Figure 5.

 Model Structure of SKP2A and Identification of the Auxin Binding Site.

(A) Superposition of the LRR domain of SKP2A (green) and the half side of TIR1 protein that binds auxin (gray) shown as ribbon diagrams. Residues of SKP2A in a neighborhood of 4.0 Å around auxin molecule (magenta) of the TIR1 structure are depicted as sticks.

(B) Auxin molecule (green frame) docked to a binding site composed of the 11 residues (cyan frames) indicated. Numbers colored cyan refer to β-strands of the LRR domain, which is shown as a ribbon diagram in dark green. Lines represent bonds between backbone atoms, and sticks correspond to bonds between side chain atoms involved in the interaction. The bottom panel shows a close-up view of the nearest groups to auxin revealed by quantum calculations. Yellow dashed lines represent hydrogen bonds with numerical labels indicating H···X distances in Å. Cyan dashed lines represent hydrogen bonds that depend on internal rotation of S151 OH group and C177 SH group. Red line represents a putative interaction of the type N-H···π between a NH₂ side group of N202 and the π electron cloud of the aromatic ring of auxin. The red label gives the distance in Å between N atom and the center of this ring. Atoms are colored: C, light green and cyan; N, blue; O, red; S, yellow; H, white.

(C) Recombinant MBP, MBP-SKP2A, and two SKP2A mutants, which have replaced the Ser-151 for Ala (MBP-SKP2A[S151A]), the Leu-128 for Ser (MBP-SKP2A[L128S]), or double mutant (MBP-SKP2A[L128S; S151A]), were incubated in the presence of 50 nM [³H]-IAA. The retained [³H]-IAA in the amylose beads after three washes was measured by scintillation counting. Each value is the mean of three independent measures, and the errors bars correspond to the sd. The results were normalized relative to the amount of [³H]-IAA retained in the MBP-SKP2A beads.

(D) Recombinant MBP, MBP-SKP2A, MBP-SKP2B, and a SKP2B mutant that has replaced the Ser-128 for Leu (MBP-SKP2B[S128L]) were incubated in the presence of 50 nM [³H]-IAA. The retained [³H]-IAA in the amylose beads after three washes was measured by scintillation counting. Each value is the mean of three independent measures, and the errors bars correspond to the sd. The results were normalized relative to the amount of [³H]-IAA retained in the MBP-SKP2A beads and are presented as a percentage.