Glucokinase (GK) plays a major role in the regulation of blood glucose homeostasis in both the liver and the pancreas. In the liver, GK is controlled by the GK regulatory protein (GKRP). GKRP in turn is activated by fructose 6-phosphate (F6P) and inactivated by fructose 1-phosphate (F1P). Disrupting the GK-GKRP complex increases the activity of GK in the cytosol and is considered an attractive concept for the regulation of blood glucose. We have determined the crystal structure of GKRP in its inactive F1P-bound form. The binding site for F1P is located deeply buried at a domain interface, and H-D exchange experiments confirmed that F1P and F6P compete for this site. The structure of the inactive GKRP-F1P complex provides a starting point for understanding the mechanism of fructose phosphate-dependent GK regulation at an atomic level.

Introduction of specific point mutations has been an effective strategy in enhancing the thermal stability in detergents that aid the purification of mono-dispersed G-protein coupled receptors (GPCRs). Our previous work showed that a specific residue position on transmembrane helix 6 (TM6) in class A GPCRs consistently yields thermostable mutants. The crystal structure of human chemokine receptor CCR5 also showed increased thermostability at two positions, A233D6.33 and K303E7.59. With the goal of testing the transferability of these two thermostabilizing mutations in the other chemokine receptors, we tested the mutations A237D6.33 and R307E7.59 in human CCR3 for thermostability and aggregation properties in DDM detergent solution. Interestingly, the double mutant exhibited a 6–10 fold decrease in the aggregation propensity of the wild type protein. This is in stark contrast to the two single mutants whose aggregation properties resemble more to the wild type (WT). Moreover, Unlike in CCR5, the two single mutants separately showed no increase in thermostability compared to the wild type CCR3, while the double mutant A237D6.33/R307E7.59 confers an increase of 2.6°C in the melting temperature compared to the WT. Extensive all-atom molecular dynamics (MD) simulations in detergent micelles show that a salt bridge network between transmembrane helices TM3, TM6 and TM7 that is absent in the two single mutants confers stability in the double mutant. Free energy surface of the double mutant shows conformational homogeneity compared to the single mutants. An annular n-dodecyl maltoside (DDM) detergent layer packs tighter to the hydrophobic surface of the double mutant CCR3 compared to the single mutants providing additional stability. The purification of other C-C chemokine receptors lacking such stabilizing residues may benefit from the incorporation of these two point mutations in appropriate TM regions.