Structural basis for membrane binding specificity of the Bin/Amphiphysin/Rvs (BAR) domain of Arfaptin-2 determined by Arl1 GTPase - PubMed (original) (raw)
SPR analysis of the binding modes of Arl1 and Rac1 to Arfaptin-2 BAR. A, structure of the Rac1·Arfaptin-2 BAR complex superimposed on that of the Arl1·Arfaptin-2 BAR complex. The Rac1 molecule is shown in gray and colors used for representing Arl1 and Arfaptin-2 BAR regions are the same as those in Fig. 1. Rac1 binds mainly to α1 and weakly to α2, and the Arfaptin-2 residues involved in Rac1 binding do not overlap with those involved in Arl1 binding. However, Pro-106 and Asn-107 of Rac1 clash with Asn-60 and Leu-61 of the proximal Arl1 molecule. B and C, binding of Arl1 and Rac1 to Arfaptin-2 BAR. A concentration series of His-tagged Arl1(Q71L:14–181) (0–18 μ
m
) (B) or Rac1(G12V) (0–10 μ
m
) (C) was injected over GST-Arfaptin-2 BAR. The set of curves corresponding to the concentration series was prepared for the evaluation by subtracting the running buffer signal. Resonance units at equilibrium were plotted against the analyte concentrations and the plots were fitted to the equation described under “Experimental Procedures.” D, binding of Arl1 to Arfaptin-2 BAR when co-injected with Rac1. A concentration series of His-Arl1(Q71L:14–181) (0–18 μ
m
) and His-Rac1(G12V) (0–10 μ
m
) were simultaneously injected over GST-Arfaptin-2 BAR. Resonance units at equilibrium were plotted from the set of curves corresponding to the concentration series of His-Arl1(Q71L:14–181) at the given concentration of His-Rac1(G12V) from which buffer signals alone had been subtracted. The black, red, green, blue, and light blue lines represent injection of series of His-Rac1(G12V) with 0, 3, 6, 12, and 18 μ
m
His-Arl1(Q71L:14–181), respectively. E, models of possible interactions of Arfaptin-2, Arl1, and Rac1. The green crescents, blue diamonds, and gray hexagons represent the Arfaptin-2 BAR dimer, Arl1, and Rac1, respectively. Models a–c are binding modes characterized by the crystal structures. Models d–f are possible binding models derived from models a–c. The resonance units obtained from the simultaneous injection of His-Arl1(Q71L:14–181) and His-Rac1(G12V) clearly exclude the existence of the model f complex.