Solution Structure of a GSK 3b Binding Motif, AXINpep
Solution Structure of a GSK 3 b ？ Binding Motif, AXINpep
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초록
Axin is a scaffold protein of the APC/axin/GSK complex, binding to all of the other signalling components. Axin interacts with Glycogen synthase kinase 3β (GSK3β) and functions as a negative regulator of Wnt signalling pathways. To determine the solution structure of the GSK3β binding regions of the axin, we initiated NMR study of axin fragment comprising residues Val388 - Arg401 using circular dichroism (CD) and two-dimensional NMR spectroscopy. The CD spectra of axinpep in the presence of 30% TFE displayed a standard α-helical conformation, exhibiting the bound structure of axinpep to GSK3β. On the basis of experimental restraints including NOEs, and 3JHNα coupling constants, the solution conformation of axinpep was determined with program CNS. The 20 lowest energy structures were selected out of 50 final simulated-annealing structures in both water and TFE environment, respectively. The RMSDs for the 20 structures in TFE solution were 0.086 nm for backbone atoms and 0.195 nm for all heavy atoms, respectively. The Ramachandran plot indicates that the φ, ψ angles of the 20 final structures is properly distributed in energetically acceptable regions. Axinpep in aqueous solution consists of a stable -helix spanning residues from Glu391 to Val399, which is an interacting motif with GSK3β.
Abstract
Axin is a scaffold protein of the APC/axin/GSK complex, binding to all of the other signalling components. Axin interacts with Glycogen synthase kinase 3β (GSK3β) and functions as a negative regulator of Wnt signalling pathways. To determine the solution structure of the GSK3β binding regions of the axin, we initiated NMR study of axin fragment comprising residues Val388 - Arg401 using circular dichroism (CD) and two-dimensional NMR spectroscopy. The CD spectra of axinpep in the presence of 30% TFE displayed a standard α-helical conformation, exhibiting the bound structure of axinpep to GSK3β. On the basis of experimental restraints including NOEs, and 3JHNα coupling constants, the solution conformation of axinpep was determined with program CNS. The 20 lowest energy structures were selected out of 50 final simulated-annealing structures in both water and TFE environment, respectively. The RMSDs for the 20 structures in TFE solution were 0.086 nm for backbone atoms and 0.195 nm for all heavy atoms, respectively. The Ramachandran plot indicates that the φ, ψ angles of the 20 final structures is properly distributed in energetically acceptable regions. Axinpep in aqueous solution consists of a stable -helix spanning residues from Glu391 to Val399, which is an interacting motif with GSK3β.
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