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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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TF spectra for 0.3 μM GKRP in the presence or absence of 5 μM GK at 20°C showing the fluorescence increase (vertical arrows) and blue shift of the spectra upon GK binding for GKRP-WT (A) and GKRP-P446L (B) (λexc=295 nm)GK-Wfree, GK-W99R/W167F/W257F.
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Figure 3: TF spectra for 0.3 μM GKRP in the presence or absence of 5 μM GK at 20°C showing the fluorescence increase (vertical arrows) and blue shift of the spectra upon GK binding for GKRP-WT (A) and GKRP-P446L (B) (λexc=295 nm)GK-Wfree, GK-W99R/W167F/W257F.

Mentions: The high average quantum yield of GKRP TF allowed for accurate measurements at 0.3 μM GKRP-WT and GKRP-P446L. The two proteins showed comparable spectra and quantum yields with the same TF maxima at 332 nm (Figures 2 and 3, and Supplementary Table S4 at http://www.biochemj.org/bj/459/bj4590551add.htm). Saturating amounts of GK (5 μM) increased the TF of GKRP-WT by 29% and GKRP-P446L by 44% (Figure 3 and Table 1). GK addition also caused a comparable blue shift in the TF maxima by 3–5 nm (Figure 3, and Supplementary Figures S5 and S6 at http://www.biochemj.org/bj/459/bj4590551add.htm). These results imply that GKRP undergoes a conformational change during complex assembly, which has been previously observed for the hGK–rGKRP (rat GKRP) complex crystallographically [49] and for the hGK–hGKRP complex by MD [47], but not with xGK (Xenopus GK) and xGKRP by crystallography [47]. Glucose, F6P, F1P and GKA had differential effects on the TF of GKRP-WT and GKRP-P446L. Glucose had no effect on the fluorescence of GKRP-WT, but lowered the TF signal observed for GKRP-P446L, suggesting that this amino acid substitution modifies the sugar phosphate-binding site of GKRP in a manner that specifically affects binding of unphosphorylated hexoses (Supplementary Figures S7 and S8 at http://www.biochemj.org/bj/459/bj4590551add.htm). An increase and decrease in the TF signal for both GKRP-WT and GKRP-P446L was observed in the presence of F6P and F1P respectively. Remarkably, positive as well as negative ΔTF values for GKRP-P446L were twice that of WT-GKRP for both phosphate esters (Table 1). Also noteworthy is the sigmoidal shape of the TF concentration-dependency curve for F6P contrasting with the hyperbolic shape of that for F1P as indicated by the different Hill coefficients. A corollary for these opposite effects of the two fructose-phosphate esters on GKRP TF are crystallographic observations showing that the Lid domain/SIS-2 interface of GKRP is differentially affected by the two ligands [49]. The TF changes encountered in the present study ranged in magnitude from 4 to 44% and therefore allowed for accurate ligand concentration-dependency studies, as well as assessment of the structural stability and refolding capacity of GKRP-WT and GKRP-P446L following denaturation by urea.


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)

TF spectra for 0.3 μM GKRP in the presence or absence of 5 μM GK at 20°C showing the fluorescence increase (vertical arrows) and blue shift of the spectra upon GK binding for GKRP-WT (A) and GKRP-P446L (B) (λexc=295 nm)GK-Wfree, GK-W99R/W167F/W257F.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4109836&req=5

Figure 3: TF spectra for 0.3 μM GKRP in the presence or absence of 5 μM GK at 20°C showing the fluorescence increase (vertical arrows) and blue shift of the spectra upon GK binding for GKRP-WT (A) and GKRP-P446L (B) (λexc=295 nm)GK-Wfree, GK-W99R/W167F/W257F.
Mentions: The high average quantum yield of GKRP TF allowed for accurate measurements at 0.3 μM GKRP-WT and GKRP-P446L. The two proteins showed comparable spectra and quantum yields with the same TF maxima at 332 nm (Figures 2 and 3, and Supplementary Table S4 at http://www.biochemj.org/bj/459/bj4590551add.htm). Saturating amounts of GK (5 μM) increased the TF of GKRP-WT by 29% and GKRP-P446L by 44% (Figure 3 and Table 1). GK addition also caused a comparable blue shift in the TF maxima by 3–5 nm (Figure 3, and Supplementary Figures S5 and S6 at http://www.biochemj.org/bj/459/bj4590551add.htm). These results imply that GKRP undergoes a conformational change during complex assembly, which has been previously observed for the hGK–rGKRP (rat GKRP) complex crystallographically [49] and for the hGK–hGKRP complex by MD [47], but not with xGK (Xenopus GK) and xGKRP by crystallography [47]. Glucose, F6P, F1P and GKA had differential effects on the TF of GKRP-WT and GKRP-P446L. Glucose had no effect on the fluorescence of GKRP-WT, but lowered the TF signal observed for GKRP-P446L, suggesting that this amino acid substitution modifies the sugar phosphate-binding site of GKRP in a manner that specifically affects binding of unphosphorylated hexoses (Supplementary Figures S7 and S8 at http://www.biochemj.org/bj/459/bj4590551add.htm). An increase and decrease in the TF signal for both GKRP-WT and GKRP-P446L was observed in the presence of F6P and F1P respectively. Remarkably, positive as well as negative ΔTF values for GKRP-P446L were twice that of WT-GKRP for both phosphate esters (Table 1). Also noteworthy is the sigmoidal shape of the TF concentration-dependency curve for F6P contrasting with the hyperbolic shape of that for F1P as indicated by the different Hill coefficients. A corollary for these opposite effects of the two fructose-phosphate esters on GKRP TF are crystallographic observations showing that the Lid domain/SIS-2 interface of GKRP is differentially affected by the two ligands [49]. The TF changes encountered in the present study ranged in magnitude from 4 to 44% and therefore allowed for accurate ligand concentration-dependency studies, as well as assessment of the structural stability and refolding capacity of GKRP-WT and GKRP-P446L following denaturation by urea.

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