Fig 2: Sequence analysis of Nef binding site and in vitro binding studies of GPM6B and Nef.A. Amino acid sequence of the Nef binding motif of CD4. B. Clustal W [70] based alignment of the cytoplasmic loops of GPM6B, GPM6A and PLP1. Note, for GPM6B, GPM6A and PLP-DM20/PLP1 isoform 2, the sequences of the complete cytoplasmic loop regions are shown. Additionally, positions of the indicated proteolipid proteins with homologies to a sequence region in the cytoplasmic tail of T-cell surface glycoprotein CD4 (shown at the top) that includes the core Nef binding motif of CD4 [20], [21] are highlighted. Positions identical to the respective CD4 residue are shaded black, those with high similarity are shaded grey and those with lower similarity are boxed. The indicated residue numbers show the beginning and the end of the amino-terminally fluoresceinylated GPM6B and PLP1 peptides used for the Nef binding studies shown in D. Numbering of GPM6B is based on UniProtKB entry Q13491-1 throughout this figure. C. Sequence of the cytoplasmatic portion of CD320/TCblR, homologies to CD4 indicated as described above. D. Fluorescence titration of 0.5 µM of fluoresceinyl-labeled peptides, GPM6B112–127, GPM6BAA, or PLP193–108 with recombinant HIV-1SF2 Nef2–210 protein (prepared as described in [68]). The fluorescence signals are shown as a function of the Nef2–210 protein concentration. Values result from the fluorescence of the peptides in the presence of the indicated concentration of Nef2–210 in comparison with a buffer control titration. Assuming a simple bimolar interaction between the peptide and Nef2–210, the data were described by a model based solely on the law of mass action which accounts for ligand depletion [71]. Nonlinear curve fitting of the model to the fluorescence data (lines) yielded dissociation constants of 0.64±0.06 µM for GPM6B112–127 and of 0.71±0.03 µM for PLP193–108.