Divergence of Genbank and human tumor Bcl-2 sequences and implications for binding affinity to key apoptotic proteins.
Heterodimerization of antiapoptotic and pro-apoptotic Bcl-2 family of proteins provides an important mechanism for apoptosis regulation. Knowledge about key amino acids in the binding groove of native Bcl-2 contributing to this interaction will greatly facilitate the design of Bcl-2-specific inhibitors. There are two different Bcl-2 sequences, M13994 and M14745, in Genbank. Chimeric proteins Bcl-2(1) and Bcl-2(2) derived from the above sequences, although similar in structure, showed different binding affinities to Bak and Bad BH3 peptides (Petros et al., 2001). In this study, we show that the Bcl-2(1) sequence in normal and tumor human tissue samples differs from M13994 and M14745, and contains P59, T96, R110, S117 and G237. The actual sequence in the binding pocket matches the Bcl-2-Ig fusion sequence X06487, originally identified in a t(14:18) translocation of the Bcl-2 gene, associated with follicular lymphoma. The possible effects of the observed amino acid differences compared to M13994 and M14745 were investigated by combining structural data with fluorescence anisotropy. G110R substitution confers on Bcl-2(1) substantially increased binding affinity to Bak, Bad and Bax BH3 peptides, demonstrating that R110 is a key contributor to the BH3 binding affinity of Bcl-2. Although NMR structure did not predict R110 involvement in binding to these BH3 peptides, fluorescence anisotropy data clearly points to a critical role for this residue in binding to pro-apoptotic Bcl-2 family members.
Joseph
Mary K
MK
Cancer Research, Global Pharmaceutical Research Division, Abbott Laboratories, Abbott Park, IL 60064, USA. mary.joseph@abbott.com
Solomon
Larry R
LR
Petros
Andrew M
AM
Cai
Jill
J
Simmer
Robert L
RL
Zhang
Haichao
H
Rosenberg
Saul
S
Ng
Shi-Chung
SC
D000595:Amino Acid Sequence; D017209:Apoptosis; D001483:Base Sequence; D017931:DNA Primers; D030561:Databases, Nucleic Acid; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D019253:Proto-Oncogene Proteins c-bcl-2