Fig. 1. Three-dimensional modeling and site-directed mutagenesis of NRP/B. (A) Schematic presentation of primary sequence analysis of NRP/B. IVS, intervening sequence. (B) Three-dimensional modeling and mutation in the BTB domain. BTB mutant A includes mutation of Asp(D)47 to Ala(A). BTB mutant A also has mutations in specific conserved residues His(H)60 to Ala(A) and Arg(R)61 to Asp(D). Red color indicates -helix and yellow color indicates the ß-sheet structure. Mutation sites are shown as white balls. (C) Equilibrium binding analysis of NRP/B. Purified NRP/B-(His)6 bound to a HisSorb 96 plate and total cell lysate obtained from mutant NRP/B-transfected cells were incubated in the same plate. The dimerized NRP/B was detected by anti-GFP antibody (upper panel). Equal amounts of NRP/B protein bound to the HisSorb 96-well plate were probed by monoclonal anti-NRP/B antibody, VD2. (D) Dimer formation between the BTB domains of NRP/B as revealed by protein crosslinking. GST sequence was proteolytically removed from recombinant NRP/B-GST, and then recombinant NRP/B was incubated in the presence (+) (lanes 1 and 2) or absence (-) (lane 3) of DSP. After quenching the residual DSP with Tris-HCl, pH 7.4, samples were run on reducing 8% SDS-PAGE (lane 2) or non-reducing 8% SDS-PAGE (lanes 1 and 3), and then visualized by staining with Coomassie Blue. Arrows indicate monomeric (mono) and dimeric (di) NRP/B. BTB mtA, BTB mutant A. Positions of molecular size markers are indicated in kDa on the right.