Apoptotic Regulation by MCL-1 through Heterodimerization

Abstract

Myeloid cell leukemia 1 (MCL-1), an anti-apoptotic BCL-2 family member active in the preservation of mitochondrial integrity during apoptosis, has fundamental roles in development and hematopoiesis and is dysregulated in human cancers. It bears a unique, intrinsically unstructured, N-terminal sequence, which leads to its instability in cells and hinders protein production and structural characterization. Here, we present collective data from NMR spectroscopy and titration calorimetry to reveal the selectivity of MCL-1 in binding BCL-2 homology 3 (BH3) ligands of interest for mammalian biology. The N-terminal sequence weakens the BH3 interactions but does not affect selectivity. Its removal by calpain-mediated limited proteolysis results in a stable BCL-2-like core domain of MCL-1 (cMCL-1). This core is necessary and sufficient for BH3 ligand binding. Significantly, we also characterized the in vitro protein-protein interaction between cMCL-1 and activated BID by size exclusion chromatography and NMR titrations. This interaction occurs in a very slow manner in solution but is otherwise similar to the interaction between cMCL-1 and BID-BH3 peptides. We also present the solution structure of complex cMCL-1⅐hBID-BH3, which completes the family portrait of MCL-1 complexes and may facilitate drug discovery against human tumors.The BCL-2 family of proteins plays a pivotal role in regulating apoptosis mainly by regulating the outer mitochondrial membrane integrity (1). They are classified into two functional groups: anti-apoptotic and pro-apoptotic. They each share conserved BH regions. The anti-apoptotics (BCL-2, BCL-w, BCL-X L , MCL-1, and A1) share up to four BH regions named BH1-4, 4 and prevent cells from entering apoptosis. The pro-apoptotics can be further grouped into the effectors and BH3-only proteins. The effectors, BAX and BAK, contain three BH (BH1-3) regions and promote cell death by oligomerization and mitochondria outer membrane permeabilization. The BH3-only proteins share the BH3 region of sequence similarity. Members of this group include BID, BIM, BAD, BMF, BIK, PUMA, Noxa, HRK/DPS (Harakiri), NIP3, bNIP3, and MULE. The BH3 region is 16 to 25 amino acid residues long and can promote apoptosis when introduced into cells. The three groups of BCL-2 proteins form a delicately balanced network of opposing functions that regulates the fate of the cell. MCL-1 belongs to the anti-apoptotic family but is unique due to its short half-life, the presence of an extra N-terminal sequence, and its particular selectivity in binding BH3 peptides. Its concentration in vivo is tightly regulated at multiple levels, and its N-terminal sequence presents many sites for post-translational modifications, including polyubiquitination (2, . These modifications are involved in regulating its activity in binding pro-apoptotic proteins, or in regulating its concentration by either promoting or blocking its degradation. A deregulated, high level of MCL-1 has been correlated with various hematopoietic and lymphoid cancers (8 -10)....

Heterodimerization of BAK and MCL-1 Activated by Detergent Micelles

“…Upon the addition of a range of IGEPAL concentrations from four times below its critical micelle concentration (ϳ0.02% v/v) to 20 times above, only slight chemical shift changes were observed in 1 H- 15 N HSQC spectra of 15 N-labeled cMCL-1 (Fig. 3A and data not shown).…”

“…FLAG-BAK-HMK-⌬TM-His 6 (residues 2-186 from NCBI: AAA74466), calpain-proteolysed BAK (cBAK, residues 16 -186 from NCBI: AAA74466), cBAK-L78A, core domain of MCL-1 (also calpain-proteolysed MCL-1, cMCL-1, residues 163-326 from NCBI: AAF64255) and BID-BH3 peptide (residues 76 -106 from NCBI: NP_001187 with an N-terminal extension of GPLG) were purified as described previously (6,15,34). Escherichia coli strain BL21 (DE3) containing the corresponding plasmid was grown in LB media at 37°C to an optical density of 0.8 at 600 nm and then induced by 1 mM isopropyl-␤-Dthiogalactoside at 30°C for 4 h. For NMR samples, cells were grown in M9 media supplemented with 15 N ammonium chloride. The soluble proteins were purified by batch Ni 2ϩ -NTA or glutathione S-transferase affinity chromatography (Qiagen), followed by Sephadex G-75 size exclusion chromatography (GE Healthcare) and Q-Sepharose anion-exchange chromatography (GE Healthcare).…”

“…Aliquots of unlabeled binding partners such as cMCL-1, cBAK, cBAK-L78A, BID-BH3, BAK-BH3-L78A, IGEPAL, and CHAPS stock solutions were titrated in until the saturation was achieved. The 1 H- 15 N HSQC spectra were recorded on a Bruker Avance DRX600 MHz spectrometer at 30°C, processed by NMRPipe (35), and visualized by NmrViewJ (version 8.0) (36). The backbone assignments of cMCL-1 and BAK in free form were determined previously (6,15).…”

“…The 1 H- 15 N HSQC spectra were recorded on a Bruker Avance DRX600 MHz spectrometer at 30°C, processed by NMRPipe (35), and visualized by NmrViewJ (version 8.0) (36). The backbone assignments of cMCL-1 and BAK in free form were determined previously (6,15). Assignments in detergent were made by following chemical shift changes during titrations.…”

“…1). The competitively activating model (CAM) (15) proposes that BH3-only proteins release BAK from complexes with MCL-1 or BCL-X L by competitively binding to the pocket of the antiapoptotic proteins (19,28,29). The directly activating model (DAM) posits that BH3-only activators including tBID, BIM, and PUMA directly bind and activate BAK, and the role of the antiapoptotic proteins is to sequester the BH3-only activators to prevent apoptosis (4,(25)(26)(27)30).…”