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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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Effects of glucose, fructose-phosphate esters, GKAs and GKRPIs on the GK–GKRP complexThe Figure summarizes the structural information on the GK–GKRP complex under the influence of glucose and known allosteric ligands of the two participating proteins ([21,22,38,47–49] and the present study). (A and B) The results of mutational analyses [21]. (C and D) The combined results of mutational [21] and crystallographic [22,47–49] analyses of these interactions [note that the orientation differs from that of (A) and (B) (an approximately 90° rotation towards the face of the page) in order to emphasize that the site of GK–GKRP interaction is distinct from that of GK–GKA]. All structural information is projected on to the GK structures published by Kamata et al. [38], and the tryptophan residues are depicted as black stick structures for improved orientation. (A) The allosteric modifier region of GK with two distinct binding sites for GKRP and GKA in the open conformation. Amino acids that affect interaction with GKRP are shown as space-filled structures in cyan. Arg186 of GK is presented in magenta [47,49] to show its close vicinity to other amino acids in the small lobe of GK that have been implicated in GKRP binding. GKA-contacting amino acids are shown as red stick models. Activation of GK by glucose (green) combined with a GKA (yellow) profoundly alters the proposed GKRP-binding site by affecting groups of amino acids involved in binding including the critical Trp99 for binding (B). (C and D) The allosteric binding site for GKRP is indicated in grey space-filled structures for eight of the 15 identified contact amino acids [47,49]. Amino acids immediately surrounding the GKRP contact site are those identified by mutational analysis to influence GKRP binding to GK and are presented as space-filled structures in cyan (see also A and B) [21]. The GK-binding site for GKRP is drawn schematically as a small circular patch in (C) to delineate where the two proteins interact in the complex and a larger patch in (D) to delineate the conformational change that dislodges the inhibitor when glucose and/or GKA are bound to GK.
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Figure 9: Effects of glucose, fructose-phosphate esters, GKAs and GKRPIs on the GK–GKRP complexThe Figure summarizes the structural information on the GK–GKRP complex under the influence of glucose and known allosteric ligands of the two participating proteins ([21,22,38,47–49] and the present study). (A and B) The results of mutational analyses [21]. (C and D) The combined results of mutational [21] and crystallographic [22,47–49] analyses of these interactions [note that the orientation differs from that of (A) and (B) (an approximately 90° rotation towards the face of the page) in order to emphasize that the site of GK–GKRP interaction is distinct from that of GK–GKA]. All structural information is projected on to the GK structures published by Kamata et al. [38], and the tryptophan residues are depicted as black stick structures for improved orientation. (A) The allosteric modifier region of GK with two distinct binding sites for GKRP and GKA in the open conformation. Amino acids that affect interaction with GKRP are shown as space-filled structures in cyan. Arg186 of GK is presented in magenta [47,49] to show its close vicinity to other amino acids in the small lobe of GK that have been implicated in GKRP binding. GKA-contacting amino acids are shown as red stick models. Activation of GK by glucose (green) combined with a GKA (yellow) profoundly alters the proposed GKRP-binding site by affecting groups of amino acids involved in binding including the critical Trp99 for binding (B). (C and D) The allosteric binding site for GKRP is indicated in grey space-filled structures for eight of the 15 identified contact amino acids [47,49]. Amino acids immediately surrounding the GKRP contact site are those identified by mutational analysis to influence GKRP binding to GK and are presented as space-filled structures in cyan (see also A and B) [21]. The GK-binding site for GKRP is drawn schematically as a small circular patch in (C) to delineate where the two proteins interact in the complex and a larger patch in (D) to delineate the conformational change that dislodges the inhibitor when glucose and/or GKA are bound to GK.

Mentions: Kinetic studies of GKRP-P446L have demonstrated a reduced ability of this variant protein to inhibit GK as a consequence of reduced responsiveness to F6P, and cellular studies have shown impaired GK binding and nuclear storage [28,30,31]. Subsequent alterations in GK localization and activity may explain the association of this GKRP variant with metabolic traits such as fasting glucose and triacylglycerol levels [33]. The present study expands the characterization of GKRP-P446L. First, we show that structural instability or impaired protein folding are unlikely to be major contributors to GKRP-P446L dysfunction (Figure 4). Secondly, the increase in the GK S0.5 value for binding to GKRP-P446L, the reduced affinity of GK for GKRP-P446L in the presence of high glucose and GKA, the abnormal constancy of TF maxima under various conditions, and, finally, the reduced co-operativity with GK, suggest a narrower conformational range for this variant protein (Figures 5–8 and Table 1, and Supplementary Figure S9). There is also evidence for reduced fructose-phosphate ester binding by GKRP-P446L (Supplementary Figure S12). Taken together, these data indicate significant conformational differences as a result of the P446L substitution, which could limit the ability of GKRP to facilitate nuclear uptake of GK via the NPC and act as an effective storage partner in the nucleus. The recently disclosed crystal structure of human GKRP and the binary xGK–xGKRP and hGK–rGKRP complex [47–49] suggests that Pro446 is located in one of the surface loops of the SIS-2 domain, a region with high surface entropy, that may explain both the conformational and functional alterations observed in the present study (Figures 1 and 9). Proline is a helix-disrupting amino acid [70], and replacing it with leucine may destabilize this region.


