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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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Structure of GKRP(A) Locations of tryptophan residues in GKRP domains: Trp19 at the N-terminus (6–44; grey), Trp222 in the SIS-1 domain (45–284; red), Trp335, Trp461 and Trp483 in the SIS-2 domain (289–499; cyan), and Trp517 in the Lid (513–606; green). The linker between SIS-1 and the Lid is shown in blue. The P446L point mutation is shown in magenta. F1P bound to one of the two known allosteric sites is shown in yellow. The second allosteric site for GKRPIs, situated between the Lid and the SIS-1–SIS-2 complex [22] is not shown. (B) Magnified view of the fructose phosphate-binding site to indicate the amino acids involved in F1P and F6P binding: Asn512, Lys514, Leu515 and Arg518 in the Lid (green), Glu348 and His351 in the SIS-2 domain (cyan), and Gly107, Thr109, Ser110, Glu153, Leu178, Ser179, Val180, Gly181, Leu182, Ser183, Gly256, Ser258 and Arg259 in the SIS-1 domain (red) (PDB code 4BB9). (C) The widely dispersed location of functional variants associated with triacylglycerol levels in GKRP. (D) The close proximity of the common mutant P446L to Asp413 [47] and Gln443 [49] forming a critical salt bridge to Arg186 of GK is illustrated.
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Figure 1: Structure of GKRP(A) Locations of tryptophan residues in GKRP domains: Trp19 at the N-terminus (6–44; grey), Trp222 in the SIS-1 domain (45–284; red), Trp335, Trp461 and Trp483 in the SIS-2 domain (289–499; cyan), and Trp517 in the Lid (513–606; green). The linker between SIS-1 and the Lid is shown in blue. The P446L point mutation is shown in magenta. F1P bound to one of the two known allosteric sites is shown in yellow. The second allosteric site for GKRPIs, situated between the Lid and the SIS-1–SIS-2 complex [22] is not shown. (B) Magnified view of the fructose phosphate-binding site to indicate the amino acids involved in F1P and F6P binding: Asn512, Lys514, Leu515 and Arg518 in the Lid (green), Glu348 and His351 in the SIS-2 domain (cyan), and Gly107, Thr109, Ser110, Glu153, Leu178, Ser179, Val180, Gly181, Leu182, Ser183, Gly256, Ser258 and Arg259 in the SIS-1 domain (red) (PDB code 4BB9). (C) The widely dispersed location of functional variants associated with triacylglycerol levels in GKRP. (D) The close proximity of the common mutant P446L to Asp413 [47] and Gln443 [49] forming a critical salt bridge to Arg186 of GK is illustrated.

Mentions: The critical role of GK and its regulation by GKRP are illustrated by the impact that genetic variation in the genes that encode these proteins have on normal glucose and triacylglycerol metabolism in humans [25–32]. Over 600 naturally occurring mutations in the GCK (glucokinase) gene have been described, which have a wide range of functional consequences including inactivation or activation of catalytic capacity, structural and functional protein instability and decreased responsiveness to GKRP [29]. Heterozygous inactivating GCK mutations cause an autosomal dominantly inherited condition characterized by mild fasting hyperglycaemia, whereas inheritance of two defective GCK alleles results in the more severe phenotype of permanent neonatal diabetes [25,27]. In contrast, heterozygous activating mutations cause the opposite phenotype of hypoglycaemia, whereas homozygous cases have not been found because they are probably lethal [26]. A common non-synonymous variant (P466L) in GCKR, the gene that encodes GKRP, present in the healthy population has been reproducibly associated with fasting serum triacylglycerol and glucose levels [33]. Functional characterization of this variant protein has demonstrated that it is a less effective inhibitor of GK and results in reduced nuclear storage of GK [28,31]. Moreover, rare variants in GCKR have been shown to be associated with serum triacylglycerol and cholesterol levels in healthy adults and to be overrepresented in individuals with hypertriglyceridaemia (Figure 1C) [30,32].


