Glucokinase (GK) is a glucose-phosphorylating enzyme that regulates insulin release and hepatic metabolism, and its loss of function is implicated in diabetes pathogenesis. GK activators (GKAs) are attractive therapeutics in diabetes; however, clinical data indicate that their benefits can be offset by hypoglycemia, owing to marked allosteric enhancement of the enzyme's glucose affinity. We show that a phosphomimetic of the BCL-2 homology 3 (BH3) α-helix derived from human BAD, a GK-binding partner, increases the enzyme catalytic rate without dramatically changing glucose affinity, thus providing a new mechanism for pharmacologic activation of GK. Remarkably, BAD BH3 phosphomimetic mediates these effects by engaging a new region near the enzyme's active site. This interaction increases insulin secretion in human islets and restores the function of naturally occurring human GK mutants at the active site. Thus, BAD phosphomimetics may serve as a new class of GKAs.
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We thank K. Robertson and P. Chen for technical assistance; F. Bernal, S. Devarakonda and C. Buettger for advice on protein purification; E. Gavathiotis, A. West and R. McNally for advice on structural studies; and M. Eck, N. Gray, S. Blacklow, G. Yellen and members of the Danial and Walensky laboratories for valuable discussions. This work was supported by US National Institutes of Health grants R01DK078081 (N.N.D.) and R01GM090299 (L.D.W.), a Burroughs Wellcome Fund Career Award in Biomedical Sciences (N.N.D.), Juvenile Diabetes Research Foundation grant 17-2011-595 (N.N.D.), a Claudia Adams Barr Award in Innovative Basic Cancer Research (N.N.D.), a Stand Up to Cancer Innovative Research Grant (L.D.W.), a National Sciences and Engineering Research Council of Canada postgraduate scholarship (C.R.B.), a Swiss National Science Foundation postdoctoral fellowship (S.L.) and a Juvenile Diabetes Research Foundation postdoctoral fellowship (M.A.O.).
L.D.W. is a consultant and member of the scientific advisory board for Aileron Therapeutics.
Integrated supplementary information
Supplementary Figure 1 Phospho-BAD BH3 peptides cross-link to GK after UV exposure in the presence or absence of glucose.
Uncropped gels related to Fig. 2a document detection of BAD SAHBA–GK crosslinked complex as visualized by western blot analysis using an anti-biotin antibody. The lower band corresponds to monomeric GK crosslinked to the BAD SAHBA compound, which was excised and subjected to MS/MS analysis.
Supplementary Figure 2 Cross-linking profile of BAD BH3 stapled peptide in the presence of glucose and RO0281675.
Spectral count frequency of BAD SAHBA (S118pS D123R F125Bpa) crosslinked along the GK protein sequence. GK residues involved in crosslinking, which were unique to that peptide (with a frequency >25%), are labeled.
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Szlyk, B., Braun, C., Ljubicic, S. et al. A phospho-BAD BH3 helix activates glucokinase by a mechanism distinct from that of allosteric activators. Nat Struct Mol Biol 21, 36–42 (2014). https://doi.org/10.1038/nsmb.2717
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