP-P446L (1.49 compared with 1.23 μM) (Supplementary Figure S11 and Table 4). F1P disrupted the 3:1 GK–GKRP complex very effectively (Supplementary Figure S12). However, the apparent Kd values for GKRP-WT and GKRP-P446L differed markedly (127 compared with 298 μM), resembling the 1:2 ratio of the ligand's binding constant for unbound GKRP (65 compared with 139 μM) (Figure 7). The lower affinity of F1P for GK-bound GKRP is likely the result of an altered phosphate ester binding site on GKRP when complexed with GK. We did not study the effect of F6P on complex assembly because the ΔTF value was too small (Figures 7C and 7D).


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)

Fructose phosphate ester binding to GKRP and 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 F6P and F1P for GKRP-WT (A) and GKRP-P446L (B). (C and D) The effect of increasing amounts of GK-W99R/W167F/W257F (GK-Wfree) on the change in TF for 0.5 μM GKRP-WT in the absence (○) or presence (●) of 500 μM F6P or 500 μM F1P for GKRP-WT (C) and for GKRP-P446L (D) (λexc=295 nm; λem=340 nm).
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Figure 7: Fructose phosphate ester binding to GKRP and 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 F6P and F1P for GKRP-WT (A) and GKRP-P446L (B). (C and D) The effect of increasing amounts of GK-W99R/W167F/W257F (GK-Wfree) on the change in TF for 0.5 μM GKRP-WT in the absence (○) or presence (●) of 500 μM F6P or 500 μM F1P for GKRP-WT (C) and for GKRP-P446L (D) (λexc=295 nm; λem=340 nm).
Mentions: Disassembly of the GK–GKRP complex was studied by TF using F1P, glucose and GKA with a GK/GKRP ratio of 3:1, as this ratio most closely mimics the ratio of these two proteins in the cytosol and provided optimal conditions to observe TF changes (see Figure 6). The concentration-dependency curves for these agents were hyperbolic for both GKRP-WT and GKRP-P446L (Tables 2–4 and Supplementary Figures S10–S12 at http://www.biochemj.org/bj/459/bj4590551add.htm). The glucose-dependency curve was monophasic for GKRP-WT, but biphasic for P446L-GKRP (Supplementary Figure S10). An apparent Kd value of 12 mM is consistent with the lower glucose Kd value of 5 mM for unbound GK-W99R/W167F/W257F under these conditions, and reflects the fact that GK-mediated glucose uptake and disposal by the intact hepatocyte is less efficient than expected based on the observed S0.5 value for purified GK [9]. The observation of two distinct glucose Kd values in the presence of GKRP-P446L (13.6 and 99.5 mM) is striking. This result is perhaps due to the altered ability of GKRP-P446L to bind glucose besides fructose phosphates (Supplementary Figure S7), which would result in increased, but separate, binding constants for the complex (one for GK and the other for GKRP). GKA was also highly effective in dissociating the GK–GKRP complex, with practically equal Kd values for GKRP-WT and GKRP-P446L (1.49 compared with 1.23 μM) (Supplementary Figure S11 and Table 4). F1P disrupted the 3:1 GK–GKRP complex very effectively (Supplementary Figure S12). However, the apparent Kd values for GKRP-WT and GKRP-P446L differed markedly (127 compared with 298 μM), resembling the 1:2 ratio of the ligand's binding constant for unbound GKRP (65 compared with 139 μM) (Figure 7). The lower affinity of F1P for GK-bound GKRP is likely the result of an altered phosphate ester binding site on GKRP when complexed with GK. We did not study the effect of F6P on complex assembly because the ΔTF value was too small (Figures 7C and 7D).

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