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. 2015 Sep 15;112(37):11553-8.
doi: 10.1073/pnas.1506664112. Epub 2015 Aug 17.

Dual Allosteric Activation Mechanisms in Monomeric Human Glucokinase

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Free PMC article

Dual Allosteric Activation Mechanisms in Monomeric Human Glucokinase

A Carl Whittington et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Cooperativity in human glucokinase (GCK), the body's primary glucose sensor and a major determinant of glucose homeostatic diseases, is fundamentally different from textbook models of allostery because GCK is monomeric and contains only one glucose-binding site. Prior work has demonstrated that millisecond timescale order-disorder transitions within the enzyme's small domain govern cooperativity. Here, using limited proteolysis, we map the site of disorder in unliganded GCK to a 30-residue active-site loop that closes upon glucose binding. Positional randomization of the loop, coupled with genetic selection in a glucokinase-deficient bacterium, uncovers a hyperactive GCK variant with substantially reduced cooperativity. Biochemical and structural analysis of this loop variant and GCK variants associated with hyperinsulinemic hypoglycemia reveal two distinct mechanisms of enzyme activation. In α-type activation, glucose affinity is increased, the proteolytic susceptibility of the active site loop is suppressed and the (1)H-(13)C heteronuclear multiple quantum coherence (HMQC) spectrum of (13)C-Ile-labeled enzyme resembles the glucose-bound state. In β-type activation, glucose affinity is largely unchanged, proteolytic susceptibility of the loop is enhanced, and the (1)H-(13)C HMQC spectrum reveals no perturbation in ensemble structure. Leveraging both activation mechanisms, we engineer a fully noncooperative GCK variant, whose functional properties are indistinguishable from other hexokinase isozymes, and which displays a 100-fold increase in catalytic efficiency over wild-type GCK. This work elucidates specific structural features responsible for generating allostery in a monomeric enzyme and suggests a general strategy for engineering cooperativity into proteins that lack the structural framework typical of traditional allosteric systems.

Keywords: allostery; diabetes; glucokinase; intrinsic disorder; monomeric cooperativity.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Conformational changes and kinetic profile of human GCK. (A) In the unliganded state, the mobile loop (cyan) displays no electron density and the small domain (orange) adopts a super-open conformation. (B) Upon glucose binding, the mobile loop folds into an antiparallel β-hairpin and K169 forms a hydrogen bonding network with glucose (green) and S151. Isoleucine residues (yellow) used as NMR reporters are evenly distributed throughout the molecule. (C) The sigmoidal kinetic response of GCK results from conformational rearrangements occurring on a timescale comparable to kcat. (D) View of the 151–180 loop, revealing the β-hairpin formed by residues 154–164 upon glucose binding (green). The thermolysin cleavage site is shown as a dashed line.
Fig. 2.
Fig. 2.
Mapping GCK intrinsic disorder via proteolysis. (A) Time course of GCK proteolysis by thermolysin monitored by SDS/PAGE. N-terminal sequencing of fragments reveals that GCK is cleaved at one site in the mobile loop between residues 170 and 171. (B) Proteolysis kinetics monitored by measuring residual GCK activity at increasing thermolysin concentrations. Curves were fitted with a single exponential decay function (error bars are ± SD) and the resulting proteolysis rates (kobs) were plotted versus thermolysin concentration to yield values for Kop (Inset; error bars are ± SE of fit).
Fig. S1.
Fig. S1.
Proteolysis profiles of human GCK. (A) Thermolysin digestion of wild-type GCK following addition of 0.2 M glucose. (B) Thermolysin digestion of the α13-helix variant. (C) Proteinase K digestion of GCK in the absence and presence of glucose. (D) Trypsin digestion of GCK in the absence and presence of glucose. Time points are listed in minutes from left to right across the top of each gel.
Fig. S2.
Fig. S2.
Positional insertion of 13C-Ile to probe the structure of the small domain β-sheet of unliganded GCK. (A) Location of Ile insertions; (BE) 1H-13C HMQC spectra of the L77I, L79I, L88I, and L146I variants of GCK demonstrate the appearance of new cross-peaks residing outside of the window expected for disordered isoleucines (boxed).
Fig. S3.
Fig. S3.
Positional insertion of 13C-Ile to probe the structure of the small domain α13-helix of unliganded GCK. (A) Location of Ile insertions; (BD) 1H-13C HMQC spectra of the G446I, V452I, L463I variants of GCK demonstrate the appearance of new cross-peaks. The boxed area denotes the region expected for disordered isoleucines.
Fig. S4.
Fig. S4.
1H-13C HMQC spectrum of glucose bound GCK with positional 13C-Ile probes in the C-terminal α13-helix of GCK. I452 is shifted away from the disordered region (B), whereas I446 and I463 (A and C) remain in the disordered region (boxed area) upon addition of glucose.
Fig. 3.
Fig. 3.
Two functionally distinct mechanisms of GCK activation. α-type activation, as exemplified by the α13-helix variant, shifts the ensemble structure toward a state resembling the glucose-bound conformation, as evidenced by increased sharpness of cross-peaks, shifting of mobile loop cross-peaks away from the disordered region of the spectrum, and the appearance of new cross-peaks. β-type activation, as exemplified by the β-hairpin variant, alters the structure and/or dynamics of the mobile loop (cyan), as evidenced by increased sharpness of mobile loop cross-peaks that remain in the disordered region of the spectrum.
Fig. S5.
Fig. S5.
Overlay of 1H-13C Ile HMQC spectra of glucose-bound wild-type GCK with the unliganded α13-helix variant. In the unliganded α13-helix variant, I159 and I163 each display two peaks. One is in the disordered region corresponding to their position in the glucose bound wild-type, whereas the other peak is shifted away from the disordered region (denoted with a prime).
Fig. 4.
Fig. 4.
Model of GCK cooperativity. Unliganded GCK undergoes millisecond exchange between E and E* with a rate constant (kex = kf + kr) comparable to kcat, which produces kinetic cooperativity (red). Cooperativity is reduced (green) by alterations in kex or kcat such that these values are no longer comparable. α-type activation results from alterations in kex that cause a population shift toward one-state, whereas β-type activation results from alterations in kex and/or kcat that do not substantially perturb the conformational equilibrium. Steps that contribute to the value of kcat are colored gray, g is glucose, g6p is glucose 6-phosphate, and values for kex, kf, kr, and kcat are from ref. .

Comment in

  • Allostery vs. "allokairy".
    Hilser VJ, Anderson JA, Motlagh HN. Hilser VJ, et al. Proc Natl Acad Sci U S A. 2015 Sep 15;112(37):11430-1. doi: 10.1073/pnas.1515239112. Epub 2015 Sep 8. Proc Natl Acad Sci U S A. 2015. PMID: 26372953 Free PMC article. No abstract available.

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