Author:

Abstract:

The small molecule macrocycle cyclotriazadisulfonamide (CADA) was identified as a signal peptide-binding inhibitor of co-translational translocation, which prevents the translocation of human CD4 across the ER membrane. Initial experiments with human/mouse CD4 hybrids used the CADA-insensitive murine CD4 signal peptide to assess the contribution of different regions in the human CD4 signal peptide towards CADA sensitivity. We performed an alanine scan to determine the contribution of individual amino acid residues in the human CD4 signal peptide towards CADA sensitivity. We also included several residues from the amino-terminus of the mature CD4 protein in this mutagenesis study. Residues crucial for CADA activity are located in the central hydrophobic region of the human CD4 signal peptide, as expected from the CD4 hybrid experiments. Remarkably, positively charged residues in the mature CD4 protein region following the signal peptide also appear to contribute significantly to the inhibitory action of CADA. We propose a crude model of the human CD4 signal peptide in a folded state inside the translocon, stabilized by CADA. Crosslinking experiments between alanine mutants and photoreactive CADA analogs will complement this model in the future.