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Figure 4 | BMC Structural Biology

Figure 4

From: Characterization of the SAM domain of the PKD-related protein ANKS6 and its interaction with ANKS3

Figure 4

Characterizing the ANKS3-SAM polymer interface and the ANKS3-SAM/ANKS6-SAM interaction interface. A) The negGFP native gel screen identifies point mutations in both the EH- and ML-surfaces of ANKS3-SAM that result in a loss of polymeric character. Point mutations in ANKS6-SAM do not impact the native gel migration or monomeric character of this SAM domain. B) Mixing negGFP-ANKS3-SAM and negGFP-ANKS6-SAM mutants in 1:1 molar ratios reveals that the EH-surface of ANKS3-SAM binds the ML-surface of ANKS6-SAM. Point mutations on the EH-surface of ANKS3-SAM inhibit interaction with ANKS6-SAM whereas point mutations on the ML-surface of ANKS3-SAM do not affect the hetero-interaction. Point mutations on the ML-surface of ANKS6-SAM and the Cy mutation (R54W according to our numbering) inhibit interaction with ANKS3-SAM. C) ANKS3-SAM precipitate is resolubilized by the addition of ANKS6-SAM but not by the addition of buffer alone. D) The binding affinity of the native ANKS3-SAM polymer interface is measured by SPR (Kd = 5.8 ± 0.4 μM). Equilibrium binding measurements were performed in triplicate and fit to a 1:1 steady-state model. The calculated Kd is an approximation since tested analyte concentrations were insufficient to reach saturation. The error bars are smaller than the data points. E) The binding affinity of the native ANKS3-SAM/ANKS6-SAM interface is measured by SPR (Kd = 249 ± 8 nM). Equilibrium binding measurements were performed in triplicate and fit to a 1:1 steady-state model. The error bars are smaller than the data points. F) The binding between both ANKS3-SAM/ANKS3-SAM and ANKS3-SAM/ANKS6-SAM exhibits a salt dependency. The Kd of each interaction was determined by SPR at four different ionic strengths. The slope of the linear fit (~2 for ANKS3-SAM/ANKS6-SAM, ~1 for ANKS3-SAM/ANKS3-SAM) indicates that each interface is salt-dependent and employs ionic interactions.

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