High-Quality NMR Structure of Human Anti-Apoptotic Protein Domain Mcl-1(171-327) for Cancer Drug Design

A high-quality NMR solution structure is presented for protein hMcl-1(171–327) which comprises residues 171–327 of the human anti-apoptotic protein Mcl-1 (hMcl-1). Since this construct contains the three Bcl-2 homology (BH) sequence motifs which participate in forming a binding site for inhibitors of hMcl-1, it is deemed to be crucial for structure-based design of novel anti-cancer drugs blocking the Mcl1 related anti-apoptotic pathway. While the coordinates of an NMR solution structure for a corresponding construct of the mouse homologue (mMcl-1) are publicly available, our structure is the first atomic resolution structure reported for the ‘apo form’ of the human protein. Comparison of the two structures reveals that hMcl-1(171–327) exhibits a somewhat wider ligand/inhibitor binding groove as well as a different charge distribution within the BH3 binding groove. These findings strongly suggest that the availability of the human structure is of critical importance to support future design of cancer drugs.


Introduction
The malfunctioning of cellular apoptosis [1] is a major hallmark of cancer. The regulation of apoptosis depends on the family of Bcl-2 proteins which contain one or several Bcl-2 homology (BH) sequence motifs. Based on their function and the similarity of their respective BH sequence motifs, these proteins can be grouped into three classes [2], [3]: (i) multi-domain pro-apoptotic proteins such as Bax and Bak, (ii) anti-apoptotic (i.e., pro-survival) proteins such as Mcl-1, Bcl-1, Bcl-x L , Bcl-w and Bfl-1/A1, all of which exhibit a similar architecture as Bax and Bak, and (iii) several pro-apoptotic proteins comprising only a single BH3 sequence motif such as Bid, Bad, Bim, Puma, Noxa, Hrk, Bmf, and Nbk/Bik ('BH3-only' proteins). The BH3 motif of class (iii) proteins forms an amphipathic a-helix which interacts specifically with a hydrophobic pocket formed in both pro-apoptotic class (i), and antiapoptotic class (ii) proteins with participation of their respective BH motifs [2], [3]. Inhibition of the resulting protein-protein complex formation offers a promising strategy to treat cancer. For example, the small molecule Bcl-2 antagonist ABT-737 [4] inhibits anti-apoptotic class (ii) proteins Bcl-x L , Bcl-w and Bcl-1, and a congener [5] that can be orally administered is currently in clinical trials.
The anti-apoptotic, pro-survival 350-residue protein Mcl-1 ('myeloid cell leukemia-1') [2] is primarily anchored in the outer mitochondrial membrane by a C-terminal trans-membrane domain and contains three BH sequence domains: BH3 (residues 209-223), BH1 (252-272) and BH2 (residues 304-319) [2]. Mcl-1 inhibits death receptor-induced apoptosis by selectively binding to truncated Bid (tBid) [6] and can sequester endogenous Bak to block Bak-mediated cell death. Moreover, Mcl-1 interacts with several BH3-only proteins (Bim, Bid and Puma, Noxa and Bak). Hence, Mcl-1 plays an early role in response to signals directing either cell survival or cell death [2] and has been shown to be upregulated in numerous malignant tumors. Approaches abrogating the Mcl-1's anti-aptototic function either by reducing its abundance or by inactivating its functional BH3-binding groove show great promise for the cancer treatment [2], [4], [6], [7]. Here we present the high-quality NMR solution structure of polypeptide segment 171-327 of human Mcl-1 (hMcl-1) which comprises the three BH motifs deemed to be crucial for structure based drug design.
The fact that the human apo protein exhibits a somewhat wider binding groove than the mouse homologue (Table 2) can be, at least partially, ascribed to the side chain of Leu 246 in the human protein which is not buried as deeply as the corresponding Phe side chain in the mouse protein. Furthermore, when comparing the human and the mouse protein, differences are observed for the charge distributions in the BH3-binding groove ( Figure 2): the human protein is negatively charged on the side of helix a3, while the corresponding surface of mouse protein is positively charged. This difference arises from Ser 255 corresponding to Lys 236 in the mouse protein. Remarkably, hMcl-1(171-327) is structurally more similar to the hMcl1(171-327)-hBim BH3 complex ( Figure 3) than to apo mMcl-1(152-308) ( Table 2).
Taken together, structural comparisons show that, in spite of the 89% sequence identity between human and mouse protein, the availability of the human hMcl-1(171-327) structure can be expected to be of critical importance for supporting future design of cancer drugs.

NMR Sample Preparation
Preliminary studies showed that hMcl-1(171-327) (UniProtKB/ Swiss-Prot ID Q07820/MCL1_HUMAN) is not stable in solution. However, the mutant Cys 286 R Ser is stable for several weeks at concentrations ,0.7 mM, and both wild-type and mutant bind the Bim-BH3 peptide with the same affinity (K d , 60 pM) in a Biacore assay. Hence, we solved the NMR structure of hMcl-1(171-327) Cys 286 R Ser referred to as hMcl-1(171-327) in this publication.