(a) IR spectra of nitrile-labeled phosphorylated AurA. The apo sample (solid black line), and the sample bound to Tpx2 (dashed black line), were measured at 5°C, and the kinase bound to ADP (colored lines) was measured at the indicated temperatures. Arrows indicate peaks assigned to the DFG-In and DFG-Out states. The inset shows the same experiments performed with unphosphorylated AurA. Single representative spectra are shown, normalized to peak maxima. (b) Overview of the structure of AurA in the active conformation bound to ADP (yellow) and Tpx2 (beige), with enlarged views of the DFG-In (right, PDB ID: 1OL5) and DFG-Out (bottom, PDB ID: 5L8K) states with the nitrile probe (Q185CN) modeled into the structures. (c) Second derivatives of IR spectra of apo WT and W277A AurA, showing the ~1.5 cm−1 spectral shift of the 2158 cm−1 peak (arrow).