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)

Effects of glucose, fructose-phosphate esters, GKAs and GKRPIs on the GK–GKRP complexThe Figure summarizes the structural information on the GK–GKRP complex under the influence of glucose and known allosteric ligands of the two participating proteins ([21,22,38,47–49] and the present study). (A and B) The results of mutational analyses [21]. (C and D) The combined results of mutational [21] and crystallographic [22,47–49] analyses of these interactions [note that the orientation differs from that of (A) and (B) (an approximately 90° rotation towards the face of the page) in order to emphasize that the site of GK–GKRP interaction is distinct from that of GK–GKA]. All structural information is projected on to the GK structures published by Kamata et al. [38], and the tryptophan residues are depicted as black stick structures for improved orientation. (A) The allosteric modifier region of GK with two distinct binding sites for GKRP and GKA in the open conformation. Amino acids that affect interaction with GKRP are shown as space-filled structures in cyan. Arg186 of GK is presented in magenta [47,49] to show its close vicinity to other amino acids in the small lobe of GK that have been implicated in GKRP binding. GKA-contacting amino acids are shown as red stick models. Activation of GK by glucose (green) combined with a GKA (yellow) profoundly alters the proposed GKRP-binding site by affecting groups of amino acids involved in binding including the critical Trp99 for binding (B). (C and D) The allosteric binding site for GKRP is indicated in grey space-filled structures for eight of the 15 identified contact amino acids [47,49]. Amino acids immediately surrounding the GKRP contact site are those identified by mutational analysis to influence GKRP binding to GK and are presented as space-filled structures in cyan (see also A and B) [21]. The GK-binding site for GKRP is drawn schematically as a small circular patch in (C) to delineate where the two proteins interact in the complex and a larger patch in (D) to delineate the conformational change that dislodges the inhibitor when glucose and/or GKA are bound to GK.
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Related In: Results  -  Collection

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Show All Figures
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Figure 9: Effects of glucose, fructose-phosphate esters, GKAs and GKRPIs on the GK–GKRP complexThe Figure summarizes the structural information on the GK–GKRP complex under the influence of glucose and known allosteric ligands of the two participating proteins ([21,22,38,47–49] and the present study). (A and B) The results of mutational analyses [21]. (C and D) The combined results of mutational [21] and crystallographic [22,47–49] analyses of these interactions [note that the orientation differs from that of (A) and (B) (an approximately 90° rotation towards the face of the page) in order to emphasize that the site of GK–GKRP interaction is distinct from that of GK–GKA]. All structural information is projected on to the GK structures published by Kamata et al. [38], and the tryptophan residues are depicted as black stick structures for improved orientation. (A) The allosteric modifier region of GK with two distinct binding sites for GKRP and GKA in the open conformation. Amino acids that affect interaction with GKRP are shown as space-filled structures in cyan. Arg186 of GK is presented in magenta [47,49] to show its close vicinity to other amino acids in the small lobe of GK that have been implicated in GKRP binding. GKA-contacting amino acids are shown as red stick models. Activation of GK by glucose (green) combined with a GKA (yellow) profoundly alters the proposed GKRP-binding site by affecting groups of amino acids involved in binding including the critical Trp99 for binding (B). (C and D) The allosteric binding site for GKRP is indicated in grey space-filled structures for eight of the 15 identified contact amino acids [47,49]. Amino acids immediately surrounding the GKRP contact site are those identified by mutational analysis to influence GKRP binding to GK and are presented as space-filled structures in cyan (see also A and B) [21]. The GK-binding site for GKRP is drawn schematically as a small circular patch in (C) to delineate where the two proteins interact in the complex and a larger patch in (D) to delineate the conformational change that dislodges the inhibitor when glucose and/or GKA are bound to GK.
Mentions: Kinetic studies of GKRP-P446L have demonstrated a reduced ability of this variant protein to inhibit GK as a consequence of reduced responsiveness to F6P, and cellular studies have shown impaired GK binding and nuclear storage [28,30,31]. Subsequent alterations in GK localization and activity may explain the association of this GKRP variant with metabolic traits such as fasting glucose and triacylglycerol levels [33]. The present study expands the characterization of GKRP-P446L. First, we show that structural instability or impaired protein folding are unlikely to be major contributors to GKRP-P446L dysfunction (Figure 4). Secondly, the increase in the GK S0.5 value for binding to GKRP-P446L, the reduced affinity of GK for GKRP-P446L in the presence of high glucose and GKA, the abnormal constancy of TF maxima under various conditions, and, finally, the reduced co-operativity with GK, suggest a narrower conformational range for this variant protein (Figures 5–8 and Table 1, and Supplementary Figure S9). There is also evidence for reduced fructose-phosphate ester binding by GKRP-P446L (Supplementary Figure S12). Taken together, these data indicate significant conformational differences as a result of the P446L substitution, which could limit the ability of GKRP to facilitate nuclear uptake of GK via the NPC and act as an effective storage partner in the nucleus. The recently disclosed crystal structure of human GKRP and the binary xGK–xGKRP and hGK–rGKRP complex [47–49] suggests that Pro446 is located in one of the surface loops of the SIS-2 domain, a region with high surface entropy, that may explain both the conformational and functional alterations observed in the present study (Figures 1 and 9). Proline is a helix-disrupting amino acid [70], and replacing it with leucine may destabilize this region.

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