Figure 1

Ala substitution of residues in the NMR-determined epitope of mAb33 decrease antibody binding. (A) Model of the HIV-1 CTD dimer. Left panel: The NMR-determined SH3-fold structure of the CTD is displayed with one subunit colored in yellow and the other in blue. DNA binding residues E246, K264, K266, and R262 are shown in ball-and-stick representation and labeled with black lettering. Residues of the putative mAb33 epitope, as determined by NMR techniques [28], are displayed in green space filling representation. K244 is shown in grey, as it is unlikely to be a critical component of the mAb33 epitope (see Panel B). The long arrow points to residue W243 at the interface between subunits in this NMR dimer of the CTD which forms a "saddle" with both K264 residues extending into the cleft proposed to bind DNA [19]. Right panel: An orthogonal view rotated 90 degrees about the displayed axis. This view shows residues known to be involved in DNA binding on one face of the subunit and adjacent to the mAb33 epitope. (B) ELISA data for CTD substituted HIV-1 sIN proteins and their interactions with mAb33. A high binding microtiter plate was coated with 50 ng of antigen (sIN or one of the Ala substituted sIN proteins) and incubated overnight at 4°C. The plates were then blocked with bovine serum albumin, washed, and incubated with the mAb33 1° antibody which was serially diluted (2-fold) from a starting concentration of 250 ng per well. A standard ELISA protocol was then followed using an alkaline phosphatase conjugated 2° antibody against the kappa chain. The relative binding efficiency of mAb33 to the IN proteins was determined by measuring the absorbance at 405 nm.