Abstract
Menin is a tumor suppressor protein that is encoded by the MEN1 (multiple endocrine neoplasia 1) gene and controls cell growth in endocrine tissues. Importantly, menin also serves as a critical oncogenic cofactor of MLL (mixed lineage leukemia) fusion proteins in acute leukemias. Direct association of menin with MLL fusion proteins is required for MLL fusion protein-mediated leukemogenesis in vivo, and this interaction has been validated as a new potential therapeutic target for development of novel anti-leukemia agents. Here, we report the first crystal structure of menin homolog from Nematostella vectensis. Due to a very high sequence similarity, the Nematostella menin is a close homolog of human menin, and these two proteins likely have very similar structures. Menin is predominantly an α-helical protein with the protein core comprising three tetratricopeptide motifs that are flanked by two α-helical bundles and covered by a β-sheet motif. A very interesting feature of menin structure is the presence of a large central cavity that is highly conserved between Nematostella and human menin. By employing site-directed mutagenesis, we have demonstrated that this cavity constitutes the binding site for MLL. Our data provide a structural basis for understanding the role of menin as a tumor suppressor protein and as an oncogenic co-factor of MLL fusion proteins. It also provides essential structural information for development of inhibitors targeting the menin-MLL interaction as a novel therapeutic strategy in MLL-related leukemias.
Highlights
(6); chromatin-associated proteins such as mSin3A [7], MLL [8, 9], and lens epithelium-derived growth factor [10]; DNA repair proteins such as the DNA damage repair protein FANCD2 [11]; and the replication protein A subunit RPA2 [12]
Menin functions as a critical oncogenic co-factor of MLL fusion proteins required for their leukemogenic activity [15]
Because human menin was recalcitrant to crystallization efforts, we crystallized a menin homolog from N. vectensis, which is highly conserved with the human protein and is expected to have a very similar structure
Summary
Expression, and Purification—The synthetic gene encoding N. vectensis menin was ordered from GenScript and subcloned into pET32a vector (Novagen). Nematostella menin was expressed in Rosetta2(DE3) cells (Novagen) and purified using affinity chromatography column HisTrap HP (GE Healthcare) followed by ion exchange employing Q Sepharose FF (GE Healthcare). Results were used to calculate binding affinity (Kd) for the MLLderived peptide with wild type menin or menin mutants using the Origin program (version 7.0). All samples were prepared in quadruplicates and contained proteins at 5 M concentrations in 50 mM Tris, pH 7.5, 50 mM NaCl buffer, and 50 M 1-anilinonaphthalene-8sulfonic acid. The initial low resolution model obtained from data collected for a crystal containing SeMet-substituted protein was improved by manual rebuilding in COOT [31]. The low resolution model was later improved in HKL-3000 using higher resolution data collected for crystal of the native protein. Model of human menin was built using Modeler 9 (version 8) [36], and analysis of sequence conservation was carried out employing ConSurf [37] based on multiple sequence alignment of 21 menin homologs
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