Role of connecting loop I in catalysis and allosteric regulation of human glucokinase

Martinez, Juliana Andrea; Larion, Mioara; Conejo, Maria S.; Porter, Carol M.; Miller, Brian G.

doi:10.1002/pro.2473

Cited by 11 publications

(9 citation statements)

References 39 publications

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“…This region is located within a loop/β-strand motif that connects the large and small domains. 38 Notably, previous studies have identified several activating substitutions within this region, including S64P, a variant that displays NMR features characteristic of the α-type activation mechanism. 11,17 HDX-MS revealed increased deuterium uptake in this region, in agreement with a comparison of the unliganded and glucose bound crystal structures, demonstrating that this region is more solvent-exposed in the glucose-bound state (Figure 4, bottom).…”

Section: Resultsmentioning

confidence: 99%

Mechanistic Origins of Enzyme Activation in Human Glucokinase Variants Associated with Congenital Hyperinsulinism

et al. 2018

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Human glucokinase (GCK) acts as the body’s primary glucose sensor and plays a critical role in glucose homeostatic maintenance. Gain-of-function mutations in gck produce hyperactive enzyme variants that cause congenital hyperinsulinism. Prior biochemical and biophysical studies suggest that activated disease variants can be segregated into two mechanistically distinct classes, termed α-type and β-type. Steady-state viscosity variation studies indicate that the kcat values of wild-type GCK and an α-type variant are partially diffusion-limited, whereas the kcat value of a β-type variant is viscosity-independent. Transient-state chemical quench-flow analyses demonstrate that wild-type GCK and the α-type variant display burst kinetics, whereas the β-type variant lacks a burst phase. Comparative hydrogen-deuterium exchange mass spectrometry of unliganded enzymes demonstrates that a disordered active-site loop, which folds upon glucose binding, is protected from exchange in the α-type variant. The α-type variant also displays increased exchange within a β-strand located near the enzyme’s hinge region, which becomes more solvent-exposed upon glucose binding. In contrast, β-type activation causes no substantial difference in global or local exchange relative to unliganded, wild-type GCK. Together these results demonstrate that α-type activation results from a shift in the conformational ensemble of unliganded GCK toward a state resembling the glucose-bound conformation, whereas β-type activation is attributable to an accelerated rate of product release. This work elucidates the molecular basis of naturally occurring, activated GCK disease variants and provides insight into the structural and dynamic origins of GCK’s unique kinetic cooperativity.

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Section: Resultsmentioning

confidence: 99%

Mechanistic Origins of Enzyme Activation in Human Glucokinase Variants Associated with Congenital Hyperinsulinism

et al. 2018

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“…Conversely, a handful of activating mutations in GCK can cause hypoglycemia through excessive insulin secretion [11]. Many of these activating mutations map to connecting loop I (variably defined as aa 47-71 [19] or aa 62-72 [20]) in the GCK hinge, thereby confirming the functional importance of this region [11]. Connecting loop I exhibits dramatic structural flexibility as the enzyme reorganizes its two lobes in response to glucose [3, 19].…”

Section: Introductionmentioning

confidence: 99%

“…Many of these activating mutations map to connecting loop I (variably defined as aa 47-71 [19] or aa 62-72 [20]) in the GCK hinge, thereby confirming the functional importance of this region [11]. Connecting loop I exhibits dramatic structural flexibility as the enzyme reorganizes its two lobes in response to glucose [3, 19]. It is critical to the enzyme’s sigmoidal dependence on glucose [19], and it provides 4 contact residues (aa 62, 63, 65 and 66) for allosteric GCK activators [3, 21], thus making this region an allosteric site.…”

Section: Introductionmentioning

confidence: 99%

“…Connecting loop I exhibits dramatic structural flexibility as the enzyme reorganizes its two lobes in response to glucose [3, 19]. It is critical to the enzyme’s sigmoidal dependence on glucose [19], and it provides 4 contact residues (aa 62, 63, 65 and 66) for allosteric GCK activators [3, 21], thus making this region an allosteric site. Within this region, the discovery of a tankyrase-binding motif (TBM), 63 RSDPEG 68 , is reported herein.…”

Section: Introductionmentioning

confidence: 99%

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Tankyrase interacts with the allosteric site of glucokinase and inhibits its glucose-sensing function in the beta cell

