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

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.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2009

Author dc:creator

• Fire, Emiko J. (Emiko June)

Advisor dc:contributor.advisor

• Amy E. Keating.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/46808

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/46808