The Molecular Mechanism of Substrate Engagement and Immunosuppressant Inhibition of Calcineurin
Figure 3
CN-A238L interactions: The PxIxIT substrate binding site.
(A) Close-up view of the CN PxIxIT substrate binding site. A238L PKIIIT is shown as magenta sticks and labeled; CNA is shown as a grey surface. (B) Same view as (A), with a transparent CNA surface. Individual CNA residues that participate in the interaction with PKIIITA238L are shown as grey sticks. (C) Superposition of the PKIIITA238L motif (purple) with a synthetic PVIVIT peptide (orange) and the IAIIIT CN docking site of AKAP79 (green) bound to CN. Pro13PVIVIT and Cys213A238L are shown as sticks. (D) Overlay as in (C) but illustrated as sticks. Corresponding “variable” residues Ile209A238L, Val9PVIVIT, and Ile341AKAP79 (the second “x” in PxIxIT) participate in the same hydrophobic interaction with Val328CNA (dotted lines). (E) Secondary plot of Kiapp as a function of [RII] for inhibition of CN by A238LFLCVKmut, which retains the PKIIIT site. Data show a linear dependence characteristic of competitive inhibition, with Ki = 7,700±3,000 nM. Kiapp values were obtained from the nonlinear fit of Figure S4A. Points represent averages ± s.e.m.