Chi¹,

Eisemann

Yeh³

et al. 2021

Preprint

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Insulin secretion in the pancreatic beta cell is rate-limited by glucokinase (GCK), the glucose sensor that catalyzes the first step of glucose metabolism. GCK consists of two lobes connected by a flexible hinge that allows the kinase to sample a spectrum of conformations ranging from the active, closed form to several inactive, less-compact forms. Activating GCK mutations can cause hyperinsulinemia and hypoglycemia in infants. A similar phenotype is exhibited in mice deficient in tankyrase (TNKS), prompting us to investigate whether TNKS might modulate the glucose-sensing function of GCK. We found that TNKS colocalizes and directly interacts with GCK. Their interaction is mediated by two ankyrin-repeat clusters (ARC-2 and -5) in TNKS and a tankyrase-binding motif (TBM, aa 63-68) in the GCK hinge. This interaction is conformation sensitive: human GCK variants that cause hyperglycemia (V62M) or hypoglycemia (S64Y) enhance or diminish the interaction respectively, even though they have no impact on TNKS interaction in the context of a GCK peptide (V62M) or a peptide library (S64Y). Moreover, the TNKS-GCK interaction is inhibited by high concentrations of glucose, which are known to stabilize GCK in the active (closed, glucose-avid) conformation. Conversely, glucose phosphorylation by GCK in vitro is inhibited by TNKS. To validate this in vitro inhibitory effect in the MIN6 beta cells, we showed that glucose-stimulated insulin secretion is suppressed upon stabilization of the TNKS protein and conversely is enhanced upon TNKS knockdown. Based on these findings as well as by contrasting with hexokinase-2, we propose that TNKS is a physiological GCK inhibitor in pancreatic beta cells that acts by trapping the kinase in an open (inactive) conformation.

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“…Commonly, the knowledge about the specificity‐determining residues of the certain protein is far from complete. The residues non‐involved into the direct interaction may affect the molecular recognition by allosteric manner (Hubbard et al, ; Martinez et al, ). It hampers the evaluation of prediction accuracy, including the evaluation in comparison with other methods.…”

Section: Introductionmentioning

confidence: 99%

Prediction of amino acid positions specific for functional groups in a protein family based on local sequence similarity

Karasev

Veselovsky

Oparina

et al. 2015

J of Molecular Recognition

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The exchange of single amino acid residue in protein can substantially affect the specificity of molecular recognition. Many protein families can be divided into the groups based on specificity to recognized ligands. Prediction of group-discriminating residues within the certain family is extremely necessary for theoretical studies, enzyme engineering, drug design, and so on. The most existing methods use the multiple sequence alignment. They have the limitations in prediction accuracy due to the family sequence divergence and ligand-based grouping. We developed a new method SPrOS (Specificity Projection On Sequence) for estimating the specificity of residues to user-defined groups. SPrOS compares the sequence segments from the test protein and training proteins. Contrary to other segment-comparison approaches extracting the string motifs, SPrOS calculates the scores for single positions by the similarity of their surroundings. The method was evaluated on the simulated sequences and real protein families. The high-prediction accuracy was achieved for simulated sequences, in which SPrOS detected specific positions not predicted with the alignment-based method. For bacterial transcription factors (LacI/GalR) clearly divided into functional groups, the predicted specific residues corresponded to the published experimental data. In a more complicated case of protein kinases classified by inhibitor specificity, the positions predicted with high significance were located in ligand-binding areas. As the ligand specificity is not necessary coincided with phylogeny, evolutionary-coupled mutations could disturb the detection of ligand-specific residues. Excluding proximate homologs of the test protein kinase from the training set, we improved the prediction of the ligand-specific residues. The SPrOS is available at http://www.way2drug.com/spros/

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Role of connecting loop I in catalysis and allosteric regulation of human glucokinase

Cited by 11 publications

References 39 publications

Mechanistic Origins of Enzyme Activation in Human Glucokinase Variants Associated with Congenital Hyperinsulinism

Mechanistic Origins of Enzyme Activation in Human Glucokinase Variants Associated with Congenital Hyperinsulinism

Tankyrase interacts with the allosteric site of glucokinase and inhibits its glucose-sensing function in the beta cell

Prediction of amino acid positions specific for functional groups in a protein family based on local sequence similarity

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