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-1xhBID-BH3, which completes the family portrait of MCL-1 complexes and may facilitate drug discovery against human tumors.
Highlights
We present the structure of cMCL-11⁄7hBID-BCL-2 homology 3 (BH3) complex solved by NMR spectroscopy
We turned to limited proteolysis of Myeloid cell leukemia 1 (MCL-1) ⌬N119-⌬C23 using trypsin, chymotrypsin, and calpain
As part of structure determination, a set of 182 15N-1H residual dipolar couplings (RDCs) were measured on two complexes with 15N labeling of either core domain of MCL-1 (cMCL-1) or the BID-BH3 peptide in Pf1 phage
Summary
Protein Expression and Purification—Constructs of human MCL-1 cDNA lacking the C-terminal transmembrane region were expressed as His-tagged fusion proteins (supplemental Fig. S2A). For NMR samples, cells were grown in M9 media supplemented with [15N]ammonium chloride and/or [13C]glucose to produce uniformly 15N- or 15N-,13C-labeled proteins. Purified proteins (cBAK and/or cMCL-1) were used at 0.05 M (final concentration) in the cytochrome c release reactions at 37 °C for 1 h. Experiments performed in 50 mM HEPES (pH 7.0) at 23 °C, consisted of 37 injections (8 l) of 0.1 mM BH3 peptides into 1.45 ml of 0.01 mM human MCL-1 (four different constructs) to obtain a final peptide/protein ratio of 2:1. NMR Data Collection, Analysis, and Spectral Assignments— For structure calculation, a series of samples, 0.5 mM 13C,15Ndouble-labeled cMCL-1:unlabeled BID-BH3 and 0.5 mM unlabeled cMCL-11⁄713C,15N-double-labeled BID-BH3, were prepared in 20 mM HEPES (pH 6.5), 1 mM dithiothreitol in either 10% D2O or 100% D2O. Structure figures were generated with MacPymol [31]
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