Crystal structure of the DENR-MCT-1 complex revealed zinc-binding site essential for heterodimer formation

Significance Protein synthesis or mRNA translation by ribosomes is essential for the cell’s survival. A multitude of human diseases are the direct result of disruption of translation, specifically at the initiation step. The density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein support noncanonical translation initiation, reinitiation, and ribosome recycling linked to cancer, neurological disorders, and viral infections. Here, we present the crystal structure of a DENR-MCT-1 heterodimer, which reveals atomic details of DENR and MCT-1 interactions that are crucial for understanding their function in translation. Our results provide foundation for the future research of the mechanism of regulation of noncanonical protein synthesis and may potentially be used for antiviral, anticancer, and neurological drug design. The density-regulated protein (DENR) and the malignant T cell-amplified sequence 1 (MCT-1/MCTS1) oncoprotein support noncanonical translation initiation, promote translation reinitiation on a specific set of mRNAs with short upstream reading frames, and regulate ribosome recycling. DENR and MCT-1 form a heterodimer, which binds to the ribosome. We determined the crystal structure of the heterodimer formed by human MCT-1 and the N-terminal domain of DENR at 2.0-Å resolution. The structure of the heterodimer reveals atomic details of the mechanism of DENR and MCT-1 interaction. Four conserved cysteine residues of DENR (C34, C37, C44, C53) form a classical tetrahedral zinc ion-binding site, which preserves the structure of the DENR’s MCT-1–binding interface that is essential for the dimerization. Substitution of all four cysteines by alanine abolished a heterodimer formation. Our findings elucidate further the mechanism of regulation of DENR-MCT-1 activities in unconventional translation initiation, reinitiation, and recycling.

[1]  De Novo Mutations in DENR Disrupt Neuronal Development and Link Congenital Neurological Disorders to Faulty mRNA Translation Re-initiation , 2016, Cell reports.

[2]  N. N. Joseph,et al.  Crystal Structure of the C-terminal Domain of Human eIF2D and Its Implications on Eukaryotic Translation Initiation. , 2017, Journal of molecular biology.

[3]  A. Teleman,et al.  DENR–MCTS1 heterodimerization and tRNA recruitment are required for translation reinitiation , 2018, PLoS biology.

[4]  Alan G Hinnebusch,et al.  The scanning mechanism of eukaryotic translation initiation. , 2014, Annual review of biochemistry.

[5]  T. Steitz,et al.  Supplemental Information Crystal Structure of the Human Ribosome in Complex with DENR-MCT-1 , 2022 .

[6]  M. Tainsky,et al.  drp, a novel protein expressed at high cell density but not during growth arrest. , 1998, DNA and cell biology.

[7]  A. Hinnebusch,et al.  Please do not recycle! Translation reinitiation in microbes and higher eukaryotes , 2017, FEMS microbiology reviews.

[8]  Nathan A. Baker,et al.  Electrostatics of nanosystems: Application to microtubules and the ribosome , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[9]  D. Slamon,et al.  Identification of differentially expressed genes associated with HER-2/neu overexpression in human breast cancer cells. , 1999, Nucleic acids research.

[10]  Colin Echeverría Aitken,et al.  Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex , 2015, Molecular cell.

[11]  R. Gartenhaus,et al.  A novel candidate oncogene, MCT-1, is involved in cell cycle progression. , 1998, Cancer research.

[12]  T. Steitz,et al.  The initiation of mammalian protein synthesis and the mechanism of scanning , 2013, Nature.

[13]  Klaus Wethmar,et al.  The regulatory potential of upstream open reading frames in eukaryotic gene expression , 2014, Wiley interdisciplinary reviews. RNA.

[14]  T. Preiss,et al.  Translation initiation by cap‐dependent ribosome recruitment: Recent insights and open questions , 2018, Wiley interdisciplinary reviews. RNA.

[15]  Fei Long,et al.  REFMAC5 dictionary: organization of prior chemical knowledge and guidelines for its use. , 2004, Acta crystallographica. Section D, Biological crystallography.

[16]  Richard J Jackson,et al.  Termination and post-termination events in eukaryotic translation. , 2012, Advances in protein chemistry and structural biology.

[17]  N. Ban,et al.  Structural and Functional Insights into Human Re-initiation Complexes. , 2017, Molecular cell.

[18]  Evan T. Geller,et al.  Patterns and rates of exonic de novo mutations in autism spectrum disorders , 2012, Nature.

[19]  J. Coleman,et al.  Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins. , 1992, Annual review of biochemistry.

[20]  Y. Tong,et al.  Crystal structure of human multiple copies in T‐cell lymphoma‐1 oncoprotein , 2013, Proteins.

[21]  C. Shu,et al.  MCT-1 oncogene downregulates p53 and destabilizes genome structure in the response to DNA double-strand damage. , 2007, DNA repair.

[22]  W. Merrick,et al.  GTP-independent tRNA Delivery to the Ribosomal P-site by a Novel Eukaryotic Translation Factor* , 2010, The Journal of Biological Chemistry.

[23]  K. Henrick,et al.  Inference of macromolecular assemblies from crystalline state. , 2007, Journal of molecular biology.

[24]  A. Komar,et al.  Activities of Ligatin and MCT-1/DENR in eukaryotic translation initiation and ribosomal recycling. , 2010, Genes & development.

[25]  A. Teleman,et al.  DENR•MCT-1 Promotes Translation Reinitiation Downstream of uORFs to Control Tissue Growth , 2014, Nature.

[26]  J. L. Jennings,et al.  Systematic identification and functional screens of uncharacterized proteins associated with eukaryotic ribosomal complexes. , 2006, Genes & development.

[27]  Janne Jänis,et al.  Zinc coordination spheres in protein structures. , 2013, Inorganic chemistry.

[28]  K. Mazan-Mamczarz,et al.  MCT-1 protein interacts with the cap complex and modulates messenger RNA translational profiles. , 2006, Cancer research.

[29]  Randy J. Read,et al.  Overview of the CCP4 suite and current developments , 2011, Acta crystallographica. Section D, Biological crystallography.

[30]  Nicholas J. Pace,et al.  Zinc-Binding Cysteines: Diverse Functions and Structural Motifs , 2014, Biomolecules.

[31]  A. Hinnebusch,et al.  Translational control by 5′-untranslated regions of eukaryotic mRNAs , 2016, Science.