Dissection, evolution and disruption of protein-protein iinteractions

Abstract

The interaction between two or more proteins is an event of crucial importance for virtually every biological process. Although protein-protein interactions usually involve burial of large protein surface areas, only a small set of residues on either partner (defined as "hot spots") are actually responsible for the energy of the interaction. Hot spot mapping by mutagenesis is an essential aspect of the discovery and development of new therapeutic agents. Here we have studied protein-protein interactions from two distinct perspectives. In the first part of this thesis we have performed a detailed analysis of factors governing the high affinity interaction between HIV-1 antibody D5 and its protein antigen ('5-Helix') by combinatorial scanning mutagenesis. Although antigen-antibody interactions are often dictated by heavy chain interactions, the present analysis revealed several hot spot residues located in the antibody light chain that contribute to shape complementarity and electrostatic characteristics. Therefore, the affinity of D5 for its target is a result of extended interactions involving both the heavy and light chains of D5. These results provide significant insights for future antibody engineering efforts as well as for epitope assessment during HIV viral fusion.;In the second part of this thesis we have focused on the disruption of protein-protein interactions between the oncogene BCL6 and its corepressors partners. The BCL6 transcriptional repressor is the most frequently involved oncogene in diffuse large B cell lymphoma; loss of BCL6 function can result in tumor death. BCL6 is a member of the BTB/POZ family of transcription factors, which are able to recruit corepressors by protein-protein interactions through an exposed surface groove. In this study, we identified small molecules that bind to the corepressor binding groove of the BCL6 BTB domain and disrupt the BCL6/corepressor complexes in vitro and in vivo. This compound induced expression of BCL6 target genes resulting in death of BCL6-positive cell lines. We have demonstrated here that, although BCL6 recruits its corepressor partners through a large surface buried area, we were able to disrupt these protein-protein interactions using small molecular weight molecules that likely disrupt important interactions on BCL6 hot spots.