Figure 5. Determinants of the Pseudosubstrate Recognition by ILK.

(A) Overall architecture of the ILK KD (blue) bound to CH2 (yellow). The activation segment is colored in green. The ATP molecule and magnesium ion are depicted in stick (magenta) and sphere (white) models, respectively. (B) Open-book view of the binding interface of the ILK KD-CH2 complex. Left panel: CH2-binding surface (gray) on ILK KD. The bound CH2 is shown in loop model (yellow) and ILK KD in surface model (blue). Right: ILK-binding surface (gray) on CH2. The bound ILK KD is shown in loop model (blue) and CH2 in surface model (yellow). All residues in the interface are labeled. (C) Involvement of the ILK αG-helix and activation loop in binding to CH2. Left: Ribbon model of the ILK KD/CH2 complex. The CH2 is depicted by the transparent surface model. Right: Close-up view of the hydrophobic and polar interactions between the G-helix in the ILK KD and α-parvin CH2. The ILK KD-binding hydrophobic patch on the CH2 is depicted in stick models and colored in yellow on the transparent surface model of CH2. M350 and P353 in the C-terminal activation segment that interact with CH2 are depicted in green stick and transparent models. (D) Identification of the critical residues involved in the ILK-CH2 interface. Left: Normal expression of GFP-tagged wild type and the mutant forms of ILK. GFP tagged wild type and M402A/K403A mutant forms of ILK were immunoprecipitated from HeLa cells expressing the corresponding ILK proteins with an anti-GFP antibody. Right: Evaluation of the α-parvin binding. The immunoprecipitates were analyzed by Western blotting with anti-α-parvin antibody. (E-H) Focal adhesion localization of wild type and M402A/K403A mutant forms of ILK. HeLa cells expressing GFP-tagged wild type (E and F) and M402A/K403A mutant (G and H) forms of ILK were plated on fibronectin coated cover slips. The cells were stained with a mouse monoclonal antibody recognizing migfilin (as a marker of FAs) and Rhodamine Red^TX-conjugated anti-mouse IgG antibodies. The GFP-tagged wild type (E) and mutant (G) forms of ILK and migfilin (F and H) were observed under a fluorescence microscope. Bar indicates 10 μm. See also Figure S5.