Flexibility and specificity of the interaction of MCL-1 with BIM BH3

Abstract

Interactions among proteins of the BCL-2 family regulate apoptosis - the process of programmed cell death. This thesis focuses on interactions between anti-apoptotic BCL-2 proteins and BH3 peptides derived from pro-apoptotic BCL-2 proteins, observed in earlier studies to occur with significant selectivity. In order to better understand determinants of specificity in anti-apoptotic/BH3 interactions, I have employed structural and binding studies of BIM BH3 point mutants with anti-apoptotic proteins. I report X-ray crystal structures of MCL-1 in complex with peptides of wild-type and three point mutant sequences of BIM BH3. Together, these structures exhibit a range of conformations within the complex. Structural change with respect to wild-type is most dramatic for an isoleucine-to-tyrosine mutant of BIM (I2dY). The larger residue is accommodated by helix [alpha]3 of MCL-1 and the BIM BH3 peptide helix shifting away from one another. In a phenylalanine-to-glutamate mutation (F4aE), the altered side chain is accommodated by a simple rotation of the side chain out of the hydrophobic pocket, a more modest structural change overall. I have also adapted SPOT array technology to qualitatively and simultaneously measure the interactions of anti-apoptotic BCL-XL and MCL-1 with a large number of surface-bound BH3 peptides. The best results were obtained when (i) longer BH3 peptides (26-mer vs. 20-mer) were employed, and (ii) antibodies were used to detect the binding of anti-apoptotic proteins.

(cont.) The SPOT technique was used to perform substitution analyses, wherein each position of interest in a BH3 peptide is mutated to all possible amino acids (or a subset thereof). The results of these experiments identify several sites that, in the context of BIM, play a role in discriminating between MCL-1 and BCL-XL. Two of these sites are always occupied by small amino acids in known native BH3s, while two others are typically hydrophobic positions that become buried upon interaction with anti-apoptotic BCL-2 proteins. Together, these studies illustrate that the MCL-1 interaction with BIM BH3 is flexible enough to accommodate numerous point mutations, and that certain sites within BIM can be used to encode interaction specificity for both MCL-1 and BCL-XL.