The structure of the C-terminal helical bundle in glutathione transferase M2-2 determines its ability to inhibit the cardiac ryanodine receptor.
Ca(2+) release from the sarcoplasmic reticulum through cardiac ryanodine receptors (RyR2) is essential for heart function and is inhibited by the carboxy terminal domain of glutathione transferase M2-2 (GSTM2-C) and derivative fragments containing helix 6. Since a peptide encoding helix 6 alone does not fold into a helix and does not inhibit RyR2 Ca(2+) release, the importance of the structure of helix 6 and its role in stabilizing GSTM2-C was tested by inserting potentially destabilizing mutations into this helical segment. GSTM2-C preparations with D156A or L163A mutations were so insoluble that the protein could not be purified. Proteins with F157A and Y260A substitutions were soluble, but had lost their capacity to inhibit both RyR2 Ca(2+) release from vesicles and RyR2 channels in bilayers. Circular dichroism studies indicated that these mutated proteins retained their helical secondary structure, although changes in their endogenous tryptophan fluorescence indicated that the F157A and Y160A mutations caused changes in their folding. The single channel studies were conducted with 2mM ATP and 10microM Ca(2+) in the cytoplasmic solution, mimicking concentrations in the cytosol of cardiac myocytes. Wild type GSTM2-C inhibited RyR2 only at a potential of +40mV, which may develop during Ca(2+) efflux, but not at -40mV. Together, the results indicate that the structure of helix 6 in the C-terminal fold is critical to the inhibitory action of GSTM2-2 and suggest that therapeutics mimicking this structure may reduce excess Ca(2+) release during diastole, which can lead to fatal arrhythmia.
Hewawasam
Ruwani
R
Structural Biology Program, John Curtin School of Medical Research, Australian National University, PO Box 334, Canberra, ACT 2601, Australia.
Liu
Dan
D
Casarotto
Marco G
MG
Dulhunty
Angela F
AF
Board
Philip G
PG
D000818:Animals; D002121:Calcium Channel Blockers / Q000378:metabolism*; D005982:Glutathione Transferase / Q000737:chemistry* / Q000502:physiology*; D006801:Humans; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry* / Q000502:physiology*; D017433:Protein Structure, Secondary / Q000502:physiology*; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D012756:Sheep
D002121:Calcium Channel Blockers; D010446:Peptide Fragments; D019837:Ryanodine Receptor Calcium Release Channel; D005982:Glutathione Transferase; C412602:glutathione S-transferase Mu 2