Fig. 3.

Monobodies as activators and inhibitors of AurA activity. (A) The ADP/NADH coupled assay was used to monitor Lats2 peptide phosphorylation by AurA in the absence (black) or presence of TPX2 or monobodies Mb1 to Mb6. Fits to Michaelis Menten kinetics, normalized by the enzyme concentration (k_obs), show that the monobodies affect both the k_cat and K_M of AurA for Lats2. Assays were carried out at 25 °C in the presence of 1 or 0.25 μM AurA and saturating concentrations of monobody or TPX2, ATP, and MgCl₂. (B) Comparison of k_obs values of AurA at 1 mM Lats2 showing that Mb1 activates AurA 15-fold, a value comparable to allosteric activation by TPX2, whereas monobodies Mb2-Mb5 inhibit AurA between 2-fold and 20-fold, with Mb6 not significantly affecting AurA kinetics. (C) Monobodies can outcompete TPX2 from AurA in a manner consistent with their relative AurA affinities and reflective of their inhibitory/neutral (Mb2-Mb6) or activating (Mb1) propensity. Experiments were conducted in the presence of 1 μM AurA, 5 μM TPX2, 3 mM Lats2, using the same coupled assay as described earlier. Errors in B were determined from jackknifing of data in A.