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)

Structure of GKRP(A) Locations of tryptophan residues in GKRP domains: Trp19 at the N-terminus (6–44; grey), Trp222 in the SIS-1 domain (45–284; red), Trp335, Trp461 and Trp483 in the SIS-2 domain (289–499; cyan), and Trp517 in the Lid (513–606; green). The linker between SIS-1 and the Lid is shown in blue. The P446L point mutation is shown in magenta. F1P bound to one of the two known allosteric sites is shown in yellow. The second allosteric site for GKRPIs, situated between the Lid and the SIS-1–SIS-2 complex [22] is not shown. (B) Magnified view of the fructose phosphate-binding site to indicate the amino acids involved in F1P and F6P binding: Asn512, Lys514, Leu515 and Arg518 in the Lid (green), Glu348 and His351 in the SIS-2 domain (cyan), and Gly107, Thr109, Ser110, Glu153, Leu178, Ser179, Val180, Gly181, Leu182, Ser183, Gly256, Ser258 and Arg259 in the SIS-1 domain (red) (PDB code 4BB9). (C) The widely dispersed location of functional variants associated with triacylglycerol levels in GKRP. (D) The close proximity of the common mutant P446L to Asp413 [47] and Gln443 [49] forming a critical salt bridge to Arg186 of GK is illustrated.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4109836&req=5

Figure 1: Structure of GKRP(A) Locations of tryptophan residues in GKRP domains: Trp19 at the N-terminus (6–44; grey), Trp222 in the SIS-1 domain (45–284; red), Trp335, Trp461 and Trp483 in the SIS-2 domain (289–499; cyan), and Trp517 in the Lid (513–606; green). The linker between SIS-1 and the Lid is shown in blue. The P446L point mutation is shown in magenta. F1P bound to one of the two known allosteric sites is shown in yellow. The second allosteric site for GKRPIs, situated between the Lid and the SIS-1–SIS-2 complex [22] is not shown. (B) Magnified view of the fructose phosphate-binding site to indicate the amino acids involved in F1P and F6P binding: Asn512, Lys514, Leu515 and Arg518 in the Lid (green), Glu348 and His351 in the SIS-2 domain (cyan), and Gly107, Thr109, Ser110, Glu153, Leu178, Ser179, Val180, Gly181, Leu182, Ser183, Gly256, Ser258 and Arg259 in the SIS-1 domain (red) (PDB code 4BB9). (C) The widely dispersed location of functional variants associated with triacylglycerol levels in GKRP. (D) The close proximity of the common mutant P446L to Asp413 [47] and Gln443 [49] forming a critical salt bridge to Arg186 of GK is illustrated.
Mentions: The critical role of GK and its regulation by GKRP are illustrated by the impact that genetic variation in the genes that encode these proteins have on normal glucose and triacylglycerol metabolism in humans [25–32]. Over 600 naturally occurring mutations in the GCK (glucokinase) gene have been described, which have a wide range of functional consequences including inactivation or activation of catalytic capacity, structural and functional protein instability and decreased responsiveness to GKRP [29]. Heterozygous inactivating GCK mutations cause an autosomal dominantly inherited condition characterized by mild fasting hyperglycaemia, whereas inheritance of two defective GCK alleles results in the more severe phenotype of permanent neonatal diabetes [25,27]. In contrast, heterozygous activating mutations cause the opposite phenotype of hypoglycaemia, whereas homozygous cases have not been found because they are probably lethal [26]. A common non-synonymous variant (P466L) in GCKR, the gene that encodes GKRP, present in the healthy population has been reproducibly associated with fasting serum triacylglycerol and glucose levels [33]. Functional characterization of this variant protein has demonstrated that it is a less effective inhibitor of GK and results in reduced nuclear storage of GK [28,31]. Moreover, rare variants in GCKR have been shown to be associated with serum triacylglycerol and cholesterol levels in healthy adults and to be overrepresented in individuals with hypertriglyceridaemia (Figure 1C) [30,32].

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