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. 2017 Dec 12;7:17405. doi: 10.1038/s41598-017-17703-5

Figure 1.

Figure 1

Autoinhibited Itk and Src kinases preferentially bind ADP. (a) Schematic of constructs evaluated in this study. Itk32K, Src32K, and Src32KΔC contain the conserved module of SH3, SH2, and kinase (32K) domains. Src32K was truncated by 10 amino acids to remove its C-terminal tail (inclusive of the Y527 phosphorylation site), generating a construct we refer to as Src32KΔC (ΔC-tail). (b) Competition nucleotide displacement assay for Itk32K. 40 μM Itk32K was incubated with 40 μM Mant-ADP until equilibrium was established. Unlabelled ADP or AMPPNP was titrated into the reaction and fluorescence anisotropy monitored. Comparison of the equilibrium inhibition constants (K i) gives an indication of the relative strength of nucleotide binding. AMPPNP binds to Itk32K with 42-fold weaker affinity. (c) Competition nucleotide displacement assay for Src32K. 40 μM tail-phosphorylated Src32K (pY527) or doubly phosphorylated Src32K pY416 (pY527) was incubated with 40 μM Mant-ADP until equilibrium was established. Unlabelled ADP or AMPPNP was titrated into the reaction and fluorescence anisotropy monitored. Solid lines correspond to Src32K, dashed lines to Src32K pY416. AMPPNP binds to Src32K with 20-fold weaker affinity than ADP. Activation loop phosphorylation (dashed lines) does not affect the nucleotide-binding properties of Src32K. (d) Omit map showing clear electron density for ADP and a single magnesium ion in the nucleotide-binding pocket of Src. The magnesium is coordinated by Asn391, Asp404, and the α- and β- phosphates of ADP. The β-phosphate is directly coordinated by the guanidinium side chain of Arg388. (e) Thermodynamic cycle used for molecular dynamics calculations. The binding processes of ADP or ATP moving from bulk water to the bound state within Src kinase is computationally expensive to calculate. Instead we make use of a thermodynamic cycle. Because free energy is a state function, we can determine the relative binding free energy from the vertical arrows, where we convert ADP to ATP both when in solution and when bound to Src kinase.