Antidiabetic Disruptors of the Glucokinase−Glucokinase Regulatory Protein Complex Reorganize a Coulombic Interface

Abstract

The glucokinase regulatory protein (GKRP) plays an essential role in glucose homeostasis by acting as a competitive inhibitor of glucokinase (GCK) and triggering its localization to the hepatocyte nucleus upon glucose deprivation. Metabolites such as fructose 6-phosphate and sorbitol 6-phosphate promote assembly of the GCK-GKRP complex, whereas fructose 1-phosphate and functionalized piperazines with potent in vivo antidiabetic activity disrupt the complex. Here, we establish the molecular basis by which these natural and synthetic ligands modulate the GCK-GKRP interaction. We demonstrate that a small-molecule disruptor of the protein-protein interaction utilizes a two-step conformational selection mechanism to associate with a rare GKRP conformation constituting 3% of the total population. Conformational heterogeneity of GKRP is localized to the N-terminus and deleting this region eliminates the ability of sorbitol 6-phosphate to promote the GCK-GKRP interaction. Stabilizing ligands favor an extended N-terminus, which sterically positions two arginine residues for optimal coulombic interaction with a pair of carboxylate side chains from GCK. Conversely, disruptors promote a more compact N-terminus in which an interfacial arginine residue is stabilized in an unproductive orientation through a cation-π interaction with tyrosine 75. Eliminating the ability to sample this binding impaired conformation enhances the intrinsic inhibitory activity of GKRP. Elucidating the molecular basis of ligand-mediated control over the GCK-GKRP interaction is expected to impact the development and future refinement of therapeutic agents for diabetes and cardiovascular disease, which result from improper GKRP regulation of GCK.

Mechanisms of ligand binding

“…A small-molecule inhibitor of this protein-protein interaction utilizes CS by binding to a conformation of the regulatory protein that accounts for only 3% of the total population. 90 CS is the driving force in the allosteric transition of Gal3p, an allosteric monomeric protein that activates the GAL genetic switch of Saccharomyces cerevisiae in response to galactose, which may have implications in other signaling pathways involving monomeric proteins. 91 The chaperonin-containing t-complex polypeptide 1 assists protein folding in an ATP-dependent manner.…”

Molecular and cellular regulation of human glucokinase

“…[54] Recent kinetic and mutational work suggest that the differential effects of these ligands upon complex stability are dictated by the conformational state of the flexible N-terminus. [55,56] Ligands that favor an extended N-terminus poise GKRP for optimal coulombic interactions with residues located near the hinge region of GCK. Conversely, ligands that favor a more compact N-terminus disrupt these stabilizing interactions and weaken the protein-protein interaction.…”

First Direct Evidence of Interpartner Hydride/Deuteride Exchanges for Stored Sodiated Arginine/Fructose-6-phosphate Complex Anions within Salt-Solvated Structures

“…guanidinium group of arginine and phosphate group of fructose phosphate. [11][12][13][14][15][16] Mass spectrometry (MS) with its high resolution and sensitivity is the ultimate tool to investigate and understand these mechanisms. When using electrospray ionization mass spectrometry (ESI MS) to analyze mixtures of low molecular weight compounds, formation of NCC between guanidium and phosphate groups of the interacting molecules is often observed in the gas phase (GP).…”

“…Noncovalent complexes (NCC) are generated in both liquid- and gas-phase conditions by a variety of chemical interactions, such as electrostatic (e.g., ionic or hydrogen bonding) or hydrophobic interactions. − In living organisms, noncovalent interactions between biomolecules are the basis of molecular recognition mechanisms essential for regulation of biological processes . Such mechanisms often involve interactions between reactive groups of the different partners, e.g., the guanidinium group of arginine and the phosphate group of fructose phosphate. − Mass spectrometry (MS) with its high resolution and sensitivity is the ultimate tool to investigate and understand these mechanisms. When using electrospray ionization mass spectrometry (ESI MS) to analyze mixtures of low molecular weight compounds, formation of NCC between guanidium and phosphate groups of the interacting molecules is often observed in the gas phase (GP) …”

“…43 We recently studied, under resonant excitation conditions (collision induced dissociation: CID), the behavior of ESIgenerated arginine/hexose−phosphate (HexP) complexes as [(HexP+R-H+Na)-H] − , 41 as a system representative of biologically relevant protein's arginine complexes. 11,12 Complementary investigations combining CID experiments and quantum calculations revealed the existence of two sodiated NCC structures: (i) the HB form with the remote salt group and (ii) the form with the arginine salt group solvated by the phosphate group of [HexP-H] − (i.e., solvated salt, SSA). The HB form dissociates exclusively by partner splitting, while in the case of the SSA form, the free negative charge promotes cross-ring cleavage(s) of hexose.…”